American Journal of Pathology, Vol. 150, No. 6, June 199 7Copyright X) American Society for Investigative Pathology

Molecular Detection of Tumor-AssociatedAntigens Shared by Human CutaneousMelanomas and Gliomas

Dorcas D. J. Chi,* Randall E. Merchant,tRobert Rand,t Andrew J. Conrad,*Darryl Garrison,* Roderick Turner,§Donald L. Morton,* and Dave S. B. HoontFrom the National Genetics Institute,* Los Angeles,California, the Division of Neurosurgery,t Medical College ofVirginia, Virginia Commonwealth University, Richmond,Virginia, and the Departments ofMolecular Oncology andNeuro-Oncology,* John Wayne Cancer Institute, and theDepartment of Pathology,5 SaintJohn s Hospital and HealthCenter, Santa Monica, California

Both melanocytes and glial ceUs are derived em-bryologicaly from the neural ectoderm. Theirmalignant transformed counterparts, melanomaand glioma ceUs, respectively, may share com-mon antigens. Numerous tumor-associated anti-gens have been identified in melanomas but onlya few in gliomas. Using an established reversetranscriptase polymerase chain reaction plusSouthern blot assay, we compared the mRNA ex-pression of melanoma-associated antigens(MAAs) ofmelanomas to brain tumors primarilyderived from glial ceUls. The MAAs studied in-cluded tyrosinase (Tyr), tyrosinase-relatedprotein-I and -2 (TRP-i and TRP-2), gpiOO, hu-man melanoma antigen-encoding genes I and 3(MAGE-i and MAGE-3), and melanotransferrin(p97). Glioblastoma multiforme (n = 21), ana-plastic astrocytoma (n = 3), ependymoma (n =

2), meningioma (n = 3), oligodendroglioma (n =

1), and melanoma (n = 12) tumor specimenswere assayed for MAA mRNA expression. Glio-blastoma multiforme, astrocytoma, and mela-noma cell lines were also assayed. We observedthat individual MAA mRNAs were expressed inthese brain tumors and ceU lines at varying fre-quencies. The melanogenesis-pathway-relatedMAAs Tyr, TRP-I, TRP-2, andgp1OO mRNAs werealso expressed at different levels in normal braintissues but at a much lower frequency than in

glioblastoma multiforme and melanoma. MAGE-Iand MAGE-3 mRNA were expressed in differenttypes oftumor specimens and ceUl lines but neverin normal brain tissue. Tumor antigen p97 wasexpressed in aU types oftumors and also in nor-mal brain tissues. These studies demonstratethat melanomas and primary brain tumors ex-press common MAAs and could be exploited inpatients with malignantglioma by active speciftcimmunotherapy against these common MAAs.(Am J Pathol i997, 150.2143-2152)

Glial cells and melanocytes are both derived fromthe neural ectoderm.1'2 Many studies have shownthat neoplasms derived from these two types of cells,gliomas and melanomas, share many biologicalproperties.3 6 However, there is limited characteriza-tion of tumor antigens in glial-derived tumors. Al-though melanoma-associated antigens (MAAs) havebeen well characterized, immunogenic tumor-asso-ciated antigens in gliomas have not been well de-fined. Many of the MAAs assessed in melanomas arerelated to the melanogenesis pathway. Several ofthese anti-MAA monoclonal antibodies have beenshown to cross-react with gliomas.4'5 In addition,mRNAs encoding other MAAs such as human MAAmelanoma antigen-encoding gene MAGE-1, havebeen detected in tumors of neural origin such asglioblastomas and neuroblastomas.3 Melanogene-sis-related proteins involved in the synthesis of mel-anin are inherent characteristics of melanocytes andmelanomas, and neural derived tissues of the co-chlea and eye have also been reported to expressmelanin and melanogenesis-related proteins.7-9

Supported in part by grant P01 CA1038 from the National CancerInstitute, National Institutes of Health, and by a grant from theMassey Cancer Center, Richmond, VA.

Accepted for publication February 27, 1997.

Address reprint request to Dr. D. S. B. Hoon, Department of Mo-lecular Oncology, John Wayne Cancer Institute, 2200 Santa MonicaBlvd., Santa Monica, CA 90404.

2143

2144 ChietalAJPJuine 1997, Vol. 150, No. 6

Tyrosine DOPA DOPAquione

DOPAcbrome ---

DIHI DE quCA

Indole-5,6-quinone Indcat

Eumelanin

LeucoDOPAclrome

dole-s,6-qulinone-rboxylic acid

7/Figure 1. 7be schenzatic of the melanogeniesis pathuay. DOPA, 3,4-dibvdroxyphenvlalanine; DHI, 5,6-dihydroxyindole; DHICA, 5,6-dihy-droxyindole-2-carboxylic acid.

These observations have been derived primarilythrough immunocytochemistry. The melanogenesispathway is a complex cascade of enzymatic reac-

tions beginning with tyrosine and ending with mela-nin as the final product (Figure 1). All components ofthe pathway have not been identified and steps inthe pathway are still uncertain at this time. The mel-anin observed in melanocytes is suggested to bedifferent from neuromelanin in neurons of the sub-stantia nigra, locus ceruleus, and other brain stemcells. The metabolic pathway that leads to neu-

romelanin remains unknown, although it has beenpostulated that neuromelanin may be a by-productof norepinephrine and dopamine metabolism com-

monly associated with neurological functions.To date, many MAAs, including melanogenesis-

related proteins, have been demonstrated to be an-

tigenic in humans by cytotoxic T cell and antibodyassays.10 14 Several of these MAAs are currentlybeing investigated as target antigens for active spe-

cific immunotherapy against human melanoma. 15-17On the other hand, there have been only a limitednumber of antigenic antigens identified in glial tu-mors. The identification of common MAAs in mela-noma and glioma may be potentially useful in thelatter as target antigens for active specific immuno-therapy. Cancer vaccines for melanoma treatmenthas shown some promising therapeutic benefit incontrolling tumor progression.15-17 Although surgery

and radiotherapy are the primary approaches fortreating patients with glial tumors, their efficacy forcontrolling or preventing recurrent disease has beendisappointing. Adjuvant therapies such as activespecific immunotherapy that are less debilitating tothe patient need to be developed to prevent or sta-bilize recurrent disease. To develop these therapeu-

tics, however, tumor antigen targets need to be char-acterized in these brain tumors.Due to the similar embryological origin of melano-

cytes and glial cells, we examined mRNA expressionof common MAAs in their respective tumors. Molec-ular diagnosis of cancer by reverse transcriptasepolymerase chain reaction (RT-PCR) has recentlybeen shown to be a rapid, efficient method to exam-ine expression of genes with minimal amounts oftissue.18-21 The development of RT-PCR has signif-icantly aided cancer diagnosis and characterizationof gene expression with a limited amount of tissue.Analysis of brain tumor gene expression needs to bestudied to design potential new therapeutics andanalyze tumor gene expression profiles during tumorprogression. The majority of the molecular studies todate on brain tumors have been focused on DNArearrangements, oncogenes, and tumor suppressorgene activation.22.24

In this study, we assessed four major melanogene-sis markers, tyrosinase (Tyr), tyrosinase-related protein(TRP)-1, TRP-2, and melanoma antigen (gp100), andthree tumor-associated antigens, MAGE-1, MAGE-3,and melanotransferrin (p97), as the MAA markers in aRT-PCR plus Southern blot analysis of brain tumorspecimens and glioma cell lines. Tyr is the key enzymein the initial segment of the melanin synthesis path-way. 5,26 TRP-1 and TRP-2 are involved in the latersteps of melanin synthesis.27 29 gplOO, related to thePmel-17 gene family is associated with the melanoso-mal-related matrix.30'31 The tumor-associated antigensMAGE-1 and MAGE-3 are immunogenic in humans,and their function is unknown.12 13 They are expressedin melanomas and many other types of tumors of dif-ferent embryological origin and have not been found innormal tissues except testes and placenta.32'33 p97 ispresent in most melanomas, and has been shown tobe expressed in other tumors and fetal tissues.3435This study demonstrates that gliomas and melanomasexpress common MAA genes in which a subset ofthese markers is part of the melanogenesis pathway.

Materials and Methods

Cell Lines and Tumor SpecimensHuman melanoma cell lines M12, M24, and M101were established and characterized at the JohnWayne Cancer Institute."1836 Human glioblastomamultiforme cell lines U-87 MG and U-118 MG andastrocytoma lines CCF-STTG1 and SW1088 wereobtained from American Type Culture Collection(Rockville, MD). Human glioblastoma multiformelines RC1909 and RC1913 as well as the brain tumor

Melanoma-Associated Antigens in Gliomas 2145AJPJune 1997, Vol. 150, No. 6

Table 1. Oligonucleotide Primers of Melanoma Molecular Markers and 13-Actin Gene

Gene Primer sequences Size (bp)

1-Actin 5' CCT TCC TGG GCA TGG AGT CCT G 2013' GGA GCA ATG ATC TTG ATC TTC

Tyr 5' TTG GCA GAT TGT CTG TAG CC 2843' AGG CAT TGT GCA TGC TGC TT

TRP-1 5' AGA GAT GAT CGG GAG GTC TG 4253' CTG TGC CAT GTG AGA AAA GC

TRP-2 5' GAG GTG CGA GCC GAC ACA AG 4763' CGG TGC CAG GTA ACA AAT GC

gp100 5' TGG ACC TTG CCC ATC TGG CTC TTG G 5343' TGC CCA TCT GTG GTG CCT GGA ACT G

MAGE-1 5' CGG CCG AAG GAA CCT GAC CCA G 4213' GCT GGA ACC CTC ACT GGG TTG CC

MAGE-3 5' GAA GCC GGC CCA GGC TCG 4233' GGA GTC CTC ATA GGA HTG GCT CC

p97 5' TAC CTG GTG GAG AGC GGC CGC CTC 2863' AGC GTC TTC CCA TCC GTG T

specimens were obtained from patients undergoingtreatment at Virginia Commonwealth University. Celllines were maintained as monolayer cultures in RPMI1640 supplemented with 10% heat-inactivated fetalcalf serum (Gemini, Calabasas, CA), penicillin, andstreptomycin. Total RNA was extracted from cellswhen cultures reached 70 to 80% confluency.

Brain tumor specimens studied were from surgi-cally resected tissue and were immediately frozen inthe operating room and stored at -80°C until used.Other specimens from these operations were as-sessed by pathologists to determine the type andgrade of tumors. Pathologically defined normal braintissues from patients undergoing operation for a non-tumor-related problem were studied for comparison.These brain specimens were taken from the frontalcortex and temporal lobe of the brain. Pathologicallydefined melanoma specimens, along with normalbreast and lung tissues from cancer patients werealso assessed for comparison. Normal breast andlung tissues were used in this study as negativeRT-PCR controls.

Normal male and female volunteer donor bloodwas used for negative controls. Ten milliliters ofblood was collected in sodium-citrate-containingtubes and centrifuged using a hypotonic densitygradient solution (Dot Kit, National Genetics Institute,Los Angeles, CA).19'20 Total peripheral blood lym-phocytes (PBLs) in the blood were used for RNAisolation.

RNA PreparationTotal cellular RNA was extracted using Tri-Reagentaccording to the manufacturer's protocol (MolecularResearch Center, Cincinnati, OH) as previously de-scribed.19'20 All specimens were kept on ice during

processing. The tissues were minced and lysed in 1ml of Tri-Reagent. The nucleic acid extracts werewashed once with 75% ethanol, vacuum-dried, andresuspended in 10 mmol/L Tris/HCI with 1 mmol/LEDTA solution (pH 7.4). The total amount of RNA andits purity were assessed by ultraviolet spectropho-tometry. The integrity of mRNA was assessed byRT-PCR of ,B-actin expression and analyzed by gelelectrophoresis plus ethidium bromide staining. AllRNA extraction was carried out in a designated ster-ile laminar flow hood with RNAse-free labware. Sep-arate rooms and building facilities were designatedfor tissue processing, RNA isolation, RT-PCR set-up,and Southern blot analysis to avoid post-PCR con-tamination.

Oligonucleotide Primers and cDNA Probes

Oligonucleotide primers were synthesized and puri-fied at the National Genetics Institute. Optimal primersequences were designed with the Oligo PrimerAnalysis Software, version 5.0, by National Biomed-ical Systems, Plymouth, MN (Table 1).

Reverse Transcriptase Polymerase ChainReactionAn aliquot containing 1 jig of total RNA was mixedwith diethyl pyrocarbonate-treated double-distilledwater to bring the volume to 11.5 Atl. The sampleswere placed at 70°C for 5 minutes and then chilledon ice. The RT reaction mixture consisted of 4 ,ul offirst-strand RT buffer (USB, Cleveland, OH), 2 ,tl of10 mmol/L deoxynucleotide triphosphate mixture(dNTPs), 20 U of RNAsin, 500 ng of oligo dT15primer, and 200 U of Moloney murine leukemia virus

2146 Chi et alAJPJune 1997, Vol. 150, No. 6

Table 2. mRNA Expression of Molecular Markers in Glioma Cell Lines

Cell line Type Tyr TRP-1 TRP-2 gp100 MAGE-1 MAGE-3 p97

RC1909 G + + - - - - -RC1913 G - + - - - - -CCF-STTG1 A - + + + - + +SW1088 A + + + + + + +U-87 MG G + + + + + + +U-118MG G + + - + + + +% Positive 67% 100% 50% 67% 50% 67% 67%

Analysis of cell lines (1 ,ug of RNA) was by RT-PCR and Southern blot. A, astrocytoma cell line; G, glioblastoma multiforme cell line.

reverse transcriptase (Promega, Madison, WI). TheRT mixture was added to the samples to a finalvolume of 20 Al, and the reaction was incubated at370C for 2 hours, heated to 950C for 5 minutes, andthen chilled on ice. All RT reactions were carried outwith oligo dT priming.

The PCR mixture was prepared as previously de-scribed.l8-20 Briefly, the mixture consisted of 10 ,ul of1OX thermophilic DNA polymerase reaction buffer, 8 ,uiof 10 mmol/L dNTP, 6 ,ul of MgCl2 (25 mmol/L), 100pmol/L each primer, 5 U of AmpliTaq DNA polymerase(Promega), and 20 ,ul of RT mixture. Double-distilledwater was added to bring the reaction volume to 100,ul. The PCR program was set as follows: 1 cycle of950C for 5 minutes; 35 cycles of 950C for 1 minute,55°C for 1 minute (for all primers except p97 at 650C),and 720C for 1 minute; and final extension at 720C for10 minutes. PCR was performed in an OmniGene tem-perature cycler (Hybaid, Middlesex, England).

Automated Southern Blot AnalysisThe RT-PCR cDNA products were first run on 2%agarose gel and then alkaline denatured and trans-ferred onto nitrocellulose membrane with 20X stan-dard saline citrate (SSC) buffer as previously de-scribed.la.19 Specific cDNA probes for Tyr, TRP-1,TRP-2, gplOO, MAGE-1, MAGE-3, and p97 were de-veloped from corresponding RT-PCR cDNA prod-ucts and labeled with digoxigenin.18 Southern blot-ting was performed using automated Southern blotinstrumentations under standard operation proce-dures (National Genetics Institute). The use of anautomated Southern blot procedure allows analysisof multiple specimens and controls under highlystringent quality-controlled conditions at the sametime. Southern blot analysis is very essential in veri-fying the RT-PCR cDNA product and can enhancesensitivity. For each Southern blot run, negative con-trols included RT-PCR performed with all reagentsexcept for mRNA and two normal PBL samples thatwere carried throughout the entire RT-PCR proce-dure. Positive controls included two positive mela-

noma cell lines. The blots were scanned on an elec-tronic imager and verified by at least two readers forpositive and negative results. Any discrepancies,questionable blots, and false positives were re-peated for verification.

ResultsMelanoma cell lines, melanoma specimens, gliomalines, and brain tumor specimens were assayed forexpression of MM mRNA. We have demonstrated theexpression of the MM in melanoma specimens (to bepublished).l' In this study, we assessed melanomas toprovide a representative analysis for comparison tobrain tumor cell lines and tumor specimens. Melanomaspecimens were run at the same time and with theidentical conditions as the brain tumor specimens forassay comparison purposes. As negative controls,mRNA of PBLs from 15 normal donors were assessedfor the MM. None of these normal PBLs was positivefor the MM molecular markers after Southern blotting,although there was an occasional related weak positiveband for gplOO (<2%) observed near the gplOO RT-PCR cDNA band.

MAA mRNA Expression in Melanoma andGlioma Cell LinesAll three representative cutaneous melanoma celllines assessed (M12, M24, and M101) were positivefor each of the MAA markers studied: Tyr, TRP-1,TRP-2, gplOO, MAGE-1, MAGE-3, and p97. The sixglioma cell lines (four glioblastoma multiforme andtwo astrocytoma lines) expressed one or more ofthese MAA markers. The overall expression level ofMAA mRNA in glioma lines, however, was lower thanthat observed for melanomas. TRP-1 was expressedin all six glioma lines, whereas Tyr, gplOO, MAGE-3,and p97 were expressed in four (67%) of the lines.TRP-2 and MAGE-1 were expressed in three (50%)of the lines (Table 2). The glioma cell lines wereassessed to show relative MAA marker distribution.

Melanoma-Associated Antigens in Gliomas 2147AJPJune 1997, Vol. 150, No. 6

Tyrosinase

TRP-1

TRP-2

gplOO

1 2 3 4 5 6

Figure 2. Representative RT-PCR and Southern(tyrosinase, 7TP-1, TRP-2, andgplOO) mRNA enlines and biopsy specimens. Lanes 1 and 2, Alanes 3 to 6, glioma biopsies positivefor MAAdonor PBLs; lane 8, positive marker SouthernBands on top of the specific cDNA bandsfor TR?are genomic products.

Representative examples of RT-PCblots are shown (Figures 2 and 3).ular markers in general are overanlished cell lines,37 we evaluated turrget a more realistic assessment ofpression.

p97

MAGE-1

MAGE-3

1 2 3 4 5 6

Figure 3. Representative RT-PCR and Southern(MAGE-1, MAGE-3, and p97) mRNVA expressioand biopsy specimenZs. Lanes 1 and 2, glioblastto 6, glioma biopsies positive for MAA mRNA;PBLs; lane 8, positive marker Southernz blot sta,

- 284 bpMAA mRNA Expression in Melanoma andBrain Tumor SpecimensTwelve tissue specimens of malignant melanoma were

-425bp analyzed by RT-PCR plus Southern blot to detect MM

mRNA expression (Table 3). p97 was present in every

- 476bp specimen, followed by Tyr and TRP-2, which werepresent in all but one specimen (92%). gplOO, TRP-1,MAGE-3, and MAGE-1 were expressed in 83, 67, 58,and 42% of the tumor specimens, respectively. All 12

-- 534bp melanoma specimens expressed at least three MMmRNAs, and 10 (83%) were positive for at least five of

7 8 the seven MMs studied.blot analysis ofMAA

upression inglioma cell Thirty brain tumor specimens, which included twen-,tlioblastoma cell lines; ty-one glioblastoma multiforme, three astrocytomas,mRNA; lane 7, normalblot standard control. three meningiomas, two ependymomas, and one oli-P-1, TRP-2, andgplOO godendroglioma were assessed for MM mRNA ex-

pression (Tables 4 and 5). RT-PCR plus Southern blotanalysis showed that all the MM mRNAs were ex-

,R and Southern pressed in these specimens, although their expressionAs tumor molec- varied among individual tumors (Figures 2 and 3).nplified in estab- Of the 21 glioblastoma specimens analyzed fornor specimens to MAA by RT-PCR plus Southern blot, 7 (33%) wereMAA mRNA ex- positive for at least five markers, and only 3 speci-

mens were negative for all seven MAAs (Table 4).The melanoqenesis-related proteins Tyr, TRP-1,

-:: 286bp TRP-2, and gp100 mRNA were expressed in 38, 52,62, and 38% of the glioblastoma specimens, respec-tively (Table 4). The tumor antigens MAGE-1,

* *---421bp MAGE-3, and p97 were present in 38, 33, and 67%* of the glioblastoma specimens, respectively. The

overall frequency of all of the markers mRNA expres-sion in these glioblastoma specimens was lower than

>.,;; 9 ~423bp that observed in melanoma specimens.TshhIl R .chc',A/,c, tht mr\lIA Pynrqooi-n frcnr lr,,-.so

7 8 CIaUIr J ,bIl IUVVQ Li It;1 I II\n t:;A .I 'JI I1 tV U IlIy 'l

blot examples ofMAA the markers in the astrocytoma (n = 3), ependymoman in glioma cell lines (n = 2), meningioma (n = 3), and oligodendrogliomatoma cell lines, lanes 3 (n = 1) specimens. These tumor specimens were as-lane 7, normal donorndard control. sessed to determine whether other brain tumors of glial

Table 3. mRNA Expression of Molecular Markers in Metastatic Melanoma Specimens

Melanoma specimen

ABCDEFGH

JKL% Positive

Tyr TRP-1

+

+

TRP-2 gplOO MAGE-1

+

++ ++ ++

+ ++ +

92% 67%

++

+2

++++++++

83%

+

42%

MAGE-3 p97

+

+

+

58%

++++++++++++

100%

Analysis of 12 melanoma specimens (1 [kg of RNA) tested by RT-PCR and Southern blot.

2148 Chi et alAJP June 1997, Vol. 150, No. 6

Table 4. mRNA Expression of' Molecular Markers in Glioblastoma Specimens

Specimen

RC 1904RC 1908RC 1934RC 1941RC1946RC1951RC 1953RC 1955RC 1967RC 1969RC 1974RC 1977RC 1983RC 1988RC 1995RC2002RC2004RC2027RC2049RC2057RC2060% Positive

Tyr TRP-1

+

+

+

+++

+

38%

+++

++

52%

TRP-2

+

++++2

gp100

+

+

+

38%

MAGE-1

Analysis of 21 glioblastoma specimens (1 jig of RNA) tested by RT-PCR and Southern blot.

and nonglial origin express MM mRNA. All three as-trocytoma specimens expressed p97, followed by Tyr,TRP-1, and TRP-2 in two specimens and gplOO andMAGE-1 in one specimen. None of the three astrocy-tomas were positive for MAGE-3. TRP-1 and p97 wereshown in both ependymoma specimens. One ependy-moma specimen expressed all of the MAAs exceptMAGE-1. gplOO and p97 were expressed in all threemeningioma specimens, followed by TRP-1 andMAGE-1 in two specimens and TRP-2 and MAGE-3 inone specimen. None of the three meningioma speci-mens were positive for Tyr. The oligodendrogliomaspecimen was positive for TRP-2, gpl 00, MAGE-1, andp97 MAA.

Assessment ofMAA mRNA in NormalTissues

For comparison, pathology-defined normal brain(n = 1 1) tissues were also assessed in this study and

were run at the same time as the brain tumors. Themelanogenesis-related markers Tyr, TRP-1, TRP-2,and gp100 mRNA were found in some normal brainspecimens (Table 6). p97 mRNA was present in four(36%) of the normal brain specimens. However,none of the normal brain tissue samples expressedMAGE-1 or MAGE-3 mRNA. Pathology-defined nor-mal tissues of breast and lung assessed at the sametime showed no marker expression.

DiscussionThis study demonstrates by molecular analysis thatmelanoma and primary tumor of the brain cells sharecommon tumor markers. The technique used to iden-tify specific markers is highly specific and sensitiveas previously described.1920 The advantage of us-ing this molecular detection approach as opposed toother techniques such as biochemical analysis or

Table 5. mRNA Expression of Molecular Markers in Nonglioma Tumor Specimen.s

Type Tyr TRP-1

AAAEEM

M

M

0

+

TRP-2 gplOO MAGE-1

~~~+

+-_

+

+

t

MAGE-3 p97

_ ~~+

_ ~~+

_ ~~+

_ +~~

+

+

Analysis of specimens (1 ,tg of RNA) tested by RT-PCR and Southern blot. A, astrocytoma; E, ependymoma; M, meningioma; 0,oligodendroglioma.

MAGE-3

++

p97

++

+ ++

+

+

t

38%

+

++

67%+

33%

Specimen

RC2016RC2031RC2034RC2029RC2023RC2022RC2012RC2011RC 1 957

Melanoma-Associated Antigens in Gliomas 2149AJP June 1997, Vol. 150, No. 6

Table 6. mRNA Expression qf Molecular Markers in Normal Brain Specimens

Specimen

ABCDEFGH

JK% Positive

Tyr TRP-1

+

++

++

45%

+

+++

++±+

73%

TRP-2

82%

gpl 00 MAGE-1 MAGE-3 p97

++

+

55% 0% 0%

++

36%

Analysis of specimens (1 ,ug of RNA) tested by RT-PCR and Southern blot.

antibody staining is that it is less labor intensive andmore specific, it can be easily repeated severaltimes on a small amount of tissue, reagents are

readily available for all the markers and, most impor-tantly, it requires a very small amount of tissue. Usingour established technique, we were able to assess

multiple molecular markers that are common for bothmelanomas and brain tumors. As the embryologicalorigins of both tumor types are common, there was

expectancy that expression of some of these molec-ular markers would be shared. Assessment of mo-

lecular markers by RT-PCR of tumors can providerapid assessment of genotype expression of specificgenes. However, no information about expression ofthese MAAs at the protein level in the cells or theheterogeneity of each MAA expression within a le-sion is known. We have used the tumor marker RT-PCR assay to assess metastatic occult tumor cells inblood and various types of tissues successfully.18-20To date, there has not been a comprehensive

comparison analysis of antigens in both human mel-anomas and gliomas. The study is important for sev-

eral reasons because the identification of commontumor antigens in both tumor types will help in de-veloping active specific immunotherapy protocolssuch as cancer vaccines.15 Melanoma has been wellstudied by our group and others in terms of antigen-specific immune responses induced by active spe-

cific immunotherapy.17'40 The outcome of clinical tri-als of melanoma vaccines has been encouraging.However, effective treatment of malignant primarytumors of the brain by various treatments, includingactive specific immunotherapy, is far less devel-oped.41 Identification of common antigens among

gliomas, the most common of brain tumors, andmelanomas would be important to address the fea-sibility of potential therapeutic development againstthe former. Also, it is important to note that melano-mas often eventually kill the host through brain me-

tastases.42 Greater than 70% of melanoma patientswho have expired with advanced disease have me-tastases to the brain.43 Therefore, treatment of met-astatic melanoma to the brain with active specificimmunotherapy against specific antigens will requiresome knowledge of normal brain tissue expressionof these antigens as well.

Our initial focus of study was on the melanogenesismarkers. In the literature, there are indications, primar-ily by biochemical analysis, that melanogenesis-relatedproteins are present in normal brain.8 One purpose ofour study was to determine whether neural-derivedbrain tumors express melanogenesis MAA mRNA.Several of the melanogenesis-related proteins, such asgplOO, Tyr, gp75 (TRP-1), MAGE-1, and MAGE-3,have been shown to be antigenic.10-14,16,28,40 All of theMAAs were found in varying frequencies among theglioblastoma specimens. It was surprising in detectingthe melanogenesis cascade of proteins Tyr, TRP-2,TRP-1, and gplOO. However, the frequency of Tyr wassignificantly lower in these glioblastomas versus mela-nomas. TRP-1 and TRP-2 mRNAs were highly ex-pressed in gliomas but less than in melanoma speci-mens. gplOO expression in glioblastomas versusmelanomas was comparable to Tyr expression. Othertypes of primary tumors of the central nervous systemwere shown to express MAAs. However, the number ofspecimens was limited.

p97, which was highly expressed in all primarytumors of the brain we studied, was also expressedin 36% of normal brain tissue analyzed. There arelimited studies on the overall expression of thismarker in tumors.34'35 Studies suggest that it is not amelanoma-specific protein and has other functionsin various tissues.34 A similar observation has beenfound with MAGE marker expression in various tu-mors. Both MAGE-1 and MAGE-3 were expressed toa lesser degree in the glioblastomas than in themelanomas. However, expression of MAGE-1 in glio-

2150 ChietalAJP June 1997, Vol. 150, No. 6

blastomas was not that much less than in melano-mas. Overall, MAGE-3 was less expressed thanMAGE-1 in gliomas, which was opposite that of mel-anomas. Interestingly, a similar pattern was found inthe other neural tumors. Although MAGE-1 and -3were discovered in melanomas, they are not mela-noma specific, as studies have shown that they areexpressed in various frequencies in different carci-nomas. 12'13,32'23 Both MAGE-1 and MAGE-3 are im-munogenic in humans and can be potential targetsfor active specific immunotherapy. We observed thatno normal tissues of the brain expressed MAGE-1 or-3 marker mRNA.

Previously, studies have demonstrated the pres-ence of Tyr by antibody staining or biochemical anal-ysis in several neural-crest-derived cells such asSchwann cells and the satellite of spinal ganglia.8Certain parts of the brain such as the substantianigra, locus ceruleus, and brain stem are pigment-ed.44 The retina of the eye is pigmented and is thesite of origin of the ocular melanoma that expressesthe melanogenesis marker and other MAA mRNA(unpublished data). Studies have not previously doc-umented the expression of melanogenesis pathwaymarkers such as TRP-1, TRP-2, and gplOO in braintumors. It has been indicated the pigmentation of theneural tissue is due to the presence of neuromelanin,although the neuromelanin synthesis pathway hasnot been well established.44 Catecholaminergic neu-rons involved in perception, movement, emotion,and memory have pigmentation.46 The overlap, ifany, of the neuromelanin and melanocyte melaninsynthesis is not known.

The expression of several MAAs in gliomas andother types of brain tumors offers a rationale forapplying current active specific immunotherapy pro-tocols used in treating malignant melanoma to glio-mas.41'47 Currently, we have a melanoma cell vac-cine protocol that has been shown to control tumorprogression and prolong survival of melanoma pa-tients with advanced-stage disease.15 Preliminarystudies also indicate that it can prevent tumor me-tastases to the brain.15 The melanoma cell vaccinecontains all of the MAAs described in the presentstudy, and there have been no major adverse effectsparticularly to the central nervous system in patientstreated to date. One could question whether cyto-toxic T cells to immunogenic MAAs such as Tyr orgplOO would affect normal tissue. In general, normalnonhemopoietic tissue and particularly the brain donot or weakly express major histocompatibility anti-gens (MHC) on their cell surfaces.4849 Therefore,they would not be expected to be susceptible to

attack by MAA-specific MHC-restricted cytotoxic Tcells.

Our study demonstrates a rapid system of identi-fying tumor markers in neural tumors that could bepotentially useful in screening for active specific im-munotherapy patient candidates. We have also iden-tified potential new known tumor antigens in primarybrain tumors that were not previously defined. Gli-oma patients may benefit from active specific immu-notherapy targeting specific MAAs.

AcknowledgmentsWe thank the clinical staff of the John Wayne CancerClinic and Department of Pathology, Saint John'sHospital and Health Center, for their expert help. Wealso thank Dr. William C. Broaddus and Mrs. AnnieChu for their help in acquisition and preparation ofbrain tumor specimens.

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