CT-X antigen expression in human breast cancerAnita Grigoriadisa,1,2, Otavia L. Caballerob,1,3, Keith S. Hoekc, Leonard da Silvad, Yao-Tseng Chene, Sandra J. Shine,Achim A. Jungbluthb, Lance D. Millerf, David Cloustong, Jonathan Cebong, Lloyd J. Oldb,3, Sunil R. Lakhanid,Andrew J. G. Simpsonb, and A. Munro Nevillea

aLudwig Institute for Cancer Research, 605 Third Avenue, New York, NY 10158; bLudwig Institute for Cancer Research, New York Branch at MemorialSloan–Kettering Cancer Center, 1275 York Avenue, New York, NY 10021; cDepartment of Dermatology, University Hospital of Zurich, Gloriastrasse 31/F2,CH-8091 Zurich, Switzerland; dMolecular and Cellular Pathology, UQ Centre for Clinical Research and The School of Medicine, Level 6 Building 71/918,University of Queensland, The Royal Brisbane and Women’s Hospital, Herston 4029, Brisbane, Queensland, Australia; eWeill Medical College of CornellUniversity, 1300 York Avenue, New York, NY 10021; fDepartment of Cancer Biology, Wake Forest University School of Medicine, Medical Center Boulevard,Hanes Building, Winston-Salem, NC 27157; and gAustin Health and the Cancer Vaccine Laboratory at the Ludwig Institute for Cancer Research, AustinHospital, Heidelberg 3084, Australia

Contributed by Lloyd J. Old, June 18, 2009 (sent for review April 29, 2009)

Cancer/testis (CT) genes are predominantly expressed in humangerm line cells, but not somatic tissues, and frequently becomeactivated in different cancer types. Several CT antigens havealready proved to be useful biomarkers and are promising targetsfor therapeutic cancer vaccines. The aim of the present study wasto investigate the expression of CT antigens in breast cancer. Usingpreviously generated massively parallel signature sequencing(MPSS) data, together with 9 publicly available gene expressiondatasets, the expression pattern of CT antigens located on the Xchromosome (CT-X) was interrogated. Whereas a minority ofunselected breast cancers was found to contain CT-X transcripts, asignificantly higher expression frequency was detected in estrogenand progesterone receptor (ER) negative breast cancer cell linesand primary breast carcinomas. A coordinated pattern of CT-Xantigen expression was observed, with MAGEA and NY-ESO-1/CTAG1B being the most prevalent antigens. Immunohistochemicalstaining confirmed the correlation of CT-X antigen expression andER negativity in breast tumors and demonstrated a trend for theircoexpression with basal cell markers. Because of the limited ther-apeutic options for ER-negative breast cancers, vaccines based onCT-X antigens might prove to be useful.

cancer/testis antigens � estrogen receptor � therapy

Cancer/testis (CT) antigens are encoded by a unique group ofgenes that are predominantly expressed in human germ line

cells, have little or no expression in somatic adult tissues, butbecome aberrantly activated in various malignancies (1). A totalof 153 CT antigens has been described to date and are compiledin the CT database (www.cta.lncc.br/) (2, 3). Of these antigens,83 are encoded by multigene families located on the X-chromo-some and are referred to as the CT-X antigens (1). Althoughtheir possible involvement in chromosomal recombination, tran-scription, translation and signaling has been proposed, thephysiological function of the great majority of CT-X antigensremains poorly elucidated (1, 4).

The expression of CT-X antigens varies greatly between tumortypes, being most frequent in melanomas, bladder, non-small celllung, ovarian, and hepatocellular carcinomas. The occurrence ofCT-X antigens is uncommon in renal, colon, and gastric cancers(4). Where present, CT-X expression is associated with a pooreroutcome and tends to be more frequent in higher grade lesionsand advanced disease (5–8).

The combination of their restricted expression, and in somecases potent immunogenicity, has led to intense research intotheir utilization in therapeutic vaccines (9). Clinical trials ofvaccines containing the CT-X antigens MAGEA and NY-ESO-1/CTAG1B are underway in patients with several cancers,including those of the lung, ovary, and melanoma (10–16).

Relatively few studies have explored the expression pattern ofCT-X antigens in breast cancer and the few cases studied to datehave focused on NY-ESO-1/CTAG1B (17–21). The objective of

the present study was to undertake a more comprehensiveanalysis of CT-X antigen expression in primary breast cancer inthe context of clinicopathological parameters. The results pointto a restricted expression of members of the MAGEA andNY-ESO-1/CTAG1B gene families, primarily in ER negativetumors, some of which belong to the basal phenotype. Suchlesions have a poorer prognosis for which, currently, therapeuticoptions are limited. CT-X-based immunotherapy strategies maythus represent an important therapeutic option for patients withthese subtypes of breast tumors.

ResultsDetection of CT-X Antigen Expression in Massively Parallel SignatureSequencing Data. As an initial step in exploring CT-X antigenexpression in breast cancer, we interrogated our previouslypublished MPSS data (22). These data were derived from a poolof normal human breast luminal epithelial cells, a pool ofpredominantly ER-positive epithelial enriched primary breasttumors and 4 breast epithelial cell lines (22, 23). Sequence tagscorresponding to 6 of the 83 CT-X antigens, including those forMAGEA (1,646 transcripts per million [tpm]), CSAG2 (680tpm), CT45 (263 tpm), PASD1 (24 tpm), CSAG1 (15 tpm), andFMR1NB/NY-SAR-35 (11 tpm), were detected in only onesample, an ER-negative breast cell line BT20.

CT-X Antigen Expression in Breast Cancer Gene Expression Studies. Tofurther examine the possible relationship of CT-X expressionwith ER status, a list of 66 Affymetrix probe sets identifying 65different CT-X-encoding genes was prepared (supporting infor-mation (SI) Table S1). Nine published microarray-based geneexpression datasets derived from a total of 1,259 primary breasttumors and 51 breast cancer cell lines were available, and the ERstatus was known in most. Four hundred three of 1,310 sampleswere ER-negative (Table 1). Using the HG-U133A platform, weinterrogated for each dataset gene expression patterns differ-entiating between ER-negative and ER-positive samples. Ap-plying multiple testing controls, a P value cut-off of 0.05 and a2-fold change filter, this analysis identified a set of 147 probe sets

Author contributions: A.G., O.L.C., A.J.G.S., and A.M.N. designed research; A.G., O.L.C.,K.S.H., L.d.S., Y.-T.C., S.J.S., A.A.J., D.C., J.C., and S.R.L. performed research; Y.-T.C., S.J.S.,L.D.M., and S.R.L. contributed new reagents/analytic tools; A.G., O.L.C., K.S.H., L.d.S.,Y.-T.C., L.D.M., S.R.L., A.J.G.S., and A.M.N. analyzed data; and A.G., O.L.C., K.S.H., L.d.S.,Y.-T.C., L.J.O., S.R.L., A.J.G.S., and A.M.N. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

1A.G. and O.L.C. contributed equally to this work.

2Present address: Breakthrough Breast Cancer Research Unit, Guy’s Hospital, King’s HealthPartners AHSC, London, United Kingdom.

3To whom correspondence may be addressed. E-mail: caballeo@mskcc.org or lold@licr.org.

This article contains supporting information online at www.pnas.org/cgi/content/full/0906840106/DCSupplemental.

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(131 genes) that showed significant differential expression be-tween ER-negative and ER-positive breast tumors in at least 5of the 9 datasets investigated. This list represents the ER specificexpression (ERSE) set (Table S2). Many of the ERSE geneshave previously been identified as differentially expressed inbreast tumors in an ER status-dependent manner, or to be directER target genes (24–26).

The ERSE set was used to cluster the samples in each dataset(Fig. S1), confirming that ER status annotation was consistentin each dataset and that a CT-X-specific analysis would probablynot be confounded by extraneous errors of annotation or datagathering. For each dataset, CT-X gene expression patterns wereassessed for their relationship with ER status. This analysis usedan identical test as was used to find the ERSE, except only theCT-X genes were tested and no fold-change filter was applied.The data are displayed as a summary (Table 2) and from thecontext of individual datasets (Tables S3–S11). To examine theenrichment of CT-X in the ER-negative tumors, we clustered thebreast tumor datasets using the normalized CT-X expressiondata (Fig. 1). This analysis shows that relatively few CT-X genesare strongly expressed in breast cancer samples. For 3 datasets[Neve (27), Boersma (28), and Hess (29)], no statistically sig-nificant associations were found until multiple testing controlswere subtracted from the analysis. The genes encodingCTAG1A/CTAG1B and CTAG2 (NY-ESO-1/CTAG1B family)showed the most consistent relationship with ER status(Table 2 and Fig. 2), being preferentially expressed in ER-negative samples (median adjusted P value �0.006). Other CT-Xantigens showed consistent, but ultimately non-significant, re-lationships with ER status–notably MAGEA3 and MAGEA6(Tables S3–S11).

A similar analysis was performed for examining the correla-

tion of CT-X expression and PR status, p53 mutation and HER2status where available (Table 1). For each metric, the completedatasets of the above mentioned 9 breast tumor cohorts wereinterrogated for a significant relationship with metric status, andprobe sets with less than a 2-fold change were discarded. Thisanalysis was repeated for the CT-X antigen list (Table S1). Thereis a very strong overlap between the Doane (30) and Minn (31)2-fold PR significant lists (137 probe sets, PRSE) (Table S12).The PRSE list also shared 105 probe sets in common with theERSE list (P value �0.001), confirming that PR and ERstatus-specific gene expression patterns are tightly linked. In theMinn (31) dataset, 3 CT-X antigens (MAGEA6, MAGEA3 and

Table 1. Datasets used in this study

Name Accession no.

Samples Available annotations

ReferenceERneg ERpos Unknown ER PR p53 HER2

Boersma GSE5847 26 21 1 Yes No No No 28Desmedt GSE7390 64 134 — Yes No No No 46Doane Website* 42 57 — Yes Yes No Yes 30Hess Website† 51 82 — Yes Yes No Yes 29Ivshina GSE4922 34 211 4 Yes No Yes No 44Minn GSE2603 42 57 22 Yes No No Yes 31Neve E-TABM-157 33 18 — Yes Yes Yes Yes 27Sotiriou GSE2990 34 85 6 Yes No No No 47Wang GSE2034 77 209 — Yes No No No 45vandeVijver Website‡ 69 226 — Yes No No No 34

*https://caarraydb.nci.nih.gov/caarray/.†http://bioinformatics.mdanderson.org/pubdata.html.‡http://microarray-pubs.stanford.edu/wound�NKI/.

Table 2. ER status-specific CT-X gene expression (summary)

Probe set Gene symbol Median P value

211674�x�at CTAG1A CTAG1B 0.005210546�x�at CTAG1A CTAG1B 0.005215733�x�at CTAG2 0.005217339�x�at CTAG1A CTAG1B 0.006209942�x�at MAGEA3 0.154220325�at TAF7L 0.239214612�x�at MAGEA6 0.263219702�at PLAC1 0.274205564�at PAGE4 0.314214254�at MAGEA4 0.317206626�x�at SSX1 0.317

Fig. 1. CT-X gene expression in breast cancer. Normalized gene expressiondata for CT-X genes were used to cluster breast cancer samples. Samples arearranged as columns and CT-X antigens in rows. Expression levels are pseudo-colored, red indicating transcript levels above the median for that probe setacross all samples and green below the median. The bar below each heatmapindicates the ER-negative (red) and ER-positive (green) status of the samples.

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MAGEA9) showed PR status-specific gene expression, but noneof these showed a �2-fold differential expression. No consistent,significant relationship was detected between CT-X antigens andany of the 3 metrics examined.

Table 3 shows the fraction of ER-negative samples thatexpresses increased levels of CT-X antigens. In this series of 403ER-negative primary breast cancers, members of the MAGEAand NY-ESO-1/CTAG1B families are expressed in 38.9% and20.1% of the cases, respectively. Forty-four percent of theER-negative tumors express members of either MAGEA orNY-ESO-1/CTAG1B families.

CT-X Antigen Expression in Molecular Subtypes of Breast Cancers.Using molecular profiling, breast cancers can be subdivided intoluminal, HER2-positive, basal-like and so-called normal breast-like tumors (32, 33). We were able to evaluate whether theexpression of CT-X antigens is subtype-specific using the van deVijver dataset (Table 1) (34). The correlation of CT antigenexpression with the different breast cancer subtypes in the vande Vijver data were tested with ANOVA analysis. A significantcorrelation of CT-X expression with the basal subgroup was

CTAG1BCXorf61CTAG1BTFDP3FATE1MAGEA1CTAG2CSAG2SSX1PLAC1MAGEA12MAGEA6MAGEA2BMAGEA5MAGEA10MAGEB1MAGEA4

GradeER

Sorlie.type

Fig. 3. CT-X antigen expression in breast tumor subtypes. The Van de Vivjerdataset (34) was used to determine distribution of CT-X antigen expression.The color bar at the bottom indicates the 5 subtypes defined by Sorlie et al.(33), whereby red indicates basal-like, purple HER2, luminal A blue and Borange, and green the normal like subtype. In the expression matrix, redindicates increased and blue decreased CT-X antigen expression. The uppercolor bars show biological and clinical aspects of the tumors. Blue and yellowrepresent a positive and negative status for ER, whereas gold, light green, anddark green represent grades 1, 2, and 3.

Fig. 2. NY-ESO-1 gene expression in breast cancer. Normalized gene expres-sion data for NY-ESO-1 genes were used to cluster breast cancer samples,highlighting the association that NY-ESO-1 genes have with ER-negativesamples. Samples are arranged as columns and CT-X antigens in rows. Expres-sion levels are pseudocolored, red indicating transcript levels above themedian for that probe set across all samples and green below the median. Thebar below each heatmap indicates the ER-negative (red) and ER-positive(green) status of the samples.

Table 3. Fraction of ER-negative samples expressing increasedlevels of CT-X genes*

Probe set Gene symbol Criteria I† Criteria II‡

209942�x�at MAGEA3 26.1% 26.7%214612�x�at MAGEA6 23.9% 25.8%211674�x�at CTAG1B; CTAG1A 18.4% 20.4%210546�x�at CTAG1B; CTAG1A 17.1% 20.2%210467�x�at MAGEA12 15.8% 18.5%217339�x�at CTAG1B 15.0% 19.6%215733�x�at CTAG2 14.1% 17.8%220445�s�at CSAG2 14.1% 17.4%214603�at MAGEA2; MAGEA2B 13.2% 14.5%214642�x�at MAGEA5 9.4% 15.6%214254�at MAGEA4 8.5% 7.7%210503�at MAGEA11 7.7% 6.7%210437�at MAGEA9 7.3% 8.6%

Calculated using 9 separate datasets (Table 1).*Top 13 probes.†Average percentage of ER-negative samples with �2-fold above mean ex-pression level of all genes.

‡Average percentage of ER-negative samples in which expression is in the toptenth percentile of all genes.

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confirmed for MAGEA4 (P value �0.001), MAGEA10 (P value0.022), MAGEA5 (P value 0.013), MAGEA2B (P value �0.001),MAGEA5 (P value 0.013), MAGEA6 (P value �0.01), MAGEA12(P value 0.046), CTAG2 (P value �0.001), MAGEA1 (P value�0.011), and NY-ESO-1/CTAG1B (P value �0.001). Such tumorsare of a higher grade and predominantly ER-negative (Fig. 3).

Immunohistochemical Demonstration of CT Antigens in Tissue Arraysof Breast Cancer. To confirm CT-X antigen expression in breastcancer at the tissue level, 3 TMA-based immunohistochemical(IHC) studies were carried out to complement the gene expres-sion studies. The second and third studies built on the resultsobtained from the first. The salient features are summarized inTables 4–7, and detailed results are shown in Dataset S1. In afirst analysis, a series of 153 unselected cases of infiltrating breastcarcinomas were examined revealing 12/153 (8%) tumors posi-tive for MAGEA, and/or NY-ESO-1/CTAG1B or both (Fig. 4,Tables 4–7). CT-X antigen expression was taken as positive whenat least 1–2% of the tumor cell population was positively stained.Heterogeneity was a feature for both CT-X antigens. Whereas103 of the 153 tumors in the series were ER-positive, all but oneof the CT-X antigen positive tumors (11/12) fell into theER/PR-negative category. It was notable that p53 expression wasmore prominent in the CT-X group (Fig. 4, Tables 4–7) and thatmost had a high proliferative index as assessed by Ki-67 staining(Dataset S1). The second series comprised a highly selectedgroup of 19 triple negative breast tumors (ER, PR, and HER2-negative). Antigens of the MAGEA or NY-ESO-1/CTAG1Bfamily were present in 9/19 (47%) of these breast tumors (Tables4–7). Thirteen of these 19 cases were of the basal type, of which5 were positive for CT-X antigens (Tables 4–7). The final IHCseries consisted of 29 matched pairs of primary breast tumorsand 53 corresponding brain metastases. These breast tumors hadspread preferentially and/or initially to the brain, a feature notinfrequently associated with the basal subtype (35). Fourteen of29 (48%) of these primary tumors showed MAGEA and/orNY-ESO-1/CTAG1B expression (Tables 4–7). Of these 14 CT-Xantigen positive tumors, 9 were ER-negative, 7 of which werealso PR- negative (Tables 4–7). Thirty-five of 53 (66%) breastcancer metastases to the brain showed the presence of MAGEAand/or NY-ESO/CTAG1B at the protein level. MAGEA andNY-ESO-1/CTAG1B expression individually and combined, was

observed in 29, 2, and 4 deposits, respectively. Twenty-one ofthese CT-X positive metastases were ER-negative, of which 12were also PR-negative (Tables 4–7).

The overall distribution of ER-positive and ER-negative tu-mors positive for MAGEA and NY-ESO-1/CTAG1B is shown inTable 7. The data confirm the relative frequency of expressionand distribution by ER status found by transcriptional analysis,with NY-ESO-1/CTAG1B being expressed more particularly inER-negative tumors.

DiscussionThe present study suggests that CT-X antigen expression isfrequent in ER-negative breast cancer. The single result in theoriginal MPSS data where CT-X antigens were found in anER-negative breast cell line, and not generally in breast cancersamples or in normal breast tissue, stimulated the subsequentmore detailed analysis aimed at determining whether the rela-tionship between ER status and CT-X expression was of broadsignificance in breast cancer. The ability to access publishedmicroarray datasets and carry out a series of defined analysesproved to be invaluable. The analyses of 51 breast cell lines andthe 1,259 breast tumors highlighted the association of steroidreceptor negative breast cancer and a propensity to express CT-Xantigens. The previous assumption of a general low expression ofCT antigens in breast cancer is a result of studying unselected seriesin which ER-negative tumors usually comprise only �25% of cases.Indeed, the very first immunopathology study of this work exem-plifies this conclusion where less than one-third of the tumors wereER-negative. As a result, only 8% (12/153) of the lesions were CT-Xpositive, but 11 of the cancers with CT-X expression lacked estrogenreceptors (series 1, Tables 4–7).

On the basis of molecular profiling, Perou and Sorlie and theircolleagues have classified breast cancer into 5 groups, namelyluminal A, luminal B, basal-like, HER2 positive, and so-callednormal breast-like (27, 28). Neve et al. (27) have delineated someof the breast cell lines used in the present study (Fig. 1) as beingbasal-like due to their expression of cytoplasmic componentstypically found in the basal cells of the normal breast. In ourvarious analyses of the gene arrays, basal-like cell lines andtumors both exhibited higher expression of CT-X antigens.Breast cancers with basal–like features are a recently recognizedentity of increasing importance. These lesions are generally of

Table 4. Immunohistochemistry expression of CT antigens in breast cancer

Series Tumor Description n

No. of tumors with expression No. (%)

MAGEA NY-ESO-1 MAGEA/ NY-ESO-1 MAGEA/ NY-ESO-1

1 Primary 153 6 3 3 12 (8)2 Primary 19 3 4 2 9 (47)3a Primary 29 13 0 1 14 (45)3b Brain mets from 3a 53 29 2 4 35 (66)

Table 5. Characteristics of tumors positive for MAGEA or NY-ESO-1 antigens by immunohistochemistry

Series Tumor description

Tumor characteristics

MAGEA or NY-ESO-1 positive ER neg P53 pos Basal ER neg Basal

1 Primary 12 11 10 ND ND2 Primary 9 9* ND 5 53a Primary 14 9† 9 10 73b Brain mets from 3a 35 21‡ 16 25 12

ND, not done.*Also PR-negative.†7/9 also PR-negative.‡12/21 also PR-negative.

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higher grade, have a lower propensity to metastasize to locallymph nodes, exhibit a distinct tendency to spread to brain, andcarry a very poor prognosis (35–37). Usually they are ER/PR-negative and do not overexpress HER2. Therefore, these tumorsconstitute a subset of the so-called triple negative breast cancers.

Consequently, we addressed the question of a potential asso-ciation of CT-X expression with hormone receptor and/or HER2status by an immunohistochemical analysis of 2 more collectionsof breast tumors (series 2 and 3) comprising a high number ofER-negative breast cancers, many of which resembled the basal-like type. We clearly show that MAGEA and NY-ESO/CTAG1Bare frequently expressed in such tumors (Tables 4–7).

Current clinical management of breast cancer (early detection,surgery, and cytotoxic drug regimens often in the adjuvantsetting) has resulted in significant gains in disease-free andoverall survival in recent times (38). Some additional advanceshave been achieved through the use of targeted forms of therapysuch as Tamoxifen and aromatase inhibitors for those breastcancers possessing estrogen receptors (39, 40). Trastuzumab(Herceptin), a humanized monoclonal antibody against theextracellular domain of HER2, has been shown recently tobenefit patients with HER2-positive primary and metastaticdisease (41, 42). Vaccine studies with HER2 peptide in breastcancers expressing ERBB2 are also in progress (43). Thereremains a need to develop further tumor-specific targets, par-ticularly for those tumors that lack steroid receptors and do nothave amplification of HER2.

To date, immunotherapeutic regimens for breast cancer havealso been used mainly in end-stage disease and have generallyused antigens expressed in normal tissues with elevated expres-sion or expression of mutated forms in tumor cells. Included inthis category are antigens such as MUC1, CEA, and the carbo-hydrate antigens (39). By contrast, current thinking places therole of immunotherapy as being most likely to be effective whenpatients only have minimal residual disease after initial treat-ment. CT-X antigens through their restricted distribution in thetestis and cancer cells offer a more specific opportunity forvaccine development and therapy. Currently, vaccines compris-ing members of the MAGEA and NY-ESO-1 families are inclinical trials in patients with melanoma and lung cancer, wheresuch antigens are frequently expressed (11–18).

The present results, therefore, highlight a group of CT-Xantigen-expressing steroid receptor-negative breast cancers forwhich therapeutic options are limited. From the data of this

series, it would seem that there is a restricted expression ofmembers of the MAGEA family as well as NY-ESO-1/CTAG1Bin almost half of all ER-negative breast cancers, including triplenegative and basal-like cancers. In conclusion, our study suggeststhat a high percentage of ER-negative breast cancer patients maybenefit from CT-X antigen-based vaccine treatment.

Materials and MethodsDatasets, Gene Annotations, and Expression Analyses. The CT antigen database(www.cta.lncc.br/) (2) was used as a reference to extract data corresponding tothe CT-X-encoding genes from the 9 microarray datasets analyzed in this studyusing mRNA accession numbers as cross-references (27–31, 34, 44–47). Whenseveral probe sets were available for the same gene, all were used for analysis.Each dataset was subjected to a standard normalization procedure. Values �0.01were set to 0.01. Each measurement was divided by the 50th percentile of allmeasurements in that sample. Each probe set was divided by the median of itsmeasurements in all samples. A statistical analysis (ANOVA) was used to identifyprobe sets with class-specific expression patterns using unfiltered data (22,283probe sets). For determining the ER-specific expression (ERSE) set, the statisticalanalysis used the Student 2-sample t test, a P value cut-off of 0.05, and theBenjamini and Hochberg false discovery rate (48) to control for multiple testing

A D

B E

C F

Fig. 4. Expression of MAGEA, NY-ESO-1/CTAG1B and p53 in primary andmetastatic breast tumors. Shown is IHC staining demonstrating the proteinexpression of MAGEA, NY-ESO-1/CTAG1B, and p53 in primary (A, 10�; B, 20�;C, 100�) and breast cancer metastases to the brain (D, 10�; E, 20�; F, 100�),NY-ESO-1/CTAG1B revealing a mostly cytoplasmic presence of CT-X antigensand the typical nuclear expression of p53.

Table 6. Characteristics of tumors included in the immunohistochemistry series

Series Tumor description n

Tumor characteristics

ER neg P53 pos HER2 neg Basal EGFR pos

1 Primary 153 50 66 ND ND ND2 Primary 19 19 ND 19 13 163a Primary (initial mets to brain) 29 16 17 12 19 13b Brain mets from 3a 53 30 23 42 34 4

ND, not done.

Table 7. Overall distribution of MAGEA and NY-ESO-1/CTAG1Bpositive tumors by ER status

Tumor description CT-X positivity

No.

MAGEA NY-ESO-1/CTAG1B

Primaries ER-negative 23 3Primaries ER-positive 5 0Metastases ER-negative 19 2Metastases ER-positive 11 0

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error. A 2-fold change filter was then applied, and probe sets that met theseconditions in at least 5 of the 9 datasets investigated were retained. The proba-bility of probe sets meeting these criteria by chance can be estimated usingbinomial distribution to be �P � 3.32 � 10�5. This analysis was repeated sepa-rately for the CT-X antigen-encoding genes (66 probe sets).

CT-X Antigen Expression in Molecular Subtypes of Breast Cancers. An analysisof variance (ANOVA) was performed to examine the correlation between theCT-X antigen expression level and the 5 breast tumor subtypes of the van deVijver dataset as categorized in their original study (34). The P values wereadjusted for multiple comparisons using the method of Benjamini and Hoch-berg, whereby P values �0.05 were considered significant.

Immunohistochemistry of CT-X Antigens and Tissue Microarray Analysis. Rou-tinely fixed paraffin-embedded tissue blocks containing mammary carcinomasexcised at the time of surgery were extracted from the files of the Department ofSurgical Pathology, Weill-Cornell Medical College (IHC series 1), from the files ofthe Department of Pathology, Austin Hospital, Melbourne (IHC series 2), or fromthe files of the Department of Pathology, University of Brisbane, Medical Facultyof Charles University in Plzen-Czech Republic, Instituto Nacional do Cancer-Brazil,andLaboratorioSalomaoZoppi-Brazil (IHCseries3).Series1and3servedasdonor

blocks for the TMAs. The TMAs and series 2 samples were dewaxed in xylene andrehydrated through alcohols. Antigen retrieval was performed by microwaveboiling in 100 mM citrate buffer (pH 6.0) for 15 min or for series 2 and 3 in EDTA(pH 8.0) buffer for 2 min. Endogenous peroxidase activity was quenched with0.3% hydrogen peroxide for 5 min. Sections were then incubated with affinitypurified NY-ESO-1/CTAG1B-specific rabbit polyclonal antibody (NY45) (series 1)or monoclonal antibodies ES121 or E978 specific to NY-ESO-1/CTAG1B (series 2and 3) and monoclonal antibody specific to MAGEA (detecting multiple MAGE-Aantigens, including MAGE-A1, -A3, -A4, and -A6) (6C1, Santa Cruz Biotechnology)diluted in Tris-buffered saline with 10% BSA at 1:1,000 for 1 h at room temper-ature. The slides were then processed using Dako Envision� HRP (DakoCytoma-tion,Glostrup,Denmark), followingthemanufacturer’sprotocol, counterstainedbriefly with Mayer’s hematoxylin (Amber Scientific, Belmont, WA), and coverslipped. Specimens of known antigen-positive tumors were used as a positivecontrol, and negative controls were prepared by omission of the primary anti-body or by using a relevant subclass negative control. The various antibodies andtheir source used to demonstrate breast cancer features are shown in Table S13.

ACKNOWLEDGMENTS. This work was conducted as part of the Hilton–LudwigCancer Metastasis Initiative, funded by the Conrad N. Hilton Foundation andthe Ludwig Institute for Cancer Research Ltd.

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