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Molecular strategy for detecting metastatic cancers with use of multiple tumor-specific MAGE-A genes.

The detection of metastatic tumor cells in tumor-draining lymph nodes and blood has been studied as a tool for the assessment of various cancers (1-6). Immunohistochemistry has improved detection of metastatic tumor cells, but may leave melanoma, breast, and colorectal cancer (CRC) [5] patients understaged (7-9). The detection of cancer cells in the blood by reverse transcription-PCR (RT-PCR) is attractive because it is sensitive and blood can be repetitively sampled (1-6).

Although RT-PCR can detect a few cancer cells among millions of healthy cells (7), there are limitations on the specificity and amount of mRNA detected by individual molecular marker(s) (10). Differentiation marker mRNA transcripts have been used to detect tumor cells, but some (e.g., the melanoma differentiation antigens tyrosinase and gp100) can also be found in melanocytes (1, 11, 12). To date, only a limited number of tumor markers have been identified that are not present in healthy cells but are commonly expressed in tumor cells. mRNA for the cytokeratin family members, carcinoembryonic antigen, and mucin-1 (6, 10, 13, 14) is also produced by healthy epithelial cells in the hemopoietic compartment. The melanoma-associated antigen A (MAGE-A) genes have high tumor specificity, but their use in the detection of occult metastasis has been limited by the low frequency of expression of individual MAGE-A genes within individual types of tumors.

Of the three most studied human MAGE gene families (MAGE-A, -B, and -C), the MAGE-A family has been the most characterized and the most identified in cancers (15-17). It consists of at least 12 members, A1-A12, which have been shown to be exclusively expressed in multiple types of human cancers, as well as in the healthy tissues of male germline cells and placenta (17-26). The difference in nucleotide sequence among individual MAGE-A gene family members can vary up to 40%. Under stringently defined conditions, any one single MAGE-A family member is typically found expressed in <40% of any single type of human solid tumor. MAGE-A expression is often more frequent in cancer cell lines than in primary or metastatic tumor specimens (27). Moreover, metastatic tumors can demonstrate higher MAGE-A mRNA abundance than that of the corresponding primary tumors (19, 22).

We have demonstrated that a multimarker approach offers advantages over the use of a single marker for the detection of occult tumor cells (1, 7, 27, 28). However, the assessment of multiple MAGE-A mRNAs by use of RTPCR with specific primer sets for each mRNA is logistically cumbersome and offers only limited sensitivity with gel electrophoresis-based detection systems. In the present study, we investigated the utility of detecting expression of multiple major MAGE-A genes in a tumor-specific RT-PCR/electrochemiluminescence (ECL) assay. We refer to this MAGE-A RT-PCR/ECL assay as the universal MAGE-A (uMAGE-A) assay.

The uMAGE-A assay utilizes a sensitive solution-phase detection method with specific probe hybridization. Recently, we demonstrated the use of various mRNA tumor markers for the detection of occult cancer cells, using the RT-PCR assay followed by probe hybridization to verify specificity and to improve sensitivity (2, 7, 9). This technique avoids some of the limitations of gel-based assays, such as the subjectivity of analysis and the lower sensitivity in detecting low concentrations of PCR cDNA products. The assessment of these multiple cDNA products with similar fragment size would be difficult by gel-based methods. To circumvent this problem, we have adopted a sensitive solution-phase probe hybridization assay (29) and used it to assess tumor specimens and to detect occult metastatic cancer cells in blood from patients with various malignancies.

Materials and Methods


The breast cancer cell lines BT-20, MCF-7, T47D, and MDA-MB-231 and the CRC cell lines HT-29 and SW480 were obtained from the American Type Culture Collection and cultured according to instructions. The 734B breast cell line is an established subclone of MCF-7 and was also used. In addition, other cell lines established and characterized at the John Wayne Cancer Institute (JWCI) were used: breast cancer cell lines JM992Br and PM277Br; CRC cell lines JWCI-0361, JWCI-0427, and JWCI-1100; and melanoma cell lines Matt, M10, M12, M14, M15, M24, M101, M111, and M112. All established cell lines were grown in RPMI-1640 medium supplemented with 100 mL/L heat-inactivated fetal calf serum (Gemini Bioproducts), penicillin, and streptomycin (Life Technologies) in T75 [cm.sup.2] flasks as described previously (10). Total RNA was extracted from cells when cultures reached 70-80% confluence.

Tumor specimens were obtained in consultation with the surgeon and pathologist at Saint John's Health Science Center (Santa Monica, CA). Informed consent was obtained from patients for the use of all specimens. Pathology-verified primary or metastatic lesions from cancer patients undergoing elective surgeries at the JWCI clinics were used. All tissues were collected and dissected under stringent sterile conditions to prevent RNA contamination. Tissue specimens obtained from surgery were immediately processed for RNA or were cryopreserved at -80[degrees]C until processed at a later time.


Blood samples from cancer patients were collected as described previously from the JWCI Clinic and the Martin Luther King, Jr./Drew Medical Center, Breast Cancer Clinic (Los Angeles, CA) (10, 27). Blood (10 mL) was obtained from patients and collected in sodium citrate-containing tubes. The first 1-2 mL of blood at the initial venipuncture were discarded to eliminate skin plug contamination. Blood samples from patients were obtained before surgery. Blood samples collected from 20 healthy volunteer donors were used as negative controls in the RT-PCR assay.


Total cellular RNA from tissue specimens, blood, and cell lines was extracted using Tri-Reagent according to the manufacturer's instructions (Molecular Research Center Inc.). Purity and quality were assessed by ultraviolet spectrophotometry. The same amount of total RNA (1 [micro]g) was used in all reactions for all samples in the study, including the control samples. All reverse transcription reactions were carried out with oligo(dT) priming as described previously (7, 10). Porphobilinogen deaminase (PBGD) gene expression was assessed by RT-PCR as a housekeeping gene on all samples to verify mRNA integrity (27). The PCR conditions were as follows: 1 cycle of denaturing at 95[degrees]C for 5 min, followed by 35 cycles of 95[degrees]C for 1 min, 62[degrees]C (for uMAGE-A), 60[degrees]C (for MAGE-A1), or 55[degrees]C (for MA GE-A3 and PBGD) for 1 min, and 72[degrees]C for 1 min before a final primer sequence extension incubation at 72[degrees]C for 10 min. PCR products were then evaluated by automated ECL analysis on the ORIGEN[R] Analyzer (IGEN Intl., Gaithersburg, MD). In each experiment set-up, samples of RT-PCR reagents without mRNA, molecular-grade water alone, and healthy donor peripheral blood leukocyte (PBL) RNA were used as negative controls throughout all RT-PCR steps and in ECL analysis. Positive controls in each experiment consisted of tumor cell lines with known expression of each specific MAGE-A gene.


Biotinylated oligonucleotide primers and Tris(2,2'-bipyridine) ruthenium(II) chelate (TBR)-labeled nucleic acid hybridization probes for the RT-PCR/ECL assay were synthesized by Gemini Biotech and The Midland Certified Reagent Company, respectively. Primers and probe sequences were designed for optimal use in the PCR and ECL assay system using Oligo Primer Analysis Software, Ver. 5.0, by National Biomedical Systems. Oligonucleotide sequences from MAGE-A1 cDNA (5'-000CGAAGGAACCTGACCC-3', primer uMAGE-A1 m-U) and MAGE-A3 cDNA (5'-CTCGGTGAGGAGGCAAGGT-3', primer uMAGE-A3 m-U) were used as sense strand primers, and an oligonucleotide sequence from MAGE-A3 cDNA (5'-biotin-CTGGA000TCCCTGAGGACT-3', primer uMAGE-A3 m-L) was used as antisense strand primer for the PCR assay. Primer sites shown in Fig. 1 are relative to the alignment of MAGE-A1, -A3, and -A12 genes.


The uMAGE-A3 m-U primer was designed to hybridize not only with MAGE-A3 and -A6, but also with MAGE-A1, -A5, and -A12. There is a single mismatched by G in the primer with respect to each of the cDNA products. The uMAGE-A3 m-L primer was designed to target MAGE-A3, -A6, -A1, and -A12. The uMAGE-A3 m-U and the uMAGE-A3 m-L primers have mismatched sequences relative to the other MAGE-A genes. However, the mismatched sequences consist of only one or two nucleotides near the 5' segment, thus the oligomers are still expected to amplify other MAGE-A cDNAs as well. The combination of the two sense strand primers and one antisense primer is expected to produce a MAGE-A3/A6 cDNA fragment of 357 bp, a MAGE-A12 cDNA fragment of 301 bp, a MAGE-A5 cDNA fragment of 378 bp, and a MAGE-A1 cDNA fragment of 365 bp. However, the combination of these primers does not exclude amplifying other MAGE-A genes such as -A2 and -A11.

Additionally, RT-PCR assays using individual MAGE-A1, -A3, and -A12 primers were performed for comparison with the uMAGE-A assay. 6-Carboxy-fluorescein-labeled uMAGE-A primers were also synthesized for polyacrylamide gel electrophoresis (PAGE) verification of the amplified MAGE-A genes. The primer sequences and predicted by sizes of the RT-PCR products are listed in Table 1.

Hybridization internal probe sequences extending through one exon junction were selected to ensure detection of only RT-PCR-specific cDNA products. This prevented nonspecific detection of corresponding genomic regions under optimal ECL assay conditions. The probe sequences for uMAGE-A, MAGE-A1, and MAGE-A3 were as follows: uMAGE-A, 5'-TBR-TGAGCAGAGGAGTCAGCACTG-3'; MAGE-A3, 5'-TBR-AGTGCTCCT000GGGGCCTCTGGTCCTC-3'; and MAGE-A1, 5'-TBR-TGGTGCTCCTCTGTGGCCTCCAGGGAATCC-3'. The PBGD probe sequence is 5'-TBR-GTAT000AGCAAGCTGGCTCTTGCGG-3'.


For PAGE analysis, cDNA (~1 [micro]g) was amplified by PCR in 25-[micro]g reactions containing 1 X PCR buffer (6.7 mM Tris, 16.6 mM ammonium sulfate, 6.7 mM EDTA, 10 mM (3-mercaptoethanol), 6 pmol of each primer, 1 U of Taq DNA polymerase, 0.8 mM each dNTP, and 1.5 mM MgCl,. PCR was performed with 35 cycles of 30 s at 94[degrees]C, 30 s at 60[degrees]C, and 90 s at 72[degrees]C, followed by a final extension step of 72[degrees]C for 5 min. A 5-[micro]g portion of each PCR product was mixed with 2 [micro]L of loading dye (2 mmol/L EDTA, 20 g/L dextran blue in formamide) and boiled at 95[degrees]C for 15 min. The resulting samples were electrophoresed on a 6% denaturing PAGE containing 7.7 mol/L urea for 2 h. The 6-carboxy-fluorescenn-labeled PCR product images were scanned by a fluorescent/ optical Genomyx SC scanner (Beckman) and assessed using Genomyx software. After the image acquisition was completed, image files were analyzed by Adobe Photoshop TM software.


To verify that PCR cDNA products amplified by the uMAGE-A primers were the expected members of the MAGE-A family, PCR products from the M12 melanoma cell line corresponding to putative MAGE-A PCR products (~300-400 bp) were extracted from 2% agarose gels using the QIAquick gel extraction method (Qiagen) according to the manufacturer's instructions. After ligation into pBlueScript (Stratagene), the cDNA clones were transformed into Escherichia coli DH5-[alpha] cells. Ninety-six clones were detected, and DNA was extracted from the clones by alkaline SDS. The DNA was digested with restriction enzymes BamHI and HindIII. The cDNA clone PCR products were then run on gel electrophoresis and detected by ethidium bromide. Eighty-four clones corresponding to the predicted uMAGE-A gene PCR product by sizes were isolated. Of the 84 clones, 23 were sequenced with T3 or T7 primers on an ABI Prism AutoSequencer A377 (Perkin-Elmer).


PCR products were detected using an Origen analyzer as described previously (29). Briefly, 5 [micro]L of PCR product was hybridized with 10 pmol of a TBR-labeled internal probe in 1 X PCR buffer for 30 min by denaturing at 95[degrees]C for 10 min, followed by a 20-min hybridization incubation at 50[degrees]C for uMAGE-A, 55[degrees]C for MAGE-A3 and PBGD, and 60[degrees]C for MAGE-A1. The cDNA-probe hybrids were then captured by M-280 streptavidin-coated Dynabeads (Dynal, Inc.). The Origen Analyzer was used to measure ECL activity. Reagent-negative controls included reverse transcription reaction solutions, PCR reaction solutions, beads, probes, and buffers for the ECL reaction. In addition, at least four healthy donor PBL RNA samples (negative controls) were incorporated throughout each RT-PCR/ECL assay for determining a positive cutoff point. A sample was considered positive if the ECL units detected were >2 SD above the mean of ECL units of the healthy donor PBL samples. All assays were repeated to verify results.



The study was focused on melanoma, breast cancer, and CRC because these tumors strongly express multiple MAGE-A genes and because of previous reports on the use of the MAGE-A family mRNA as tumor detection markers. During the initial assessment, uMAGE-A, MAGE-A1, and MAGE-A3 mRNA was measured in established cancer cell lines from different cancers and in PBLs from healthy volunteer donors by the RT-PCR/ECL assay. A comparison of uMAGE-A to individual MAGE-A1 and -A3 detection rates was performed because the latter two genes were the most frequently expressed members of the MAGE-A gene family in the cancers studied.

As shown in Table 2, of the seven breast cancer cell lines, six (86%) produced uMAGE-A mRNA compared with three (43%) for MAGE-A1 mRNA, and five (71%) for MAGE-A3 (Table 2). uMAGE-A mRNA was detected in all of the melanoma and CRC cell lines. In contrast, the number of cell lines that produced MAGE-A1 mRNA varied from 78% (7 of 9) in the melanoma cell lines to 40% (2 of 5) in the CRC cell lines; and MAGE-A3 mRNA was produced by all 9 melanoma cell lines, but only 3 of 5 CRC lines. Detection sensitivity and specificity of individual MAGE-A gene expression were further verified by PAGE and RT-PCR/Southern blot analysis using specific cDNA probes (data not shown).



We compared the RT-PCR/ECL assay using the uMAGE-A primers with individual MAGE-A1 and MAGE-A3 primers by assessing 26 primary breast tumors, 24 melanoma tumors, and 12 CRC tumors (Table 3). All samples assessed were PBGD positive by RT-PCR/ECL analysis. Of the 26 breast tumor specimens, 14 of 26 (54%) expressed uMAGE-A compared with 6 of 26 (23%) tumors for MAGE-A1, and 9 of 26 (35%) for MAGE-A3. Expression of all three markers was higher in melanoma tumors, ranging from 46% (11 of 24) and 50% (12 of 24), using MAGE-A3 and MAGE-A1 individual primers, respectively, to 63% (15 of 24), using the uMAGE-A primers. uMAGE-A was expressed in 5 of 12 (43%) CRC tumors, which was lower than that for patients with melanoma or breast cancer. Compared with uMAGE-A, production of individual MAGE-A1 and -A3 mRNA was also comparatively lower in CRC tumors: 17% (2 of 12) and 17% (2 of 12), respectively.


To further verify that the uMAGE PCR cDNA products detected by ECL were present in different cancer specimens, we performed PAGE analysis on cell lines and tumor specimens (Fig. 2). We were able to demonstrate, by PAGE analysis, unique and specific cDNA products that corresponded to expression of individual MAGE-A genes in each specimen. PAGE analysis allowed better differentiation of specific PCR cDNA products; however, the MAGE-A gene product bands were still situated in close proximity to one another. This illustrates the difficulty of performing a gel analysis for multiple markers in which the cDNA products are similar in size.

Sequencing of the individual PCR cDNA products was performed on each product that matched the estimated by size of the MAGE-A gene in question. The 23 clones picked for sequencing were verified to be MAGE-A3/A6, MAGE-A1, MAGE-A12, and MAGE-A11. This was a representative analysis to verify that specific MAGE-A gene family members could be amplified by the uMAGE primer set.


Total RNA was isolated from several representative cancer cell lines and serially diluted from 1 1,g to 10-7 [micro]g. The RT-PCR/ECL assay detected uMAGE-A mRNA in the melanoma, breast cancer, and CRC cell lines at -10-3, 10-1, and 1 ng of total RNA, respectively (Fig. 3). The lower detection limit for melanoma was not surprising in that melanoma is known to express several MAGE-A genes more highly than either breast cancer or CRC tumors.


Expression of uMAGE-A mRNA in the blood samples of patients with metastatic melanoma, breast cancer, and CRC was evaluated (Table 4). Blood specimens were coded and assays were run in a blinded fashion with respect to patient disease status. American Joint Committee on Cancer (AJCC) staging was determined retrospectively after the blood draw for the assay. In melanoma patient blood samples of different AJCC stages, 12 of 50 (24%) were positive for at least one MAGE-A mRNA, using the uMAGE-A primers. When samples were divided according to patients' AJCC stage, MAGE-A mRNAs were detected in 0%, 14%, 14%, and 40% of stage I, II, III, and IV patients, respectively.


Preoperative blood samples obtained from breast cancer and CRC patients were assessed by the uMAGE-A RT-PCR/ECL assay. Of the 16 breast cancer patients, 2 were stage I, 12 were stage II, 1 was stage III, and 1 was stage IV. Three of the stage 11 and one of the stage III patients were uMAGE-A positive. Of 22 CRC patients, 1 of 3 stage I/II, 2 of 4 stage III, and 4 of 15 stage IV patients' blood samples expressed uMAGE-A. Blood from all 20 healthy donors tested negative for uMAGE-A.


We describe a new method to detect mRNAs of multiple MAGE-A genes in a single reaction. The use of multiple markers, which takes into account the heterogeneity of the tumor and the variability of mRNA production, is one approach to improve the sensitivity and specificity of RT-PCR methods for detection of metastatic cells (7, 10). The high-throughput detection system allows for the assessment of multiple cDNA products at the same time and can be used to assess large numbers of samples.

The uMAGE-A primers reported in the present study were designed to minimize the number of primers needed to amplify multiple MAGE-A gene products. By keeping PCR product size as similar as possible for MAGE-A1, -A3, -A5, -A6, and -A12, similar amplification efficiency can be achieved for all markers. The primers were designed to also amplify MAGE-A2 cDNA, but the PCR product for the MAGE-A2 cDNA was 650 bp, which is twice as large as the other products. This could explain the inconsistent amplification and the detection difficulties experienced with this marker in the gel-based analysis. Our study of breast cancer and CRC cell lines and tumor specimens indicated that MAGE-A genes are expressed, although at lower abundance than in melanoma.

To our knowledge, there are no other major studies assessing the same MAGE-A gene members in the blood of patients with these cancers. We primarily assessed patients with early-stage breast cancer and CRC, in whom we found lower frequencies of expression than in melanoma patients. The lower expression rate of uMAGE-A genes in breast cancer and CRC cell lines and tumor specimens suggest that MAGE-A genes may be more useful for these cancers when combined with other molecular markers.

Gel electrophoresis and ethidium bromide staining or Southern blotting have been the most widely used methods to detect and characterize RT-PCR products. Ethidium bromide-staining analysis has limited sensitivity and is highly subjective, making probe hybridization important for improving both sensitivity and specificity (7). Southern blot analysis, however, is time-consuming and can be subjective, especially when assessing weak signals. Quantitative evaluation of RT-PCR products is difficult because of the variable expression of specific genes among tumor cells within a tumor or among various lesions in a single patient. The ECL assay provides a semiquantitative value and allows a cutoff to be established from control samples. The assay system described in this study provides a quick and efficient method to determine whether cancer cells in blood or tissues are expressing MAGE-A genes.

Assessment of MA GE-A-expressing tumor cells present in the blood or lymph nodes may be useful in identifying patients who may benefit from active-specific immunotherapy directed toward MAGE-A. MAGE-A gene products are immunogenic in humans (30-34). Previous studies have demonstrated both cytotoxic T-cell and antibody responses to various members of the MAGE-A family (17, 23, 31-35). Detection of MAGE-A-positive tumor cells may be important in evaluating future immunotherapy protocols directed toward MAGE-A genes (30, 31, 35). The amino acid sequences of some members of the MAGE-A family overlap considerably. Thus, there is cross-reactivity of immune responses to individual MAGE-A family members (31). Strategies of vaccination against specific MAGE-A gene-expressing tumor cells include peptides, DNA vaccines, and whole tumor cells (30, 31, 35).

In conclusion, we have presented a new approach to the assessment of multiple MAGE-A genes as RT-PCR markers to detect occult metastatic cancer cells. The assay was applied to cancers that are highly metastatic and in which MAGE-A genes are known to be expressed.

This work was supported in part by a grant from FANY; Grant P01 CA12582, CA29605 Project II from the National Cancer Institute, NIH; and a grant from the Osaka Medical Research Foundation for Incurable Diseases, Osaka, Japan. We would like to acknowledge IGEN International for technical support U. Heroux) and the provision of the Origen Analyzer. We thank the clinical staff of the John Wayne Cancer Clinic for their help in collecting blood. We also thank Dr. Robert Wascher for expert review.

Received October 9, 2000; accepted December 28, 2000.


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[5] Nonstandard abbreviations: CRC, colorectal carcinoma; RT-PCR, reverse transcription-PCR; MAGE-A, human melanoma-associated antigen family A; uMAGE-A, universal MAGE-A; ECL, electrochemiluminescence; JWC1, John Wayne Cancer Institute; PBGD, porphobilinogen deaminase; PBL, peripheral blood leukocyte; TBR, Tris(2,2'-bipyridine) ruthenium(11) chelate; PAGE, polyacrylamide gel electrophoresis; and AJCC, American Joint Committee on Cancer.


[1] Department of Molecular Oncology,

[2] John Wayne Cancer Clinic,

[3] Division Gastrointestinal Surgery, and

[4] Joyce Eisenberg Keefer Breast Center, John Wayne Cancer Institute, Santa Monica, CA 90404.

* Address correspondence to this author at: Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404. Fax 310-449-5282; e-mail
Table 1. Primer sequences.

Primer Primer sequence Target gene(s)


Primer PCR product size, bp

uMAGE-A 365
M-A1 421

M-A3 423

M-A12 420

Table 2. Precision of LC-MS method.

 CV, %

 Initial Intraassay, Interassay,
Sample (a) Ratio value n = 10 n = 5

Normal Mono-/di- 0.059 8.0 8.3
Increased Mono-/di- 0.391 9.7 3.6
Increased Mono-/di- 1.100 8.6 12
Increased A-/di- 0.203 11 18

(a) Samples selected from serum samples submitted for CDT

Table 3. Stability of Trf isoforms following sample dilution.

 Trf isoforms, relative ratio amount (a)

Serum samples (b) 0 h 48 h 72 h 96 h

1-Mono-/di- 0.059 0.053 0.048 0.053
2-Mono-/di- 0.391 0.418 0.458 0.446
3-Mono-/di- 1.100 0.954 0.985 0.859
4-A-/di- 0.203 0.216 0.242 0.239

 Trf isoforms, relative ratio amount (a)

Serum samples (b) Mean [+ or -] SD

1-Mono-/di- 0.053 [+ or -] 0.005
2-Mono-/di- 0.428 [+ or -] 0.030
3-Mono-/di- 0.975 [+ or -] 0.099
4-A-/di- 0.225 [+ or -] 0.019

(a) Samples were analyzed immediately after dilution (time 0 h) and
after the times indicated above. Between analyses, they were stored
at room temperature in the autosampler vials.

(b) Samples selected from serum specimens submitted for CDT

Table 4. Assessment of uMAGE-A mRNA in patient PBLs.

Type of AJCC No. of uMAGE-A mRNA
carcinoma stage patients detected, (a) n (%)

Melanoma All stages 50 12/50(24)
 I 2 0/2(0)
 II 14 2/14(14)
 III 14 2/14(14)
 IV 20 8/20(40)

(a) RT-PCR/ECL analysis of patients' presurgery blood.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Miyashiro, Isao; Kuo, Christine; Huynh, Kelly; Iida, Aritoshi; Morton, Donald; Bilchik, Anton; Giuli
Publication:Clinical Chemistry
Date:Mar 1, 2001
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