Int. J. Cancer: 67,457-460 (1996) 0 1996 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer

LETTER TO THE EDITOR

Dear Sir,

MAGE, BAGE and GAGE genes expression in fresh epithelial ovarian carcinomas

Mortality from ovarian cancer accounts for 6% of cancer deaths in women, since diagnosis is often pe$ormed when the tumor is advanced and at risk of recurrence after surgical intervention. Chemotherapy has a main role in the post- surgical treutment of ovarian cancer; however, remissions resulting from treatment are often short. Relapsing patients are usually resistant to additional chemotherapy. Post-surgical treatments capable ofpreventing tumor recurrence andprogres- sion are therefore expected to play a major therapeutic role. A potential role of the immune system in controlling ovarian tumorprogression is suggested by the presence in the neoplastic lesions of lymphocytes (Allavena et al., 1988) that specijically recognize tumor cells in a major histocompatibility complex (MHC) restricted fashion (loannides et al., 1993). Until now the only antigen identified as a potential target of such specific immune recognition is the product of the HERZlneu oncogene (Peoples et al., 1995; Fisk et al., 1995) that, however, is also expressed in some adult tissues. This may limit its therapeutic potential. More promising are antigens encoded by genes ex- pressed only in tumor cells, such as MAGE, BAGE and

GAGE genes (reviewed by Boon et al., 1995). These genes are expressed in melanomas, non-small-cell lung, breast and blad- der carcinomas, whereas no expression has been observed in normal tissues, except testis. We report herein the expression of MAGE-1, MAGE-3, BAGE and GAGE-1, -2, encoding for antigenic nonapeptides recognized by cytolytic T cells, in fresh human epithelial ovarian carcinomas.

Fifty-four cases of ovarian carcinoma were analyzed; for 25 of them the histopathological features of the tumor and the data about the clinical follow-up of the patients are presented in Table I. The presence of messenger RNA (mRhLA) of MAGE-I, -3, BAGE and GAGE-1, -2 genes was evaluated by reverse po@merase chain reaction (PCR) amplification with prim- ers specific for each gene. 7heprimers used to amplifv BAGEgenes are shared between all the members of the BAGE family.

The results of the analyses pegormed on multiple neoplastic lesions of the same patient are shown as single data in Table II: the result is considered as positive if at least one of the lesions expressed the gene analyzed. Out of the 54 samples of primaly

TABLE I - CLINICAL AND PATHOLOGICAL FEATURES 0,F 25 PATIENTS WlTH PRIMARY OVARIAN CARCINOMAS

Patient: Histology

MNl MN2 MN3 MN4 MN5 MN6 MN18 MN20 MN31 MN30 MN28 MN41 MN7 MN8 MN9 MNlO MN11 MN12 MN13 MN14 MN15 MN16 MN17 MN19 MN21

Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Clear cell Clear cell Mucinous Mucinous Mucinous Endometrioid Endometrioid

Tumor stage3

111 111 I11 111 I11 I11 I11 I11 111 I11 111 I11 IV IV IV IV IV IV 111 IV I11 111 111 I1

IV

Grading3

2

3 3 3

3 3

BL 2 3 3

3

2 2 3

3 3

Minimal residual disease4

> 10 2-5 2-5

< I 2-5 > 10 5-10 2-5

< 2 5-10 > 10 5-10 > 10 > 10 > 10 > 10 2-5 > 10 5-10 > 10

micro > 10 5-10 5-10 5-10

Follow-up'

1 17 16 35

> 65 19 56

> 26 > 49

26 21 78 35 12 2

11 33 21 35 32 50 6

11 28 19

Tumor antigens6

+ + + + + + +

-

~

-

- -

+ + - -

+ + + +

~

-

-

- -

'As determined by reverse-PCR analysis. RNA extraction and reverse transcription were performed as described (Russo et al., 1995), from tumor cell suspensions frozen at the moment of surgical intervention. PCR analysis was performed by amplification of MAGE, BAGE and GAGE genes with specific oligonucleotide primers as described (Weynants et al. 1994; Boel et al. 1995; Van den Eynde et al. 1995).-zPatients are named according to the institute of origin. MN, Milan Negri In~titute.-~Classified according to FIGO. BL, b~rderline.-~Tumor burden after surgery (cm).- 5Months survivaLh+, The expression of at least one of the tumor antigens analyzed.

458 V. RUSSO ETAL.

TABLE 2 - EXPRESSION OF MAGE, BAGE AND GAGE GENES IN 55 PRIMARY OVARIAN CARCINOMAS'

Patient2 Histoloev Tumor stage? HLA haD10tvDe4 MAGE-1 MAGE-3 BAGE GAGE

MN1 MN2 MN3 MN4 MN5 MR52 MR32 MR18 MR67 MR19 MN6 MR45 MN18 MN20 MN31 MN30 MN28 MN34 MN36 MN38 MN40 MN41 MR87 MN7 MN8 MN9 MNlO MN37 MN11 MN12 MN13 MN14 MR5 MNl5 MR60 MN16 MN17 MR40 MR117 MR47 MN19 MRlOl MR103 MN21 MN22 MN23 MN24 MN25 MN26 MN27 MN29 MN32 MN33 MN35

Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Serous Clear cell Clear cell Clear cell Mucinous Mucinous Mucinous Mucinous Mucinous Endometrioid

Endome trioid Endometrioid Endometrioid Endometrioid un un un un un un un un un un

111 111 111 111 I11 I11 111 111 111 I11 I11 I11 I11 I11 I11 I11 111 111 111 111 I11 I11 I11 IV IV IV IV IV IV IV 111 IV IV 111 111 111 I11 IV I1 I11 I11 I11 I11 IV

un un un

+I un nd nd nd nd

un un un un un un

+I un un un un un un un un

+--l nd un un nd nd

+I nd nd un un un un un

*]

1- un

'As determined by reverse-PCR analysis.3Patients are named according to the institute of origin. MN, Milan Negri Institute, MR, Milan S. Raffaele In~titute.-~Classified according to FIGO. un, ~nknown.-~The expression of HLA-A1 and -Cw * 1601 alleles was ascertained by PCR analysis of the patient tumor samples as described (Gaugler et al., 1994; van der Bruggen et aL, 1994). un, unknown; nd. not done.

ovarian carcinoma, 28% express MAGE-1 and 17% MAGE-3. In contrast to what was observed in melanoma, expression of MAGE-1 and -3 is not related. In only 4 out of the 18 tumors expressing the MAGE genes, there is co- expression of the 2 members, 9 lesions are positive for MAGE-1 alone and 5 for MAGE-3 alone. For BAGE and GAGE the percentages of expression are 15% and 31 %, respectively. Normal ovarian tissue does not express members of the gene families studied (data not shown).

Due to the low number of patients examined, no statistical analysis is possible. However, no correlation between the pres- ence of tumor antigen on the tumors and the clinical course of the disease, evaluated as mean survival, was observed (Table I).

All the clear cell carcinomas analyzed were negative, whereas a positive signal for MAGE-1 and MAGE-3 was detected in one of the 5 endometrioid carcinomas tested (Table II). There were no differences between serous and mucinous ovarian

TUMOR-ANTIGEN EXPRESSION IN OVARIAN CARCINOMAS 459

Tumor

derivation' Patient? sample MAGE-1 MAGE-3 BAGE GAGE

+ + + + + +

- MN1 Solid tumor - Ascite

MN6 Solid tumor - Ascite

MN12 Solid tumor - Ascite -

MN40 Solid tumor - Ascite -

MN20 Solidtumor + Omentum +

MN7 Ascite9/86 + + + + Ascite 9/88 + + + + Ascite 7/89 Ascite 9/89 - + +

- - - -

- - - - - - - - - - - - - - - - - - - - - ..............................................

+ + - - -

'As determined by reverse-PCR analysis.JPatients are named according to the institute of origin. MN, Milan Negri Institute.- 3Ty e of tumor at the moment of samples collection. From patient M A tumor samples were collected at different time during the disease progression. Heterogeneity of expression was detected only for genes MAGE-1 and -3, that were lost in the last tumor recurrences of patient MN7.

carcinomas in the percentage of positivity for the tumor anti- gens analyzed. A n increased number of tumors expressing MAGE-1 was observed in stage IVserous carcinomas (43%), when compared with those of stage III(22%). More informa- tion about MAGE-1 function and regulation is needed to understand the biological signiJicance of these observations.

Heterogeneity in MAGE genes expression during the tumor progression has already been described for metastatic breast {Russo et al., 1995) and bladder carcinomas (Patard et al., 1995), and was confirmed in this study for ovarian tumors. In 5 patients (MNI, 6, 12, 40, 20) multiple concomitant lesions were available for analysis (Table III); in addition, samples from 4 serial relapses that occurred during a period of 3 years from 1986 to 1989 were obtained from patient MN7. The analysis of the concomitant lesions showed a homogeneous pattern of tumor antigens expression (Table III). In contrast, in patient MN7 both the prima y tumor and the first recurrence expressed MAGE-1, -3, while a second and third recurrence showed loss of expression of both the MAGE genes. It is noteworthy that although BAGE and GAGE share with the

MAGE family common features, their expression remained unchanged during tumor progression (Table III). Although there was no positive or negative correlation between the expression of the tumor-spec@ antigens and the correspond- ing human leukocyte antigen (HLA)-restriction alleles (Table 11), the presence in patient MN7 of HLA-Al, the presenting molecule of MAGE-1, -3 products, is suggestive of an active intervention of the immune system in selecting antigen-lost variants.

It is known that conventional treatments, like surgery and chemotherapy, are not eficient to prevent the development of tumor recurrences in most ovarian tumor patients. Our data indicate that about 27% of Caucasian ovarian carcinoma patients have a tumor that expresses a well-defined tumor antigen and the appropriate HLA class Ipresenting molecules. The presence of tumor associated antigens, and the ability of the immune system to recognize these tumor antigens, suggests the feasibility of clinical protocols of spec@ immunotherapy as adjuvant to standard treatments. Such vaccines may also offer protection against recurrence of tumors expressing the same antigens.

Kncenzo Russo', Pier0 DALERLIA~, Axel RICCI~, Cristina BONAZZ15, B.E. LEONE2, Costantino MANCIONI', PaOt?a AL- LAVENA~, Claudio BORDICNON~ and Catia TRAVERSARP

'Gene Therapy Program, Dipartimento di Biologia e Biotec- nologia (DIBIT), 2Dipartimento di Patologia, Universita di Milan, Milan; and 311 Divisione Chirurgia Generale, Istituto Scient$co H.S. Raffaele; 41stituto Mario Negri; 5Dipartimento di Ostetricia e Ginecologia, Ospedale S. Gerardo, Milan, Italy.

Received: March 28,1996

ACKNOWLEDGEMENTS

We thank Drs. A. Mantovani and R. Giavazzi for helpful discussion and collaboration. This work was supported by grants from the Italian Association for Cancer Research (AIRC), Regione Lombardia, and the Italian National Re- search Council (CNR), Special Projects ' ACRO" and Inge- gneria Genetica.

6To whom correspondence should be addressed, at Gene Therapy Program, DIBIT, Istituto Scientific0 H.S. Raffaele. Via Olgettina 58, 20132 Milano, Italy. FAX: 02 26434827.

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