mucin core protein expression in colorectal cancers with ...colorectal cancers stratified by...

Mucin Core Protein Expression in Colorectal Cancers with HighLevels of Microsatellite Instability Indicates a Novel Pathwayof Morphogenesis1

Anne-Eve Biemer-Huttmann, Michael D. Walsh,Michael A. McGuckin, Lisa A. Simms,Joanne Young, Barbara A. Leggett, andJeremy R. Jass2

Department of Pathology, Mayne Medical School, University ofQueensland, Herston, Queensland 4006, Australia [A-E. B-H.,M. D. W., J. R. J.]; Mater Medical Research Institute, MaterMisericordiae Hospital, South Brisbane, Queensland 4101, Australia[M. A. M.]; and Conjoint Gastroenterology Laboratory, RoyalBrisbane Hospital, Herston QLD 4029, Australia [L. A. S., J. Y.,B. A. L.]

ABSTRACTParticular mucinous phenotypes have been associated

with serrated epithelial polyps of the colon. These polypsalso show a high frequency of DNA instability. The aim ofthis study was to examine the expression of mucins in colo-rectal cancers that arise through the suppressor and muta-tor pathways. The immunohistochemical distribution of thehuman apomucins MUC1, MUC2, MUC4, and MUC5ACwas determined in 93 sporadic colorectal cancers classifiedpreviously (J. R. Jasset al., J. Clin. Pathol., 52: 455–460,1999) according to levels of DNA microsatellite instability(MSI) as 22 MSI-high (MSI-H), 24 MSI-low (MSI-L), and 47MS stable (MSS). MUC2 expression was observed in 19(86%) MSI-H, 10 (42%) MSI-L, and 15 (32%) MSS cancers(P 5 0.0001); and MUC5AC expression was observed in 17(77%) MSI-H, 8 (33%) MSI-L, and 13 (28%) MSS cancers(P 5 0.0003). There was no association between MUC1 orMUC4 expression and MSI status. The mucinous phenotypedescribed in serrated polyps (MUC21/MUC5AC1) wasseen in 15 (68%) of 22 MSI-H and only 10 (14%) of 71MSI-L/MSS cancers (P < 0.0001). Increased expression ofthe secretory mucins MUC2 and MUC5AC was observed insporadic MSI-H cancers. Identical mucin changes and DNAMSI occurred in serrated polyps of the colorectum, whichsuggests that these lesions may represent precursors ofMSI-H cancers.

INTRODUCTIONThe morphological events underlying the evolution of

colorectal cancer have been described as the adenoma-carci-noma sequence (1). The usual genetic accompaniment of thesequence has been referred to as the “suppressor” pathway inview of the loss of multiple tumor suppressor genes. The beststudied of these isAPC, implicated in adenoma initiation, andTP53, involved in the transition from adenoma to carcinoma (2,3). The underlying mechanism is thought to be an acquired stateof chromosomal instability (4).

A second genetic pathway is driven by a defect in DNArepair caused by the inactivation of DNA mismatch repairgenes. This has been described as the “mutator” pathway be-cause of the widespread accumulation of mutations in repetitivegenomic sequences (2, 5, 6). Tumor suppressor genes are alsotargets in this pathway, but the repertoire is at least in partdetermined by the preferred selection of genes possessing shortrepetitive tracts within their coding sequences. In this regardTGFbRII (7) andBAX (8) might be viewed as substitutes forAPC andTP53. DNA MSI3 is an important biomarker for themutator pathway, which applies to about 10% of sporadic colo-rectal cancers (9–11) as well as to cancers complicating theautosomal dominant condition HNPCC (12).

The adenoma-carcinoma sequence is generally thought toapply in both the suppressor and mutator pathways. MSI-H inHNPCC adenomas is well documented (13–16) but is rarelyreported in sporadic adenomas. One study found no MSI insporadic adenomas (16), but the same authors subsequentlyreported MSI-L in 3 of 49 sporadic adenomas but no MSI-H(17). Others described MSI-L in 7 of 70 sporadic adenomas, butnone was MSI-H (18). A single report documenting MSI in 20of 73 proximal adenomas was not stringent either in excludingHNPCC or in distinguishing MSI-L and MSI-H (19).

It has been suggested that a subset of colorectal cancersmay develop through a different route of morphogenesis. In1990, Longacre and Fenoglio-Preiser (20) characterized a vari-ant of conventional adenoma distinguished by a serrated archi-tecture similar to the HP. They also showed that 37% of serratedadenomas contained foci of high-grade dysplasia, and that car-cinoma-in situoccurred in 10% of serrated adenomas. Thispathway might, therefore, follow a relatively rapid course. Inshowing clonal relationships between the hyperplastic and dys-plastic components of mixed polyps, the concept of a serratedpathway has been broadened to include HPs, mixed polyps, and

Received 12/14/99; revised 2/10/00; accepted 2/16/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by a grant from the Queensland Cancer Fund andNIH Grant 1-UO1 CA74778 (Collaborative Family Registry for Colo-rectal Cancer Studies).2 To whom requests for reprints should be addressed, at Department ofPathology, Mayne Medical School, Herston, QLD 4006, Australia. Phone:61-7-3365-5340; Fax: 61-7-3365-5511; E-mail: [email protected].

3 The abbreviations used are: MSI, microsatellite instability; HNPCC,hereditary nonpolyposis colorectal cancer; MSI-H, high-level MSI;MSI-L, low-level MSI; MSS, stable MSI; HP, hyperplastic polyp; TBS,Tris-buffered saline; TGF, transforming growth factor.

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serrated adenomas as a continuum (21). The same study dem-onstrated a high frequency of MSI in dysplastic epitheliumwithin either mixed polyps or serrated adenomas; 14 (48%) of29 showed MSI-L and 5 (17%) of 29 were MSI-H (21).

Mucins are a diverse family of high-molecular-weight gly-coproteins that are widely expressed by epithelial tissues andcharacterized by the presence of tandem repeat sequences rich inserine and threonine residues that are highlyO-glycosylated(22). Mucins can be classified as either membrane-associated orsecretory glycoproteins. MUC1 and MUC4 encode transmem-brane mucins that are expressed within the intestinal tract.MUC1 is highly expressed in carcinomas and has been impli-cated in colonic tumor progression and metastasis (23, 24).MUC1 has been shown to modulate cell adhesion (25, 26) andis involved in signal transduction (27). MUC4 contains twoextracellular epidermal growth factor-like domains that mayinteract with members of the c-erbfamily of growth factorreceptors and, thereby, modulate growth control (28, 29). Thegenes encoding MUC2, MUC5AC, MUC5B, and MUC6, all ofwhich are gel-forming secretory mucins, are clustered on 11p15(30). These mucins are produced and secreted by goblet cellsand are the major constituents of mucus, which acts to lubricateand protect epithelial tissues (31).

To date, the expression of mucins has not been studied incolorectal cancers stratified by microsatellite status. The rele-vance of such a study is 2-fold: (a) with regard to the possiblehistogenesis of MSI-H cancer through a pathway of serratedneoplasia, we have previously shown (32) that serrated epithe-lial polyps are characterized by increased expression of intesti-nal mucin MUC2, inappropriate expression of gastric mucinMUC5AC, and reduced expression of MUC4; and (b) MSI-Hcancers are distinguished from MSS/MSI-L cancers by a num-ber of clinicopathological features. These include reduced ag-gressiveness and overrepresentation of mucinous cancer (33–36). It is possible that some of these features are related todifferences in mucin expression. In particular, the overrepresen-tation of mucinous cancers among MSI-H colorectal cancersmay be explained by an origin from serrated polyps that showup-regulation of secretory mucins MUC2 and MUC5AC (32).

MATERIALS AND METHODSPatients. Colorectal cancer specimens were obtained, af-

ter informed consent, from 93 patients undergoing surgery at theRoyal Brisbane Hospital between 1989 and 1997. Informationabout patient age, sex, tumor type, site, grade, and stage wasobtained from the hospital charts. There were 45 male and 48female subjects with an average age of 69 years (range, 27–88years). All of the patients were interviewed to exclude a positivefamily history of colorectal cancer. Cancers were located in theproximal colon (40 tumors), distal colon (29 tumors), and rec-tum (23 tumors). All of the cancers showed at least submucosalinvasion and ranged in size from 16–110 mm (mean size, 49mm). Tumor type and grade of differentiation were classifiedhistologically in accordance with WHO criteria (37). Of the 93primary tumors, 74 were adenocarcinomas, and 19 were muci-nous carcinomas. Tumors were staged according to the UnionInternationale Contre Cancer (UICC) tumor-node-metastasis(TNM) classification (38). There were 14 stage I, 46 stage II, 20

stage III, and 12 stage IV carcinomas. An additional femalesubject, age 56 years, with hyperplastic polyposis and six syn-chronous colorectal cancers (two stage I, three stage II, onestage III) was included in this study. Among the 27 polypsobtained from her colectomy specimen and ranging in size from5 to 12 mm were 5 HPs, 5 serrated adenomas, 13 mixed polyps,and 4 adenomas.

Monoclonal Antibodies. Monoclonal antibodies usedwere BC2 (1.5mgzml21) against MUC1 (39), 4F1 (4mgzml21)against MUC2 (40), M4.275 (5mgzml21) against MUC4 (41),and M1 (2mgzml21) against MUC5AC (Neomarkers, Fremont,CA; Ref. 42). BC2, 4F1, and M4.275 were generated usingsynthetic peptide sequences derived from the respective apomu-cin amino acid tandem repeat regions, and M1 was raisedagainst human gastric mucin and subsequently was shown torecognize a COOH-terminal sequence of the MUC5AC coreprotein (43).

Immunohistochemistry. The specimens were fixed for4–16 h in 10% neutral buffered formalin and then routinelywere paraffin-embedded. Histologically normal mucosa fromthe margins of the specimens served as control tissue.

Serial sections were stained immunohistochemically as de-scribed previously (32). Paraffin sections (3–4mm) were af-fixed to Superfrost Plus adhesive slides (Menzel-Glaser, Braun-schweig, Germany) and were air-dried overnight at 37°C.Sections were dewaxed in xylol and rehydrated through de-scending graded alcohols to TBS [0.05M Tris and 0.15M NaCl(pH 7.2–7.4)]. Sections were incubated in 1% periodic acid indistilled H2O for 30 min (MUC1) or in 0.1% porcine trypsin(ICN Biomedicals Australasia, Sydney, Australia) with 0.1%CaCl2 in PBS for 30 min (MUC2), or transferred to 0.01M citricacid buffer (pH 6) and boiled twice for 5 min each and thentransferred to TBS (MUC4, MUC5AC; Ref. 44).

The sections were then incubated in 1.0% H2O2 and 0.1%NaN3 in TBS for 10 min to block endogenous peroxidaseactivity and then were washed in three changes of TBS for 5 mineach. Nonspecific antibody-binding was inhibited by incubatingthe sections in 4% skim milk powder in TBS for 15 min,followed by a brief wash in TBS. The sections were then placedin a humidified chamber and incubated with 10% normal (non-immune) goat serum (Zymed Corp., San Francisco, CA) for 20min. Excess normal serum was decanted from the sections, andthe primary antibody was applied overnight at room temperatureexcept for BC2 (MUC1), which was applied for 60 min.

Sections were washed in three changes of TBS for 5 mineach—the first buffer change contained 0.5% v/v TritonX-100—and then were incubated with biotinylated goat-anti-mouse immunoglobulins (Zymed) for 30 min. Sections werewashed again in three changes of TBS for 5 min each (the firstwash contained 0.1% v/v Triton X-100), were incubated withstreptavidin-horseradish peroxidase conjugate (Zymed) for 15min, and were washed in three changes of TBS for 5 min each.Color was developed in 3,39-diaminobenzidine (Sigma Chemi-cal Company, St. Louis, MO) with H2O2 as substrate for 5 min;then sections were washed in running tap water, lightly coun-terstained in Mayer‘s hematoxylin, dehydrated through ascend-ing graded alcohols, cleared in xylol, and mounted using DePeX(BDH Gurr, Poole, United Kingdom).

As negative controls, serial sections were stained as above

1910Mucin Expression in MSI Colon Cancer



but incubated either with TBS alone (i.e., with omission of theprimary antibody) or with a nonspecific monoclonal antibody,401.21, which is the same immunoglobulin isotype as BC2 andM4.275, and which is directed againsta-gliadin from wheatgluten (45).

Sections were scored by two independent observers(A-E. B-H., J.R.J.) with conflicts resolved using a conferencemicroscope. The proportion of positive cancer cell staining wasgraded as follows: (a) 0 (5 negative); (b),10% (11); (c)11–25% (21).; (d) 26–50% (31); (e) 51–75% (41); and (f).75% (51). The staining intensity of cancer cells was graded asweak (11), moderate (21), or strong (31). The localization wasclassified as either cytoplasmic or cell membrane. Scoring re-sults for intensity and proportion positive were combined bymultiplication for each mucin core protein into a summaryscore. Summary scores were then classified as either high or lowstaining after consideration of the distribution within each sum-mary score variable, to allow statistical comparison with othervariables.

DNA Replication Error Assays. The specimens werederived from a set of sporadic cancers characterized previouslyfor microsatellite status (35, 46). Six additional cancers thatwere obtained from the single subject with hyperplastic poly-posis were also characterized. Briefly, DNA was extracted fromfresh cancer tissue samples for all of the cases except for thetissue from the tumors from the subject with hyperplastic pol-yposis, which were formalin-fixed and paraffin-embedded.DNA was also extracted from matching germ-line control sam-ples derived from either normal colonic mucosa or peripheralblood lymphocytes. The DNA samples were amplified usingPCR at the following loci:MYCL, AT3, D2S123,F13B (47),BAT-26andBAT-40(48). The PCR reactions were performed ina final volume of 20ml, containing 100 ng of genomic DNA, 20pmol of each oligonucleotide primer, 1.25mM dATP, 10 mM

dCTP, dTTP, and dGTP, 6mCi [a-35S]dATP, and 1 unit Taqpolymerase (Boehringer Mannheim, Mannheim, Germany). Re-action conditions consisted of 3 min at 92°C, followed by 31cycles (45 s at 94°C, 1.5 min at 55°C, 1.5 min at 72°C) followedby a final extension for 5 min at 72°C. The PCR products wereelectrophoresed on denaturing 5% polyacrylamide (19:1) gelsand visualized by autoradiography. Cancers were classified asMSI-H if bandshifts occurred in at least 40% of loci (11).

Data Analysis. The x2 test was used to test for signifi-cant associations between variables.

RESULTSImmunohistochemical Localization of MUC1, MUC2,

MUC4, MUC5AC. In the normal mucosa, MUC1 was ex-pressed along the apical membrane and focally in the cytoplasmof goblet and columnar cells of the lower two-thirds of crypts.MUC1 immunoreactivity was often increased in cancers andseen within the glycocalyx intraluminal material and in intracy-toplasmic lumina (invaginations of the apical cell membrane;Fig. 1A). The MUC2 precursor detected by the monoclonalantibody 4F1 was expressed focally in the perinuclear cyto-plasm of normal goblet cells. In cancers, MUC2 was present inthe cytoplasm of cells assumed to be of goblet cell lineage (Fig.1B). In normal mucosa, MUC4 staining was seen in the cyto-

plasm of both goblet and columnar cells. Similar but reducedexpression was observed within the cancers (Fig. 1C). In thenormal cells, focal expression of MUC5AC was seen in thecytoplasm and mucous droplets of goblet cells in a minorproportion of crypts. MUC5AC staining was similar to that ofMUC2 in cancers, although it was more often restricted to cellswith an obvious goblet-cell theca (Fig. 1D).

Mucin Expression, Clinicopathological Features, andMSI Status. High expression of MUC1 was seen in 39 (53%)of 74 adenocarcinomas and in 12 (63%) of 19 mucinous cancers.MUC4 was highly expressed in 25 (34%) of 74 adenocarcino-mas and in 9 (47%) of 19 mucinous cancer. There was nostatistically significant correlation between MUC1 expressionand age, sex, tumor stage, grade, site, type, and MSI-status (seeTable 1). Similarly, there was no significant association betweenMUC4 expression and these features with the exception oftumor stage, in which there was high expression of MUC4 in 11(79%) of 14 stage I tumors compared with only 22 (28%) of 78higher-stage lesions (P5 0.003).

Forty-four (47%) and 38 (41%) of the 93 colorectal cancersamples showed positive staining for MUC2 and MUC5AC,respectively. High levels of MUC2 expression were not signif-icantly associated with age, sex, tumor stage, or grade. How-ever, there was a significant association between high MUC2expression and localization in the proximal colon (P5 0.035),and strong associations with both mucinous tumor type (P ,0.0001) and MSI-H phenotype (P , 0.0001). Similar correla-tions were seen for MUC5AC, with no significant differencebetween MUC5AC expression and tumor stage and grade butwith a trend toward higher expression of MUC5AC in theproximal colon (P5 0.056) and significant associations withmucinous cancer (P5 0.027) and MSI-H phenotype (P50.0003).

Combined MUC21/MUC4-/MUC5AC1 Phenotypeand Modified MUC21/MUC5AC1 Phenotype. The muci-nous phenotype with high expression of MUC2 and MUC5ACand low expression of MUC4 was seen in 5 (7%) of 74 adeno-carcinomas and 8 (42%) of 19 mucinous cancers. There was nostatistically significant correlation between age, sex, stage, orsite and expression of this phenotype. There was a significantassociation between this mucinous phenotype and poorly differ-entiated cancer (P5 0.02), mucinous tumor type (P , 0.0001),and the MSI-H phenotype (P 5 0.0024). Because loss of MUC4was not associated with MSI-H status, associations with themodified mucinous phenotype MUC21/MUC5AC1 were de-termined. This occurred in 15 (68%) of 22 MSI-H cancers, 3(13%) of 24 MSI-L, and 7 (15%) of 47 MSS cancers (P ,0.0001). There were significant associations between the mod-ified mucinous phenotype MUC21/MUC5AC1and grade(P 5 0.02), type (P, 0.0001) and MSI-H status (P, 0.0001).There was also a trend (P5 0.053) between expression of theMUC21/MUCSAC1phenotype and tumor site. Of the MSI-Hcancers with a mucinous phenotype, 10 of 10 showed highMUC2 staining compared with 9 of 12 MSI-H nonmucinouscancers; 9 of 10 showed high MUC5AC reactivity as opposed to8 of 12 nonmucinous tumors; and 9 of 10 showed high MUC2and MUC5AC reactivity as opposed to only 6 of 12 nonmuci-nous tumors.

1911Clinical Cancer Research



Fig. 1 Immunohistochemistry of mucin expression in a moderately differentiated mucinous cancer with MSI-H.A, MUC1 expression within theapical membrane and the abundant extracellular mucin; immunostaining with BC2.B, MUC2 expression within malignant cell cytoplasm;glycosylated MUC2 within the extracellular space is not immunostained; immunostaining with 4F1.C, weak and focal expression only of MUC4 ingoblet and columnar cells; immunostaining with M4.275.D, MUC5AC expression in malignant cells; glycosylated MUC5AC within the extracellularspace is not immunostained; immunostaining with M1.3300.




Hyperplastic Polyposis. Among the six cancers from thesubject with HPs, one was MSS, one was MSI-L, and four wereMSI-H. Three of the MSI-H cancers showed the MUC21/MUC5AC1 phenotype. The fourth was positive for MUC5ACbut lost MUC2 expression. Neither of the non-MSI-H cancerslost MUC4 or expressed MUC5AC, but both retained MUC2.

DISCUSSIONThe definition of mucinous cancer requires that at least

50% of the cancer tissue be composed of mucus (37). About10% of colorectal cancers are mucinous but a higher proportionis observed if the term mucinous is altered to imply up-regula-tion of secretory mucin regardless of the presence of abundantpools. If this more biologically relevant definition is adopted inthe case of MUC2, then 44 (47%) of 93 of cancers in the presentstudy were mucinous. This high figure is only partly explainedby enrichment of the series with MSI-H cancers, of which 19(86%) of 22 were mucinous according to the altered definition,because 25 (35%) of 71 MSS/MSI-L cancers were mucinousalso. Similar findings were observed for the aberrantly up-regulated gastric mucin MUC5AC (Table 2). These observa-tions indicate a switch toward the mucous cell lineage withinMSI-H cancers.

The increase in MUC1 expression and decrease in MUC4expression was independent of microsatellite status. Becauseneither MUC1 nor MUC4 were expressed differentially accord-ing to microsatellite status it is unlikely that either moleculewould explain the reduced aggressiveness of MSI-H cancers

(33–35). Indeed, MUC1 expression did not differ by stage,although loss of MUC4 was more evident in higher-stage tu-mors (P5 0.003).

Although the mucinous phenotype MUC21/MUC4-/MUC5AC1 was associated with MSI-H cancers, the effect isexplained by the changes in MUC2 and MUC5AC rather thanMUC4. The association of the modified phenotype (MUC21/MUC5AC1) with poor differentiation (Table 1) fits with theknown tendency for MSI-H cancers to be poorly differentiated(33–36). Ten (45%) of 22 MSI-H cancers were poorly differ-entiated in this study, and 8 (80%) of 10 showed the MUC21/MUC5AC1 phenotype. The modified phenotype MUC21/MUC5AC1 was also highly correlated with typing as mucinouscarcinoma. Twelve (63%) of 19 mucinous cancers showed thisphenotype, which was seen in only 13 (18%) of 74 adenocar-

Table 2 Comparison of high mucin expression with MSI Status

High mucin expressionMSS

(n 5 47)MSI-L

(n 5 24)MSI-H

(n 5 22) P

MUC1 23 (49%) 14 (58%) 14 (64%) NSa

MUC2 15 (32%) 10 (42%) 19 (86%) 0.0001MUC4 20 (43%) 6 (25%) 8 (36%) NSMUC5AC 13 (28%) 8 (33%) 17 (77%) 0.0003MUC21/MUC42/

MUC5AC13 (6%) 2 (8%) 8 (36%) 0.002

MUC21/MUC5AC1 7 (15%) 3 (13%) 15 (68%) 0.0001a NS, not significant.

Table 1 Clinicopathological features and mucin expression

Variables MUC1 MUC2 MUC4 MUC5AC MUC21/5AC1

Age,60 (n 5 17) 9 (53%) 6 (35%) 8 (47%) 6 (35%) 3 (18%).60 (n 5 76) 42 (55%) 38 (50%) 26 (34%) 32 (42%) 22 (29%)P NSa NS NS NS NS

SexMale (n 5 45) 25 (56%) 22 (49%) 14 (31%) 18 (40%) 10 (22%)Female (n5 48) 26 (54%) 22 (46%) 20 (42%) 20 (42%) 15 (31%)P NS NS NS NS NS

TNM stageI (n 5 14) 8 (57%) 9 (64%) 11 (79%) 7 (50%) 6 (43%)II (n 5 46) 24 (52%) 19 (41%) 14 (30%) 20 (44%) 13 (28%)III (n 5 20) 11 (55%) 8 (40%) 4 (20%) 8 (40%) 4 (20%)IV (n 5 12) 8 (67%) 7 (58%) 4 (33%) 2 (17%) 1 (8%)P NS NS 0.003 NS NS

GradeWell (n 5 6) 4 (67%) 3 (50%) 3 (50%) 3 (50%) 3 (50%)Moderate (n5 65) 32 (49%) 27 (42%) 26 (40%) 23 (35%) 12 (18%)Poor (n5 22) 15 (68%) 14 (64%) 5 (23%) 12 (55%) 10 (50%)P NS NS NS NS 0.02b

SiteProximal (n5 40) 25 (63%) 25 (63%) 18 (45%) 22 (55%) 16 (40%)Distal (n 5 29) 13 (45%) 12 (41%) 11 (38%) 8 (28%) 5 (17%)Rectum (n5 23) 12 (52%) 7 (30%) 5 (22%) 8 (35%) 4 (17%)P NS 0.035 NS 0.056 0.053

TypeAdenoca (n5 74) 39 (53%) 27 (36%) 25 (34%) 26 (35%) 13 (18%)Mucinous (n5 19) 12 (63%) 17 (89%) 9 (47%) 12 (63%) 12 (63%)P NS 0.0001 NS 0.027 0.0001

a NS, not significant; TNM, tumor-node-metastasis.b Well and moderateversuspoor.




cinomas (P, 0.0001). This fits with the overrepresentation ofmucinous cancer among the MSI-H subset (33–36). There wasa tendency for MUC21, MUC5AC1, and MUC21/MUC5AC1 cancers to be proximal, although this reached sta-tistical significance only for MUC2 (Table 1). Again, this fitswith the predilection by MSI-H cancers for the proximal colon.The mechanism for up-regulation of the mucinous phenotype inMSI-H cancers is unknown. One possibility could be abrogationof the TGF-b signaling pathway resulting in an uncontrolledautocrine effect. TGF-bis known to down-regulate colonocytedifferentiation, whereas goblet cells are resistant to the effects ofTGF-b (49). Hakomori (50) described significant perturbationsin O-glycosylation of tumor-associated mucins, predominantlyin the form of carbohydrate chain truncation and aberrant siala-tion or fucosylation. The core protein epitopes that are recog-nized by many antimucin antibodies, including those used in thisstudy, are potentially masked by glycosylation. It is conceivablethat the increased immunoreactivity in tumors is attributable, atleast in part, to the exposure of core protein that is available forantibody binding without necessarily having increased proteinsynthesis. However, there is no evidence to date that posttrans-lational modifications differ in colorectal cancers according tomicrosatellite status. It is more likely that the differences in thedetection of core protein relate to actual mucin expressionchanges associated with secretory lineage differentiation (32).

In this study, the finding that the mucinous phenotypeMUC21/MUC5AC1 is seen in 15 (68%) of 22 MSI-H cancersbut only 10 (14%) of 71 of the MSS/MSI-L cancers (P ,0.0001) is unexpected insofar as it associates the mucinousphenotype of serrated adenomas with MSI-H colorectal cancer(32). Nevertheless, the data fit with our earlier observations in aseries of sporadic serrated polyps in which 5 (17%) of 29dysplastic samples from mixed hyperplastic/adenomatous pol-yps or serrated adenomas were MSI-H (21).

It could be argued that sporadic MSI-H cancers do arisefrom conventional adenomas as is the case in HNPCC adenomas(13–15, 51). This would be feasible if MSI-H were acquired atthe point of transition from adenoma to carcinoma (analogous toTP53 in MSS cancers). However, this is not supported bymolecular data. The tumor suppressor geneAPCplays a criticalrole in the initiation of colorectal adenoma. MSI-H cancerswould, therefore, be expected to showAPC alterations if theadenoma-carcinoma sequence applied. Yet, an extremely lowrate ofAPC mutation has been reported in MSI-H cancers (52,53), even after full-length sequencing of APC derived fromMSI-H colorectal cell lines (54). Two studies failed to show asingle example of 5q loss of heterozygosity in a total of 29MSI-H colorectal cancers (46, 52). Furthermore, aberrant (nu-clear) immunolocalization ofb-catenin is rarely observed inMSI-H cancers, which suggests that the entire WNT signalingpathway is intact (46). A higher frequency ofAPC mutation inMSI cancers (although still significantly lower then in MSScancers) has been reported (55). However, 19 of the 52 cancerswith DNA replication errors were from subjects with HNPCCand 6 of 9 DNA markers used in this study were for dinucleotiderepeats, which may show instability in MSI-L cancers.

We suggested previously that MSI-L colorectal cancermight arise through neoplastic transformation of a HP (21). Itnow seems likely that some, if not all, MSI-H cancers arise

through a similar morphogenetic pathway. This would explainthe infrequency of both sporadic MSI-H adenomas andAPCalterations in MSI-H cancers. The mechanism could be throughtransformation of a HP or a serrated adenoma arisingde novo.We do not believe that all of the colorectal cancers arisingthrough the serrated pathway would be MSI-H. Although wehave observed MSI-H cancers arising in association with hy-perplastic polyposis (56), MSS and MSI-L cancers occur in thesame condition (57). Similar findings were seen in the subjectwith multiple colorectal cancers and hyperplastic polyposis de-scribed in this study. However, it seems probable that many, ifnot all, MSI-H cancers do arise through the alternative serratedpathway. Given the numerous clinical, pathological, and molec-ular differences between MSI-positive and MSI-negative can-cers that have now been demonstrated, it would perhaps besurprising if differences in early morphogenesis did not existalso.

ACKNOWLEDGMENTSWe thank Estelle Schoch and Nigel Misso for their excellent

technical assistance, and Clay Winterford for generously contributinghis photographic skills. We also thank Dr. David Cohn of QueenslandMedical Laboratory, Brisbane, for providing the case of hyperplasticpolyposis.

REFERENCES1. Morson, B. C., Bussey, H. J., Day, D. W., and Hill, M. J. Adenomasof the large bowel. Cancer Surv.,2: 451–477, 1983.2. Kinzler, K. W., and Vogelstein, B. Lessons from hereditary colorec-tal cancer. Cell,87: 159–170, 1996.3. Vogelstein, B., Fearon, E. R., Kern, S. E., Hamilton, S. R., Preis-inger, A. C., Nakamura, Y., and White, R. Allelotype of colorectalcarcinomas. Science (Washington DC),244: 207–211, 1989.4. Lengauer, C., Kinzler, K. W., and Vogelstein, B. Genetic instabilitiesin human cancers. Nature (Lond.),396: 643–649, 1998.5. Perucho, M., Peinado, M. A., Ionov, Y., Casares, S., Malkhosyan, S.,and Stanbridge, E. Defects in replication fidelity of simple repeatedsequences reveal a new mutator mechanism for oncogenesis. ColdSpring Harb. Symp. Quant. Biol.,59: 339–348, 1994.6. Boland, C. R., Sato, J., Saito, K., Carethers, J. M., Marra, G., Laghi,L., and Chauhan, D. P. Genetic instability and chromosomal aberrationsin colorectal cancer: a review of the current models. Cancer Detect.Prev.,22: 377–382, 1998.7. Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutter-baugh, J., Fan, R. S., Zborowska, E., Kinzler, K. W., and Vogelstein, B.Inactivation of the type II TGF-breceptor in colon cancer cells withmicrosatellite instability. Science (Washington DC),268: 1336–1338,1995.8. Rampino, N., Yamamoto, H., Ionov, Y., Li, Y., Sawai, H., Reed,J. C., and Perucho, M. Somatic frameshift mutations in theBAXgene incolon cancers of the microsatellite mutator phenotype. Science (Wash-ington DC),275: 967–969, 1997.9. Ionov, Y., Peinado, M. A., Malkhosyan, S., Shibata, D., and Perucho,M. Ubiquitous somatic mutations in simple repeated sequences reveal anew mechanism for colonic carcinogenesis. Nature (Lond.),363: 558–561, 1993.10. Thibodeau, S. N., Bren, G., and Schaid, D. Microsatellite instabilityin cancer of the proximal colon. Science (Washington DC),260: 816–819, 1993.11. Boland, C. R., Thibodeau, S. N., Hamilton, S. R., Sidransky, D.,Eshleman, J. R., Burt, R. W., Meltzer, S. J., Rodriguez-Bigas, M. A.,Fodde, R., Ranzani, G. N., and Srivastava, S. A National CancerInstitute Workshop on Microsatellite Instability for cancer detection and




familial predisposition: development of international criteria for thedetermination of microsatellite instability in colorectal cancer. CancerRes.,58: 5248–5257, 1998.

12. Aaltonen, L. A., Peltomaki, P., Mecklin, J. P., Jarvinen, H., Jass,J. R., Green, J. S., Lynch, H. T., Watson, P., Tallqvist, G., and Juhola,M. Replication errors in benign and malignant tumors from hereditarynonpolyposis colorectal cancer patients. Cancer Res.,54: 1645–1648,1994.

13. Akiyama, Y., Iwanaga, R., Saitoh, K., Shiba, K., Ushio, K., Ikeda,E., Iwama, T., Nomizu, T., and Yuasa, Y. Transforming growth factorb type II receptor gene mutations in adenomas from hereditary non-polyposis colorectal cancer. Gastroenterology,112: 33–39, 1997.

14. Yagi, O. K., Akiyama, Y., Nomizu, T., Iwama, T., Endo, M., andYuasa, Y. Proapoptotic geneBax is frequently mutated in hereditarynonpolyposis colorectal cancers but not in adenomas. Gastroenterology,114: 268–274, 1998.

15. Jacoby, R. F., Marshall, D. J., Kailas, S., Schlack, S., Harms, B.,and Love, R. Genetic instability associated with adenoma to carcinomaprogression in hereditary nonpolyposis colon cancer. Gastroenterology,109: 73–82, 1995.

16. Young, J., Leggett, B., Gustafson, C., Ward, M., Searle, J., Thomas,L., Buttenshaw, R., and Chenevix-Trench, G. Genomic instability oc-curs in colorectal carcinomas but not in adenomas. Hum. Mutat.,2:351–354, 1993.

17. Young, J., Searle, J., Buttenshaw, R., Thomas, L., Ward, M.,Chenevix-Trench, G., and Leggett, B. An Alu VpA marker on chromo-some I demonstrates that replication errors manifest at the adenoma-carcinoma transition in sporadic colorectal tumors. Genes Chromo-somes Cancer,12: 251–254, 1995.

18. Lothe, R. A., Andersen, S. N., Hofstad, B., Meling, G. I., Peltomaki,P., Heim, S., Brøgger, A., Vatn, M., Rognum, T. O., and Børresen, A. L.Deletion of 1p loci and microsatellite instability in colorectal polyps.Genes Chromosomes Cancer,14: 182–188, 1995.

19. Grady, W. M., Rajput, A., Myeroff, L., Liu, D. F., Kwon, K. H.,Willis, J., and Markowitz, S. Mutation of the type II transforminggrowth factor-breceptor is coincident with the transformation of humancolon adenomas to malignant carcinomas. Cancer Res.,58: 3101–3104,1998.

20. Longacre, T. A., and Fenoglio-Preiser, C. M. Mixed hyperplasticadenomatous polyps/serrated adenomas. A distinct form of colorectalneoplasia. Am. J. Surg. Pathol.,14: 524–537, 1990.

21. Iino, H., Jass, J. R., Simms, L. A., Young, J., Leggett, B., Ajioka,Y., and Watanabe, H. DNA microsatellite instability in hyperplasticpolyps, serrated adenomas, and mixed polyps: a mild mutator pathwayfor colorectal cancer? J. Clin. Pathol.,52: 5–9, 1999.

22. Gendler, S. J., and Spicer, A. P. Epithelial mucin genes. Annu. Rev.Physiol.,57: 607–634, 1995.

23. Aoki, R., Tanaka, S., Haruma, K., Yoshihara, M., Sumii, K.,Kajiyama, G., Shimamoto, F., and Kohno, N. MUC-1 expression as apredictor of the curative endoscopic treatment of submucosally invasivecolorectal carcinoma. Dis. Colon Rectum,41: 1262–1272, 1998.24. Hiraga, Y., Tanaka, S., Haruma, K., Yoshihara, M., Sumii, K.,Kajiyama, G., Shimamoto, F., and Kohno, N. Immunoreactive MUC1expression at the deepest invasive portion correlates with prognosis ofcolorectal cancer. Oncology,55: 307–319, 1998.25. Wesseling, J., van der Valk, S. W., Vos, H. L., Sonnenberg, A., andHilkens, J. Episialin (MUC1) overexpression inhibits integrin-mediatedcell adhesion to extracellular matrix components. J. Cell Biol.,129:255–265, 1995.26. Suwa, T., Hinoda, Y., Makiguchi, Y., Takahashi, T., Itoh, F.,Adachi, M., Hareyama, M., and Imai, K. Increased invasiveness ofMUC1 cDNA-transfected human gastric cancer MKN74 cells. Int. J.Cancer,76: 377–382, 1998.27. Zrihan-Licht, S., Baruch, A., Elroy-Stein, O., Keydar, I., andWreschner, D. H. Tyrosine phosphorylation of the MUC1 breast cancermembrane proteins. Cytokine receptor-like molecules. FEBS Letts.,356: 130–136, 1994.

28. Rossi, E. A., McNeer, R. R., Price-Schiavi, S. A., Van den Brande,J. M. H., Komatsu, M., Thompson, J. F., Carraway, C. A. C., Fregien,N. L., and Carraway, K. L. Sialomucin complex, a heterodimeric gly-coprotein complex-expression as a soluble, secretable form in lactatingmammary gland and colon. J. Biol. Chem.,271: 33476–33485, 1996.

29. Moniaux, N., Nollet, S., Porchet, N., Degand, P., Laine, A., andAubert, J. P. Complete sequence of the human mucin MUC4: a putativecell membrane-associated mucin. Biochem. J.,338: 325–333, 1999.

30. Pigny, P., Guyonnet-Duperat, V., Hill, A. S., Pratt, W. S., Galiegue-Zouitina, S., D’Hooge, M. C., Laine, A., van Seuningen, I., Degand, P.,Gum, J. R., Kim, Y. S., Swallow, D. M., Aubert, J. P., and Porchet, N.Human mucin genes assigned to 11p15.5—identification and organiza-tion of a cluster of genes. Genomics,38: 340–352, 1996.

31. Neutra, M. F., and Forstner, J. F. Gastrointestinal mucus: synthesis,secretion, and function.In: L. R. Jonson (ed.), Physiology of theGastrointestinal Tract, pp. 975–1009. New York: Raven Press, 1987.

32. Biemer-Huttmann, A. E., Walsh, M. D., McGuckin, M. A., Ajioka,Y., Watanabe, H., Leggett, B. A., and Jass, J. R. Immunohistochemicalstaining patters of MUC1, MUC2, MUC4, and MUC5AC mucins inhyperplastic polyps, serrated adenomas, and traditional adenomas of thecolorectum. J. Histochem. Cytochem.,47: 1039–1048, 1999.

33. Kim, H., Jen, J., Vogelstein, B., and Hamilton, S. R. Clinical andpathological characteristics of sporadic colorectal carcinomas with DNAreplication errors in microsatellite sequences. Am. J. Pathol.,145:148–156, 1994.

34. Lothe, R. A., Peltomaki, P., Meling, G. I., Aaltonen, L. A.,Nystrom-Lahti, M., Pylkkanen, L., Heimdal, K., Andersen, T. I., Moller,P., and Rognum, T. O. Genomic instability in colorectal cancer: rela-tionship to clinicopathological variables and family history. CancerRes.,53: 5849–5852, 1993.

35. Jass, J. R., Do, K. A., Simms, L. A., Iino, H., Wynter, C., Pillay,S. P., Searle, J., Radford-Smith, G., Young, J., and Leggett, B. Mor-phology of sporadic colorectal cancer with DNA replication errors. Gut,42: 673–679, 1998.

36. Messerini, L., Vitelli, F., De Vitis, L. R., Mori, S., Calzolari, A.,Palmirotta, R., Calabro, A., and Papi, L. Microsatellite instability insporadic mucinous colorectal carcinomas: relationship to clinico-path-ological variables. J. Pathol.,182: 380–384, 1997.

37. Jass, J. R., and Sobin, L. H. World Health Organization Interna-tional Histological Classification of Tumours. Histological typing ofintestinal tumours. Berlin: Springer-Verlag, 1989.

38. Sobin, L. H., and Wittekind, C. Colon and rectum.In: L. H. Sobinand C. Wittekind (eds.), UICC International Union Against Cancer.TNM Classification of Malignant Tumours, pp. 66–69. New York:Wiley-Liss, 1997.

39. Xing, P. X., Tjandra, J. J., Stacker, S. A., Teh, J. G., Thomps, C. H.,McLaughlin, P. J., and McKenzie, I. F. Monoclonal antibodies reactivewith mucin expressed in breast cancer. Immunol. Cell Biol.,67: 183–195, 1989.40. Devine, P. L., McGuckin, M. A., Birrell, G. W., Whitehead, R. H.,Sachdev, G. P., Shield, P., and Ward, B. G. Monoclonal antibodiesreacting with the MUC2 mucin core protein. Br. J. Cancer,67: 1182–1188, 1993.41. Xing, P. X., Prenzoska, J., Apostolopoulos, V., Karkaloutsos, J.,and McKenzie, I. F. C. Monoclonal antibodies to a MUC 4 peptide reactwith lung cancer. Int. J. Oncol.,11: 289–295, 1997.42. Bara, J., Gautier, R., Daher, N., Zaghouani, H., and Decaens, C.Monoclonal antibodies against oncofetal mucin M1 antigens associatedwith precancerous colonic mucosae. Cancer Res.,46: 3983–3989, 1986.43. Bara, J., Chastre, E., Mahiou, J., Singh, R. L., Forgue-Lafitte, M. E.,Hollande, E., and Godeau, F. Gastric M1 mucin, an early oncofetalmarker of colon carcinogenesis, is encoded by theMUC5ACgene. Int.J. Cancer,75: 767–773, 1998.44. Shi, S. R., Key, M. E., and Kalra, K. L. Antigen retrieval informalin-fixed, paraffin embedded tissues: an enhancement method forimmunohistochemical staining based upon microwave oven heating oftissue sections. J. Histochem. Cytochem.,39: 741–748, 1991.




45. Hill, A. S., and Skerritt, J. H. Monoclonal antibody-based two-siteenzyme-immunoassays for wheat gluten proteins. 1. Kinetic character-istics and comparison with other ELISA formats. Food Agric. Immunol.,1: 147–160, 1989.46. Jass, J. R., Biden, K. G., Cummings, M. C., Simms, L. A., Walsh,M., Schoch, E., Meltzer, S. J., Wright, C., Searle, J., Young, J., andLeggett, B. A. Characterisation of a subtype of colorectal cancer com-bining features of the suppressor and mild mutator phenotypes. J. Clin.Pathol.,52: 455–460, 1999.47. Souza, R. F., Lei, J. Y., Yin, J., Appel, R., Zou, T. T., Zhou, X. L.,Wang, S., Rhyu, M. G., Cymes, K., Chan, O., Park, W. S., Krasna, M. J.,Greenwald, B. D., Cottrell, J., Abraham, J. M., Simms, L., Leggett, B.,Young, J., Harpaz, N., and Meltzer, S. J. A transforming growth factorb1 receptor type II mutation in ulcerative colitis-associated neoplasms.Gastroenterology,112: 40–45, 1997.48. Parsons, R., Myeroff, L. L., Liu, B., Willson, J. K. V., Markowitz,S. D., Kinzler, K. W., and Vogelstein, B. Microsatellite instability andmutations of the transforming growth factorb type II receptor gene incolorectal cancer. Cancer Res.,55: 5548–5550, 1995.49. Deng, X. B., Bellis, S., Yan, Z. F., and Friedman, E. Differentialresponsiveness to autocrine and exogenous transforming growth factor(TGF) b1 in cells with nonfunctional TGF-breceptor type III. CellGrowth Differ., 10: 11–18, 1999.50. Hakomori, S. I. Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv. Cancer Res.,52: 257–333,1989.51. Tsao, J. L., Tavare, S., Salovaara, R., Jass, J. R., Aaltonen, L. A.,and Shibata, D. Colorectal adenoma and cancer divergence— evi-

dence of multilineage progression. Am. J. Pathol.,154: 1815–1824,1999.

52. Olschwang, S., Hamelin, R., Laurent-Puig, P., Thuille, B., DeRycke, Y., Li, Y. J., Muzeau, F., Girodet, J., Salmon, R. J., and Thomas,G. Alternative genetic pathways in colorectal carcinogenesis. Proc. Natl.Acad. Sci. USA,94: 12122–12127, 1997.

53. Salahshor, S., Kressner, U., Påhlman, L., Glimelius, B., Lindmark,G., and Lindblom, A. Colorectal cancer with and without microsatelliteinstability involves different genes. Genes Chromosomes Cancer,26:247–252, 1999.

54. Heinen, C. D., Richardson, D., White, R., and Groden, J. Micro-satellite instability in colorectal adenocarcinoma cell lines that havefull-length adenomatous polyposis coli protein. Cancer Res.,55: 4797–4799, 1995.

55. Huang, J., Papadopoulos, N., McKinley, A. J., Farrington, S. M.,Curtis, L. J., Wyllie, A. H., Zheng, S., Willson, J. K., Markowitz, S. D.,Morin, P., Kinzler, K. W., Vogelstein, B., and Dunlop, M. G. APCmutations in colorectal tumors with mismatch repair deficiency. Proc.Natl. Acad. Sci. USA,93: 9049–9054, 1996.

56. Jass, J. R., Cottier, D. S., Pokos, V., Parry, S., and Winship, I. M.Mixed epithelial polyps in association with hereditary non polyposiscolorectal cancer providing an alternative pathway of cancer histogen-esis. Pathology,29: 28–33, 1997.

57. Jeevaratnam, P., Cottier, D. S., Browett, P. J., Van de Water, N. S.,Pokos, V., and Jass, J. R. Familial giant hyperplastic polyposis predis-posing to colorectal cancer—a new hereditary bowel cancer syndrome.J. Pathol.,179: 20–25, 1996.




2000;6:1909-1916. Clin Cancer Res Anne-Eve Biemer-Hüttmann, Michael D. Walsh, Michael A. McGuckin, et al. Pathway of MorphogenesisHigh Levels of Microsatellite Instability Indicates a Novel Mucin Core Protein Expression in Colorectal Cancers with

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