Combined Use of Oligonucleotide and Tissue Microarrays Identifies by wuxiangyu

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									[CANCER RESEARCH 62, 3971–3979, July 15, 2002]


Combined Use of Oligonucleotide and Tissue Microarrays Identifies Cancer/Testis
Antigens as Biomarkers in Lung Carcinoma1
Michio Sugita, Mark Geraci, Bifeng Gao, Roger L. Powell, Fred R. Hirsch, Gary Johnson, Razvan Lapadat,
Edward Gabrielson, Roy Bremnes, Paul A. Bunn, and Wilbur A. Franklin2
Departments of Pathology [M. S., R. L. P., W. A. F.], Medicine [M. G., B. G., F. R. H., R. B., P. A. B.], and Pharmacology[G. J., R. L.], University of Colorado Health Sciences
Center, Denver, Colorado 80262, and Department of Pathology, F. S. Key Medical Center, Baltimore, Maryland 21224 [E. G.]


ABSTRACT                                                                                   of sites from which tumors may arise. Several approaches to over-
                                                                                           coming these problems are currently being tested ranging from new
    High density oligonucleotide microarrays (OMAs) have been used re-
                                                                                           imaging technologies (helical CT; Refs. 3, 4) to aggressive efforts to
cently to profile gene expression in lung carcinoma tissue homogenates.
                                                                                           identify high-risk cohorts (5). Biological properties of the tumor cells
The length of the lists of potentially interesting genes generated by these
studies is daunting, and biological and clinical relevance of these lists                  themselves (biomarkers) may also be exploited to identify subjects
remains to be validated. Moreover, specific identification of individual                   who might harbor clinically inapparent tumors. Molecules that are
biomarkers that might be used for early detection and surveillance has not                 expressed uniquely or at high level by tumor cells in comparison to
been the objective of these early studies. We have developed a schema for                  normal tissues and that may be secreted into accessible fluids such as
combining the data derived from the OMA analysis of a few lung cancer                      blood, urine, or sputum may be useful as lung cancer biomarkers.
cell lines with immunohistochemical testing of tissue microarrays to rap-                     It might be expected that, because of their stark morphological
idly identify biomarkers of potential clinical relevance. Initially, we pro-               distinction from normal lung cells and their aggressive biological
filed gene expression in lung tumor cell lines using the Affymetrix HG-                    behavior, lung cancer cells may exhibit many molecular differences
U95Av2 OMA. RNA from 2 non-small cell lung cancer (NSCLC) cell lines
                                                                                           from non-neoplastic lung cells. To date there have been numerous
(A549 and H647) and 2 small cell lung cancer (SCLC) cell lines (SHP-77
and UMC-19) were tested. Cells from 1 histologically and cytogenetically                   attempts to identify such molecules with limited success. Lung cancer
normal bronchial epithelial primary culture from a volunteer who had                       biomarkers measurable in the peripheral blood have included carbo-
never smoked and 10 samples of histologically unremarkable lung tissue                     hydrate-rich cell matrix molecules such as carcinoembryonic antigen
from resection specimens served as normalization controls. Array results                   (6), cytokeratin-derived intermediate filament molecules such as CY-
were analyzed with Gene Spring software. Results were confirmed by                         FRA-21.1 (7, 8), tissue polypeptide antigen (TPA) (9), and tissue
reverse transcription-PCR in an expanded number of cell lines. We then                     polypeptide specific antigen (TPS) (10), peptides such as proGRP
validated the cell line data by immunohistochemical testing for protein                    (11), neural markers such as neuron specific enolase (12, 13) and
using a tissue microarray containing 187 NSCLC clinical samples. Of the                    chromogranin A (14, 15), and antibodies to immunogenic molecules
20 most highly expressed genes in the tumor lines, 6 were members of the
                                                                                           such as Hu (16), calcium channel proteins (17), and p53 (18, 19). Thus
cancer/testis antigen (CTAG) gene group including 5 MAGE-A subfamily
members and NY-ESO-1. SCLC lines strongly expressed all of the
                                                                                           far tests for these molecules have had limited clinical impact because
MAGE-A genes as well as NY-ESO-1, whereas NSCLC lines expressed a                          of low specificity or low frequency of positive results in early stage
subset of MAGE-A genes at a lower level of intensity and failed to express                 patients. However, it is likely that the list of biomarkers already tested
NY-ESO-1. Reverse transcription-PCR of an extended series of 25 lung                       represents only a fraction of the molecular changes that occur in tumor
cancer cell lines including 13 SCLC, 9 NSCLC, and 3 mesothelioma lines                     cells, and that more sensitive and specific biomarkers remain to be
indicated that MAGE-A10 and NY-ESO-1 were expressed only by SCLC,                          discovered.
and that MAGE-A1, 3, 6, 12, and 4b were expressed by both SCLC and                            Recently, high-density OMAs3 have been introduced that permit
NSCLC. By immunohistochemistry using the monoclonal antibody 6C1                           rapid analysis of expression levels simultaneously for large numbers
that recognizes several MAGE-A gene subfamily members, 44% of
                                                                                           of genes (20). This approach overcomes limitations inherent in ex-
NSCLC clearly expressed MAGE-A proteins in cytoplasm and/or nucleus.
Expression of MAGE-A genes did not correlate with survival but did
                                                                                           pression analysis of single genes. With completion of human genome
correlate with histological classification with squamous carcinomas more                   (21) sequencing, comprehensive OMA expression profiles can be
frequently MAGE-A positive than other NSCLC types (P < 0.00002). We                        created for individual tumors as well as for large classes of tumors.
conclude that expression of CTAG gene products, whereas apparently not                     Early OMA analyses of lung cancers have centered on phenotypic
of prognostic importance, may be useful for early detection and surveil-                   classification of specific tumor type (22) and have not specifically
lance because of a high level of specificity for central airway squamous                   focused on biomarker discovery.
and small cell carcinomas.                                                                    Our objective in the present study was to discover potentially useful
                                                                                           biomarkers for lung cancer by first identifying large gene expression
INTRODUCTION                                                                               differences between tumor cell lines and normal lung using high
                                                                                           density OMAs. The microarray used (Affymetrix HG-U95Av2) in-
   Late stage at detection significantly and adversely affects survival                    corporates 12,600 probes accounting for a large fraction of the ex-
in lung cancer (1, 2). The most difficult obstacles to earlier detection                   pressed human genome. We searched for biomarkers that were over-
are the inaccessibility of the sites of tumor origin and the multiplicity                  expressed in relation to normal tissue, because they are more likely to
                                                                                           be useful for detection and screening of accessible specimens such as
   Received 2/20/02; accepted 5/17/02.                                                     sputum, peripheral blood, or urine than biomarkers that are underex-
   The costs of publication of this article were defrayed in part by the payment of page   pressed. We confirmed the expression levels of the gene group
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.                                       (CTAG) most frequently represented on a list of highly expressed
   1
     Supported by NIH Grants U01 CA85070, Early Detection Research Network, and            genes by testing a broader series of cell lines using relatively inex-
P50-CA85070.
   2
     To whom requests for reprints should be addressed, at Departments of Pathology,
Box B216, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Denver,          3
                                                                                                The abbreviations used are: OMA, oligonucleotide microarray; RT-PCR, reverse
CO 80262. Courier Address: Wilbur A. Franklin, MD, EDRN Biomarker Development              transcription-PCR; IHC, immunohistochemistry; NSCLC, non-small cell lung carcinoma;
Laboratory, Administrative Office Building, Room 065, 4210 E. 11th Avenue, Denver,         SCLC, small cell lung carcinoma; TMA, tissue microarray; CT, cancer/testis; ASH,
CO 80262.                                                                                  Achete-Scute Homologue.
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pensive RT-PCR methodology. Finally, results of this preliminary              Double-stranded cDNA was synthesized from 16 to 20 g total
testing were confirmed at the protein level by IHC using a TMA             RNA using an oligodeoxythymidylic acid 24 primer with a T7 RNA
containing 187 early stage NSCLCs. This algorithm for biomarker            polymerase promoter site added to the 3 end (Superscript cDNA
development takes advantage of two high throughput microarray              Synthesis System; Life Technologies, Inc., Rockville, MD). After
technologies to rapidly identify potentially important biomarkers          second-strand synthesis, in vitro transcription was performed using a
linked to clinical outcomes and prognostic importance.                     T7 Megascript kit (Ambion, Austin, TX) in the presence of biotin-
                                                                           11-CTP and biotin-16-UTP (Enzo Diagnostics, Farmingdale, NY) to
MATERIALS AND METHODS                                                      produce biotin labeled cRNA. Twenty g of the cRNA product was
                                                                           fragmented at 94°C for 35 min into 35–200 bases in length. The
   Samples and RNA Extraction Procedures. Four established cell            sample was then added to a hybridization solution containing 100
lines including 2 SCLC (SHP-77 and UMC-19) and 2 NSCLC (A549               mmol/liter 4-morpholinepropanesulfonic acid, 1 mol/liter Na , and
and H647) lines were analyzed. Before harvesting, SCLC and NSCLC           20 mmol/liter of EDTA in the presence of 0.01% Tween 20 to a final
cells were grown in RPMI 1640 supplemented with 5–10% fetal                cRNA concentration of 0.05 mg/ml. Hybridization was performed for
bovine serum. Substrate adherent cultures (SHP-77, A549, and H647)         18 –20 h by incubating 200 l of the sample to HG-U95Av2 microar-
were grown to subconfluence and harvested by rapid removal of              rays, and each microarray was stained with streptavidin-phycoerythrin
medium and application of RNeasy extraction medium (Qiagen, Va-            and scanned at 6- m resolution by Gene Array scanner G2500A
lencia, CA) containing guanidinium isothiocyanate. Nonadherent cells       (Hewlett Packard, Boise, ID) according to procedures developed by
were harvested at a concentration of 1 million cells/ml after 1            Affymetrix.
week in culture after thawing.                                                Statistical Analysis. Detailed protocols for data analysis of Af-
   Two controls were used for comparison to expression profiles of         fymetrix microarrays, and extensive documentation of the sensitivity
tumor cell lines. For one control, bronchial epithelial primary cell
                                                                           and quantitative aspects of the method have been described (20, 25).
cultures were obtained from a bronchoscopic biopsy of a healthy
                                                                           Briefly, mismatch probes act as specificity controls that allow the
48-year-old female who had never smoked and who had volunteered
                                                                           direct subtraction of both background and cross-hybridization signals.
under a Colorado Combined Institutional Review Board-approved
                                                                           To determine the quantitative RNA abundance, the average of the
protocol. The biopsy was explanted onto a T25 culture flask contain-
                                                                           difference representing perfect match         mismatch for each gene-
ing bronchial epithelial cell growth medium (Clonetics, Inc., Walkers-
                                                                           specific probe family is calculated. This data were transferred to
ville, MD) and epithelial cells were allowed to grow from the explant
                                                                           GeneSpring software (Silicon Genetics, Redwood City, CA) for ad-
to a diameter of 1 cm (10 days). Cells were then passaged into a
                                                                           ditional analysis.
second T25 flask and grown to 90% confluence (4 days). The
                                                                              Using the GeneSpring software package, a two step filtering algo-
culture cells were again split onto glass coverslips to perform spectral
karyotyping on metaphase cells according to the manufacturer’s pro-        rithm was implemented to select genes highly expressed by tumor
tocol (Applied Spectral Imaging, Inc., Carlsbad, CA). A second ali-        cells in comparison with non-neoplastic lung cells and tissue. In the
quot was split into two T75 flasks and again grown to 90% confluence       first step, cultured normal epithelial cells were compared with tumor
(4 days). One flask was additionally split into three T75 flasks and       cell lines using the following settings: the 80th percentile of all
expanded for an additional 3 days. Finally, 90% confluent cells were       measurements was used as a positive control for each sample, and
harvested by removal of culture medium followed by immediate               each measurement was divided by this control. The 0.1% measure-
addition of RNeasy extraction medium as described above. The total         ment was used as a control for background correction. The measure-
time from biopsy date to RNA harvest was 21 days. Primary cultures         ment for each gene was then divided by the corresponding value for
processed in this way grow as substrate-adherent monolayers, which         the sample of normal bronchial epithelium. A list was compiled of all
are 100% cytokeratin positive on immunohistochemical staining (23).        of the genes expressed by at least two tumor cell lines at 100 over
Spectral imaging karyotype was diploid with no detectable subchro-         the normal control. This filtering step resulted in the identification of
mosomal abnormalities.                                                     42 genes. In the second filtering step, the 50th percentile of all
   A second set of controls consisted of archival data obtained from       measurements was used as a positive control for each sample, and
experiments in which RNA was extracted from benign lung tissue             each measurement was divided by this control. The measurement for
obtained at the time of surgical resection for carcinoma elsewhere in      each gene was then divided by the corresponding value for 19 samples
the lung. For these experiments, duplicate tissue samples from 10 lung     from 10 non-neoplastic lung specimens. A list was then compiled of
specimens were snap frozen and stored in liquid nitrogen until use.        107 genes that were expressed in the tumor cell lines at 20 over
For RNA extraction, frozen tissue fragments were placed in RNeasy          the non-neoplastic tissue. The contents of the two lists was then
extraction medium and homogenized with a Tissue Tearor homoge-             compared using the GeneSpring Venn diagram feature and a list of 20
nizer (Biospec Products, Bartlesville, OK) followed by filtration          highly overexpressed genes common to the two lists was compiled.
through a QIAshredder column. The filtrate was used for RNA ex-               The selected genes were annotated using the GeneOntology data-
traction using the Qiagen RNeasy Mini protocol.                            base4 within the NetAffx5 analysis system offered by Affymetrix.
   Total RNA extracted from each sample described above was tested         GeneOntology stores a dynamic controlled vocabulary organized on
for degradation and applied to a separate HG-U95Av2 microarray.            molecular function, cellular component, and biological process that
Each control RNA from cultured normal bronchial cells or whole lung        can be applied to all organisms. The cellular component attributes
homogenate was used as a separate normalization control in the             were used to search for genes that were either extracellular (secreted)
Genespring filtering algorithms described below.                           or transmembrane molecules as potential biomarkers.
   Preparation of Labeled cRNA and Hybridization to OMAs.                     The secretory attributes of the selected genes were further investi-
Before application to test chips the quality of RNA was tested using       gated by looking at the leader sequence signal. Briefly the master
the one step duplex RT-PCR assay (24). In this assay, the ratio of         protein model sequences were obtained from the LocusLink database
short to long segment -actin PCR product is used to quantify the
extent of RNA degradation. All of the samples in this study had ratios        4
                                                                                  Internet address: http://www.geneontology.org.
of 2.6 indicating a low level of degradation.                                 5
                                                                                  Internet address: http://www.netaffx.com.
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                                                          CANCER/TESTIS ANTIGEN GENE EXPRESSION IN LUNG CANCER


and analyzed using the program SignalP6 (SignalP version2.0) that                                                        Table 2 Stage and histology for TMA
detects secretory signal peptides in amino acid sequences. The pro-                                                       Stage 1      Stage 2     Stage 3a      Stage 3b      Totals
gram splices the first 70 amino acids and runs two different types of                            Squamous                    52          14           16             9           91
detection algorithms: one based on neural network prediction and the                             Adeno                       30          18           16             7           71
                                                                                                 Large cell                   8           3            2             2           15
other based on Hidden Markov Models. Both are trained against a                                  Bronchioloalveolar           9           0            1             0           10
library of known signal peptides and calculate a final score, which will                         Total                       99          35           35            18          187
assign the protein to one of three classes: (a) nonsecretory; (b) signal
anchor (NH2 terminus of type II membrane proteins, uncleaved signal
peptides); and (c) signal peptide (secretory signal).                                        block. Normal lung and 15 other control tissues were included in each
   Spearman correlation was used for clustering of all of the hybrid-                        tissue array block. Four- m sections of the resulting microarray
ization experiments. To evaluate the expression profile for melanoma-                        blocks were cut with a Leitz microtome. Sections were transferred to
associated antigens, a list of 64 melanoma-associated genes was                              adhesive coated slides using the adhesive-coated tape sectioning sys-
compiled using the GeneSpring search feature for melanoma, and                               tem (Instrumedics Inc., Hackensack, NJ; Ref. 22). Subsequently, UV
Pearson correlation was used for clustering of this list (see Fig. 1).                       light treatment of the slides for 60 s polymerized the adhesive coating
   To evaluate the relationship between MAGE-A expression as de-                             into a plastic layer and sealed the sections to the slides. Thereafter, the
termined by IHC (see below) and survival, log rank test was per-                             tape could be removed in a solvent (Instrumedics Inc.).
formed using the SPSS statistical package, version 11.0 (SPSS, Inc.,                            The sections were then deparaffinized with standard xylene and
Chicago, IL). 2 analysis was performed using Microsoft Excel.                                hydrated through graded alcohols into water. Antigen retrieval was
   Confirmatory RT-PCR Assay. Gene expression was confirmed                                  performed using the DAKO Target Retrieval system in a Biocare
by RT-PCR in 25 cell lines with SCLC, NSCLC, and mesothelioma                                Medical decloaking chamber. Peroxide blocking was performed with
histologies (representative gel shown in Fig. 3). The RT-PCR assay                           3% hydrogen peroxide in water. After incubation of the mouse mono-
was performed using One-Step RT-PCR system (Life Technologies,                               clonal anti-MAGE-A antibody, 6C1 (Novacastra) for 1 h at room
Inc.) with MAGE A-1, 3, 4, 6, 10, 12, ASH1, PGP 9.5, and NY-ESO-1                            temperature, the DAKO Envision Plus detection was applied for 30
primers (Table 1). Reagents were mixed in a single tube for reverse                          min also at room temperature. This was followed by application of
transcription and amplification for 22 to 30 cycles including denatur-                       diaminobenzidine chromogen. The slides were then counterstained in
ation at 94°C for 1 min, annealing at 55°C for 1 min, and extension                          hematoxylin and coverslipped.
at 72°C for 2 min. The RT-PCR products were separated on 1.5%                                   Outcome data on cases used for microarray construction was ob-
agarose gels and visualized by UV transillumination of the gels                              tained from the University of Colorado tumor registry. Patients were
stained with ethidium bromide.                                                               followed for a median of 51 months (range, 18 –100).
   Preparation of Cell Lines for IHC. Protein expression was eval-                              Scoring of IHC Results. Each core on the TMA was examined by
uated in the 19 cell lines by immunoperoxidase staining of cell pellets                      conventional white light microscopy and the observed staining pattern
created by centrifugation of cultured cells that were then fixed in                          graded for each core. Percentage of tumor cells positive and intensity
formalin for 30 min and embedded in paraffin. Sections of the                                of staining was recorded for both tumor cytoplasm and nucleus. A
resulting paraffin blocks were stained with the same anti-MAGE                               grading score was obtained by multiplying the intensity of staining on
monoclonal antibody (6C1; Novacastra, Newcastle, United Kingdom)                             an arbitrary 0 – 4 scale by the percentage of cells stained separately
and by the same methods used for the TMAs described below.                                   for nuclear and cytoplasmic staining. The same grading system was
   TMA IHC. Paraffin blocks of tumor tissue from 187 patients with                           used for both TMA samples and lung cancer cell lines.
NSCLC (stages I-III) were obtained from the University of Colorado
Cancer Center and Johns Hopkins Medical Institutions according to                            RESULTS
IRB-approved protocols. Follow-up of patients represented on the
TMA ranged from 18 to 100 months. The distribution of tumor                                     OMA Detection of Overexpressed Genes. Cluster analysis of
histologies and clinical stage in this group of patients is shown in                         microarray cell line experiments in which cell lines were compared
Table 2.                                                                                     with primary cultures of normal bronchial epithelium and homoge-
   The TMAs were assembled using a tissue-arraying instrument                                nates of non-neoplastic lung tissue indicated that tumors clustered
(Beecher Instruments, Silver Spring, MD), consisting of thin-walled                          according to histological type, with the two SCLC lines clustering
stainless steel biopsy needles and stylets used to empty and transfer                        together, the two NSCLC lines clustering together, and each of the
the needle content. The assembly is held in an X-Y position guide that                       tumor types clustering separately from the cultured benign epithelium
is manually adjusted by micrometers. A large diameter stylet (1.5                            and lung tissue homogenates (Fig. 1). Each sample of non-neoplastic
mm) was used for sampling, and non-necrotic areas of the blocks were                         lung tissue invariably clustered with its corresponding duplicate sam-
routinely over-sampled with three replicate core samples of tumor                            ple. Complete hybridization data for cell lines, the primary culture,
(different areas) and normal (one, if present) regions from each donor                       and the tissue homogenates are available.7
                                                                                                The dual normalization and filtration process yielded 20 separate
                                                                                             genes (Table 3) represented by 21 probe sets that were highly over-
  6
      Internet address: http://www.cbs.dtu.dk/services/SignalP-2.0.                          expressed by at least 2 of the cell lines. Expression levels were related
                                                                                             to cell type with 14 genes overexpressed only in SCLC, 4 in NSCLC,
                               Table 1 Cell line properties                                  and 2 in both SCLC and NSCLC. With the exception of the CTAG
                                                              Degradation     Spectral
                                                                                             gene group, the chromosomal distribution of the overexpressed genes
              Cell line                  Histology              index        karyotype       appeared to be random. A wide diversity of gene functions and
 UMC19                               SCLC                        2.58       Abnormal         subcellular localizations were represented among the gene products,
 SHP77                               SCLC                        1.89       Abnormal         ranging from a membrane-associated ion transport protein to nuclear
 H647                                NSCLC                       2.01       Abnormal
 A549                                NSCLC                       1.84       Abnormal
                                                                                             transcription factors (Table 4). Four of the genes, ASH1, claudin 10,
 Bronchial epithelium                Benign Epithelium           1.42       2N (normal)
   ( day culture, nonsmoker)                                                                     7
                                                                                                     Internet address: http://uch.uchsc.edu/uccc/research/GeneExpression/index.html.
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                                                        CANCER/TESTIS ANTIGEN GENE EXPRESSION IN LUNG CANCER




    Fig. 1. Dendograms showing clustering of cell
lines, the bronchial epithelial culture, and tissue
homogenates on the vertical axis based on the anal-
ysis of all of the expressed genes (Spearman cor-
relation) and by melanoma-associated gene expres-
sion (Pearson correlation) on the horizontal axis.
High level expression is coded in the diagram as
red, low level expression as green, and intermedi-
ate expression as yellow or orange. Duplicate sam-
ple non-neoplastic lung tissue homogenates are
identified to the right of the expression diagram as
“Nl Lu” followed by a patient number and a letter
identifying the individual homogenate sample. The
cell lines and the primary culture of bronchial ep-
ithelial cells are indicated by bolding. The location
of a gene cluster containing many CTAG genes is
indicated below the diagram.




and the secretogranins I and II, contained signal peptide sequences                      9, and 11) were not overexpressed or were overexpressed in only a
suggesting the possibility that the gene products are secreted. Only 1                   single tumor. When the query was expanded to include all 64 of the
gene, ABCC2, contained a signal anchor peptide sequence.                                 melanoma-associated probes on the array, including 12 for MAGE-A,
   A notable feature of the list of overexpressed genes is the frequency                 4 for MAGE-B, 9 for GAGE, 1 for BAGE, and 4 NY-ESO-1 (LAGE-1),
of the CT group with 5 MAGE-A and the NY-ESO-1 CTAG genes                                we found only the MAGE-A and NY-ESO genes were overexpressed at
constituting 30% of the total list (Fig. 2). Here again levels of                        high levels (Fig. 1), again suggesting that MAGE profiles are tissue-
expression were related to tumor type with SCLC lines more highly                        specific, with lung cancer expressing only a fraction of the genes that
expressive of MAGE genes than NSCLC. High level overexpression                           have been associated with melanocytic differentiation.
was restricted to a subset of MAGE-A subfamily genes (Fig. 2). When                         Confirmatory RT-PCR. By RT-PCR, the differences in expres-
the database normalized to cultured bronchial epithelial cells was                       sion patterns for several of the highly expressed genes were confirmed
queried regarding levels for all of the MAGE-A genes on the HG-                          in 25 cell lines with SCLC, NSCLC, and mesothelioma histologies
U95Av2 array, MAGE-A2, 3, 6, 10, and 12 were found to be over-                           (representative gel shown in Fig. 3). As graphically depicted in Fig. 4,
expressed at 100-fold, whereas MAGE-A 1, 4b, and 5a were over-                           most of the high expression markers were detected in similar percent-
expressed at 40 –100-fold. The remaining MAGE family members (8,                         ages of cell lines, but NY-ESO-1, MAGE-A10, and ASH-1 were


                                                         Table 3 Genes highly overexpressed in lung cancer cell lines
                                                                                                                                                 Affymetrix
                                                            Gene (abbreviation)                                            Genbank no.           probe no.
        1                Achaete-Scute homologue 1 (ASH1, HASH1)                                                            L08424               40543_at
        1                Achaete-Scute homologue 1 (duplicate)                                                              L08424               40544_g_at
        2                Aldo-keto reductase family 1, member B10 (aldose reductase, AKR1B10)                               U37100               37482_at
        3                ATP-binding cassette, sub-family C (CFTR/MRP), member 2 (ABCC2, canalicular multispecific          U49248               33721_at
                           anion transporter, cMOAT)
        4                Cancer/testis antigen (CTAG1, NY-ESO-1, LAGE2)                                                     U87459               33636_at
        5                Claudin-10 (CLDN10, CPETRL3 OSP-L)                                                                 U89916               39579_at
        6                Dopa decarboxylase (Aromatic amino acid decarboxylase, AADC)                                       M76180               40201_at
        7                Insulinoma associated 1 (IA-1, INSM1)                                                              M93119               33157_at
        8                Keratin, hair, basic, 1 (KRTHB1keratin, hHKb1)                                                     X81420               36288_at
        9                KIAA0282 protein (TRIM9, tripartite motif-containing 9)                                            D87458               36903_at
       12                Melanoma antigen, family A, 2 (MAGE-A2)                                                            L18920               33518_f_at
       13                Melanoma antigen, family A, 3 (MAGE-A3)                                                            U03735               33517_f_at
       14                Melanoma antigen, family A, 6 (MAGE-A6)                                                            U10691               31599_f_at
       10                Melanoma antigen, family A, 10 (MAGE-A10)                                                          U10685               35491_at
       11                Melanoma antigen, family A, 12 (MAGE-A12)                                                          L18877               31480_f_at
       15                Na ,K -ATPase, subunit -III                                                                        M37457               35670_at
       16                Neurofilament light polypeptide (NEFL)                                                             X05608               40995_at
       17                Prostaglandin E synthase (PGE synthase; microsomal glutathione transferase homolog; p53            AF010316             38131_at
                           induced)
       18                Secretogranin I (chromogranin B)                                                                   Y00064               33426_at
       19                Secretogranin II (chromogranin C)                                                                  M25756               36924_r_at
       20                Ubiquitin COOH-terminal esterase L1 (ubiquitin thiolesterase, UCHL1, PGP 9.5)                      X04741               36990_at

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                                                       Table 4 Properties overexpressed genes and gene products
                                                                                                                                                          Chromosomal
    Gene abbreviation        Cell type                    Function                         Subcellular localization            Predicted secretion          location
   Claudin-10                Both           Caspase                                    Integral membrane protein              Signal peptide               13q31-q34
   PGP 9.5                   Both           Proteosome regulatory protein              Nuclear/cytoplasmic                    Nonsecretory protein         4p14
   Aldose reductase          NSCLC          Aldehyde dehydrogenase                     Cytoplasm                              Nonsecretory protein         7
   cMOAT                     NSCLC          Ion/peptide transport                      Integral membrane protein              Signal anchor                10q24
   hHKb1                     NSCLC          Cell size/shape/motor control              Cytoplasmic                            Nonsecretory protein         12q13
   PGE synthase              NSCLC          Prostaglandin metabolism                   Intracellular/perinuclear              Nonsecretory protein         9q34.3
   ASH1                      SCLC           Transcription factor                       Nucleus                                Signal peptide               12q22-q23
   Chromogranin B            SCLC           Protein secretion                          Secretory granule                      Signal peptide               20pter-p12
   Chromogranin C            SCLC           Calcium binding                            Secretory granule                      Signal peptide               2q35-q36
   Dopa decarboxylase        SCLC           Aromatic amino acid metabolism             Mitochrondrial outer membrane          Nonsecretory protein         7p11
   IA-1                      SCLC           Transcription factor                       Nucleus                                Nonsecretory protein         20p11.2
   KIAA0282                  SCLC           Function not defined                       Cytoplasmic                            Nonsecretory protein         14
   MAGE-A2                   SCLC           Tumor antigen                              Nucleus/plasma membrane                Nonsecretory protein         Xq28
   MAGE-A3                   SCLC           Tumor antigen                              Nucleus/plasma membrane                Nonsecretory protein         Xq28
   MAGE-A6                   SCLC           Tumor antigen                              Nucleus/plasma membrane                Nonsecretory protein         Xq28
   MAGE-A10                  SCLC           Tumor antigen                              Nucleus/plasma membrane                Nonsecretory protein         Xq28
   MAGE-A12                  SCLC           Tumor antigen                              Nucleus/plasma membrane                Nonsecretory protein         Xq28
   Na ,K -ATPase             SCLC           Sodium/potassium-transporting ATPase       Integral membrane protein              Nonsecretory protein         19q13.2
   NEFL                      SCLC           Cell size/shape/motor control              Cytoplasmic/nuclear lamina             Nonsecretory protein         8p21
   NY-ESO-1                  SCLC           Tumor antigen                              Membrane fraction                      Nonsecretory protein         Xq28



                                                                                    mic staining, and in those cases where there was discordance the
                                                                                    positive staining was weak. There was also strong correlation between
                                                                                    tumor histology and MAGE-A expression status. Squamous carcino-
                                                                                    mas were more frequently positive than adenocarcinomas, large cell
                                                                                                                                                        2
                                                                                    carcinomas, or bronchioloalveolar carcinomas (Table 5;                ,
                                                                                    P 0.00002).
                                                                                       As expected, there was a strong association between stage and
                                                                                    survival among the patients with tumors represented on the TMA, but
                                                                                    log rank test indicated no significant correlation between MAGE-A
                                                                                    expression status and survival regardless of whether cases were strat-
                                                                                    ified by tumor histology, stage, or by weak or strong (labeling score
                                                                                       100) nuclear staining, weak or strong (labeling score 100) cyto-
                                                                                    plasmic staining, or combinations of these patterns.

                                                                                    DISCUSSION

                                                                                       The schema created in this study for biomarker discovery included
                                                                                    three steps: first, a few lung tumor cell lines were analyzed for gene
                                                                                    expression using high-density oligonucleotide arrays. This step had
                                                                                    the advantages that pure tumor cells could be tested without micro-
   Fig. 2. Expression of MAGE A and NY-ESO-1 genes normalized to expression of
cultured normal bronchial epithelium. Highest levels were found in SCLC lines but
                                                                                    dissection and that usage of microarrays was efficient and minimal. At
several genes (see text) were also overexpressed in NSCLC.                          this step, a stringent filtering algorithm was used incorporating both
                                                                                    cultured epithelial cells as well as lung tissue homogenates as nor-
                                                                                    malizing controls to limit the list of highly overexpressed genes to just
expressed frequently by SCLC but infrequently or not at all by                      20. Remarkably, 30% of the genes that survived the filtration were
NSCLC. By 2 analysis, differences in expression frequencies were
significant at Ps of 0.02, 0.03, and 0.03 for MAGE-10, NY-
ESO-1, and ASH, respectively.
   Correlation of RT-PCR with IHC in Cell Lines. Of the 25 cell
lines tested by RT-PCR, 19 were also tested by IHC. IHC staining
patterns of cell lines were similar to those described below for TMA.
Cytoplasmic and nuclear staining were frequently present in the same
cells but were scored separately. There was nearly complete concord-
ance between RT-PCR and IHC results with all of the specimens with
IHC scores 1 positive by RT-PCR, and only 1 line that was negative
by IHC and was positive by RT-PCR ( 2, P 0.0003).
   TMA IHC. Nuclear and cytoplasmic staining were analyzed both
separately and combined for prognostic significance. Staining was of
variable intensity (Fig. 5) with labeling scores (described on page 13)
ranging from 0 to 387; scores were interpreted as positive if they were
1 or higher. MAGE-A was interpreted as highly overexpressed if
labeling score was 100. Of the 187 arrayed tumor samples, 44%
                                                                                       Fig. 3. RT-PCR products visualized in ethidium bromide-stained agarose gels. Expres-
exhibited some level of nuclear or cytoplasmic staining. There was a                sion of MAGE A-3/6 is visible in products prepared from a variety of cell types, whereas
strong concordance ( 2, P 0.00001) between nuclear and cytoplas-                    expression of MAGE A-10 and NY-ESO-1 is confined primarily to SCLC.
                                                                                3975
                                                          CANCER/TESTIS ANTIGEN GENE EXPRESSION IN LUNG CANCER


                                                                                             schema was strong representation of CTAG genes. This gene group
                                                                                             comprised 30% of the 20 biomarker genes identified. Expression of
                                                                                             CTAG genes is restricted to normal testicular (and ovarian) germ cells
                                                                                             and tumors of a number of cell types (26, 27). The first of these genes
                                                                                             to be identified were the MAGE-A genes, which were originally
                                                                                             named MAGE 1 through 3. They were discovered because they elicit
                                                                                             an HLA I-dependent cytotoxic response in sensitized lymphocytes
                                                                                             against the melanoma cell line MZ2-MEL (28). It is of interest that
                                                                                             this first report indicated that MAGE expression could be demon-
                                                                                             strated not only in melanoma cell lines but also in SCLC and NSCLC
                                                                                             as well. The original 3 MAGE genes were soon supplemented by 9
                                                                                             additional MAGE genes discovered by screening cosmid libraries
                                                                                             temporarily bringing the total number of MAGE genes to 12 (29), all
                                                                                             encoded at chromosome Xq28 (29, 30). Sequencing of chromosomal
                                                                                             region Xp21.3 led to the identification of a second subfamily of
                                                                                             MAGE genes named MAGE-B (31–33). In recent years, the list of
                                                                                             MAGE family genes has continued to increase, and the MAGE family
   Fig. 4. Graphic representation of RT-PCR results for tumor cell lines of various cell
types using primers for genes found to be overexpressed at high level by OMA. Markers        now is thought to contain 55 homologous members divided into 9
with specificity for SCLC include NY-ESO-1, MAGE-A10, and ASH1.                              subfamilies (34). Although structurally homologous, some recently
                                                                                             described MAGE subfamilies are ubiquitously expressed (35–37) and
                                                                                             are not members of the CTAG gene group. Early reports indicating
                                                                                             that MAGE genes may be expressed by tumors of many types (28)
                                                                                             have been confirmed in many different laboratories for many different
                                                                                             types of tumors including brain (38, 39), skeletal muscle (40), esoph-
                                                                                             agus and stomach (41), Reed-Sternberg cells (42), bladder (43), bili-
                                                                                             ary tract (44), and breast (45).
                                                                                                A second CTAG gene family, NY-ESO-1, was identified by autol-
                                                                                             ogous screening of a cDNA expression library constructed from a case
                                                                                             of esophageal carcinoma (26). A similar if not identical gene was
                                                                                             reported a short time later as LAGE-1 (46). Like the MAGE-A gene
                                                                                             family, NY-ESO-1/LAGE-1 maps to chromosome Xq28 (46).
                                                                                                Expression of CTAG genes by lung tumors has been documented by
                                                                                             IHC and RT-PCR in a limited number of studies. In two separate
                                                                                             studies using monoclonal antibodies 57B and MA454 that react with
                                                                                             MAGE-A protein, Jungbluth et al. (47) have found heterogeneous
                                                                                             expression in 32% and 56% (48) of NSCLC, respectively. IHC studies
                                                                                             are complicated by the high degree of homology among different
                                                                                             MAGE-A proteins so that many anti-MAGE antibodies cross-react
                                                                                             with several different MAGE-A subfamily members (49 –51). The
   Fig. 5. TMA stained for MAGE-A proteins using the monoclonal antibody 6C1 in a
sensitive immunoperoxidase method. On the left is an image of the microarray slide and       monoclonal antibody used in the present study, 6C1, reacts with an
on the right a high magnification of tumor cells exhibiting strong (4 ) nuclear and weaker   epitope in the COOH-terminal regions of MAGE-A1, -A2, -A3, -A4,
(2 ) cytoplasmic staining.
                                                                                             -A6, -A10, -A11, and -A12 (51) and may thus be considered an anti
                                                                                             pan-MAGE-A reagent. The use of antibody in a sensitive immunoper-
                                                                                             oxidase procedure on 187 tumors in a TMA linked to clinical and
CTAG genes. The second step consisted of confirmation by RT-PCR                              histological data allowed us to determine that 44% NSCLC express
of overexpression of selected candidate biomarkers in an expanded set                        MAGE-A protein, that expression varied according to tumor histol-
of cell lines. This provided an independent and economical way for                           ogy, and that expression is unrelated to prognosis.
assessing expression profiles in cell lines of specific histological type.                      By RT-PCR, MAGE-A1, -A3, and B2 RNA sequences have been
It also proved to be highly predictive of protein expression level. The                      found recently in 70%, 85%, and 85% of a small series of NSCLC and
third step was the application of specific monoclonal antibody to a                          is often accompanied by promoter hypomethylation (52). Also of
large TMA that was linked to clinical follow-up. This provided a rapid                       interest, this report indicated that bronchial epithelium from a large
way to assess prognostic importance of expression of the candidate                           proportion of 20 former smokers without lung carcinoma also fre-
biomarker. It also provided an opportunity to verify the distribution of                     quently expressed MAGE genes and suggested that MAGE gene
protein at a cellular level. A step beyond this discovery schema would
consist of testing blood, urine, and sputum for the presence of specific
RNA and protein in clinical cohorts with and without invasive carci-                                             Table 5 Histology versus MAGE-A status
noma or preinvasive lesions. Such clinical testing is expensive and                                                                           MAGE-A status
time consuming, and before being launched requires the maximum
rigor in estimating specificity of the putative biomarker. The schema                                    Histology                 Positive       Negative    Totals

used in this study efficiently answers many of the preliminary ques-                            Squamous carcinoma                   57              34         91
                                                                                                Adenocarcinoma                       19              52         71
tions that should be answered before proceeding to larger clinical                              Large cell carcinoma                  3              12         15
trials.                                                                                         Bronchioloalveolar carcinoma          1               9         10
   A surprising feature of the list of biomarkers that emerged from this                        Totals                               80             107        187

                                                                                         3976
                                               CANCER/TESTIS ANTIGEN GENE EXPRESSION IN LUNG CANCER


expression may occur early in lung carcinogenesis and may be a              among differing histological types, only four genes were also present
suitable target for lung cancer prevention.                                 in the current list of overexpressed genes, PGE synthase, cMOAT,
   Whether MAGE-A protein can be found in the blood, urine, or              ASH-1, and IA-1. Finally, in a recent SAGE analysis (67), 115 highly
sputum of patients with lung cancer is not known at present. The            differentially expressed genes were reported and among these was the
absence of signal sequences in the MAGE-A genes suggests that               aldoketo reductase, member B10 gene. CTAG genes were not listed in
MAGE-A proteins are not actively secreted. However, it may not be           any of these large-scale gene expression studies.
essential that a protein be actively secreted to be useful as a biomarker      This small number of overlapping genes between the current and
because protein may be released from dying tumor cells, which are           other recent studies has several possible explanations. First, the num-
frequent in lung carcinomas. Also, tumors that occur frequently in          ber of specimens examined is smaller in the present analysis than in
central airways (SCLC and squamous carcinomas) are most often               the other analyses. Second, we attached only limited importance to
MAGE-A/NY-ESO-1-positive, suggesting that these biomarkers may              intertumor heterogeneity of gene expression in this current study. We
be particularly useful for sputum testing.                                  assumed that lung cancers, because they are heterogeneous in almost
   An advantage of OMA analysis for potential biomarkers is the             every respect, are likely to also exhibit a high degree of heterogeneity
ability to interrogate microarray data for expression patterns of all or    in gene expression profiles. We expected that there would be a great
many members of entire functional pathways. In this context it is of        imbalance of many cellular pathways engendered by chromosomal
interest that melanoma genes other than the CTAG genes were found           and genetic instability, potentially resulting in high levels of over-
not to be overexpressed in lung cancer lines indicating that there are      expression of specific genes. Our objective was to identify these genes
significant differences in activation of functional pathways between        and to estimate the likelihood that their products could serve as tumor
these two tumor types. The function of CTAG genes in general and            biomarkers. Finally, we made no distinction among tumors of various
MAGE-A genes in particular is not known. Necdin, a 325 amino acid           histological origins in screening for highest level overexpression. This
protein with 30% homology to MAGE proteins (reviewed in Ref. 53)            allowed us to focus on genes that are massively overexpressed in
has been shown recently to interact with p53, inhibiting p53-induced        cancer cells in comparison to normal lung regardless of the cell type,
apoptosis. Whether or not the MAGE proteins function in a similar           an approach specifically tailored for biomarker discovery.
way is unknown at present but such a function would be consistent              We conclude that the detailed gene expression data can now be
with the frequent expression of MAGE proteins in aggressive malig-          readily obtained using OMAs. Testing of even a few suitable speci-
nancies.                                                                    mens can identify potential biomarkers for lung and other cancers that
   Several of the remaining highly overexpressed genes have proper-         can be rapidly validated by high throughput testing of TMA linked to
ties that suggest they may be useful biomarkers including signal            clinical outcome. This model should be a rich source of promising
peptide coding sequences. One of four protein products of genes             new biomarkers. To exploit these new analytical tools it will be
containing signal peptides has been tested as a lung cancer biomarker.      imperative that correlative biological materials be collected during
Chromogranin A has been found in the serum of 50% (14) of all of the        large-scale screening and treatment trials that are currently being
neuroendocrine tumors and 61–70% (15, 54) of SCLC, and is gener-            designed.
ally regarded as a promising marker for the diagnosis of neuroendo-
crine neoplasia. A second putatively secreted protein, ASH1, has been       ACKNOWLEDGMENT
associated previously with neuroendocrine neoplasia (55) but has not
been tested as a serum or urinary biomarker. That this protein is in fact      We thank Dr. Marileila Varella-Garcia for spectral karyotype analysis of the
secreted is doubtful, given its role as a transcription factor and its      bronchial epithelial cell culture.
nuclear localization. Other overexpressed genes associated with neu-
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