A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma.

Of the antigens recognized on human tumors by autologous cytolytic T lymphocytes, all those defined thus far have been identified on melanoma or renal cell carcinoma. We report here the identification of an antigen recognized by autologous cytolytic T lymphocytes on a human squamous cell carcinoma of the oral cavity. The antigen is encoded by a mutated form of the CASP-8 gene. This gene, also named FLICE or MACH, codes for protease caspase-8, which is required for induction of apoptosis through the Fas receptor and tumor necrosis factor receptor-1. The mutation, which was found in the tumor cells but not in the normal cells of the patient, modifies the stop codon and adds an Alu repeat to the coding region, thereby lengthening the protein by 88 amino acids. The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8. The antigenic peptide is a nonamer presented by HLA-B*3503. The five last amino acids are encoded by the extension of the reading frame caused by the mutation. This, together with previous observations of CDK4 and beta-catenin mutations, suggests that a significant fraction of the point mutations generating a tumor antigen also play a role in the tumoral transformation or progression.

A mutated HLA-A2 molecule recognized by autologous cytotoxic T lymphocytes on a human renal cell carcinoma.

Many human tumor cells have been shown to express antigens that are recognized by autologous cytotoxic T lymphocytes (CTL) and the molecular nature of a number of melanoma antigens has been defined recently. Here we describe the characterization of an antigen recognized on a renal cell carcinoma by autologous CTL clones. This antigen is encoded by the HLA-A2 gene present in the tumor cells. The sequence of this gene differs from the HLA-A2 sequence found in autologous peripheral blood lymphocytes by a point mutation that results in an arginine to isoleucine exchange at residue 170, which is located on the alpha-helix of the alpha 2 domain. Transfection experiments with the normal and mutated HLA-A2 cDNA demonstrated that this amino acid replacement was responsible for the recognition of the HLA-A2 molecule expressed on the tumor cells. The mutant HLA-A2 gene was also detected in the original tumor tissue from the patient, excluding the possibility that the mutation had appeared in vitro. Thus, HLA class I molecules carrying a tumor-specific mutation can be involved in the recognition of tumor cells by autologous CTL.

A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene.

A cytolytic T lymphocyte (CTL) clone that lyses many HLA-A2 melanomas was derived from a population of tumor-infiltrating lymphocytes of an HLA-A2 melanoma patient. The gene coding for the antigen recognized by this CTL was identified by transfection of a cDNA library. It is the gene which has been reported to code for N-acetylglucosaminyltransferase V (GnT-V). Remarkably, the antigenic peptide recognized by the CTL is encoded by a sequence located in an intron. In contrast to the fully spliced GnT-V mRNA, which was found in a wide range of normal and tumoral tissues, the mRNA containing the intron region coding for the antigen was not found at a significant level in normal tissues. This mRNA was observed to be present in about 50% of melanomas. Our results suggest that a promoter located near the end of the relevant intron is activated in melanoma cells, resulting in the production of an mRNA coding for the antigen.

A human minor histocompatibility antigen specific for B cell acute lymphoblastic leukemia.

Human minor histocompatibility antigens (mHags) play an important role in the induction of cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene encoding the antigenic peptide recognized by HB-1-specific CTLs. Interestingly, expression of the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus-transformed B cells. The HB-1 gene-encoded peptide EEKRGSLHVW is recognized by the CTL in association with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid substitution is critical for recognition by HB-1-specific CTLs. The restricted expression of the polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1-specific CTLs in vitro using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD.

Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.

An alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes.

We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

[The bioethics law revision: comparative analysis of contributions from different public and professional offices. Assisted Reproductive Technology, embryo and stem cells research, umbilical cord blood bank]. / La révision de la loi de bioéthique : analyse comparative des contributions de différents organismes publics ou professionnels. Assistance médicale à la procréation, recherche sur l'embryon et les cellules souches, banque de sang du cordon ombilical.

The revision of the bioethics law of 2004 must occur in a five year's time. For this revision, the authorities decided to organize general states of bioethics and requested the production of contributions by the companies, institutions or associations. These texts tackle various subjects, like the Assisted Reproductive Technologies, research on the embryo and the stem cells and the banks of umbilical cord blood. Certain opinions converge, others differ, but all take part in the great debate which will take place at the time of the general conference.

[Premature ovarian failure: which protocols?]. / L'insuffisance ovarienne débutante: quels protocoles?

This review shows the results of the various studies concerning the protocols applied to the women presenting a premature ovarian failure. Will be thus analyzed the natural cycles (or semi-natural), the increase in the dose of gonadotrophins, the clomiphene citrate and the anti-aromatases, the protocols with GnRH agonists long, short, stop or microdoses, the protocols with GnRH antagonists and the adjuvant treatments: aspirin, nitric oxyde, recombinant LH recombining, growth hormone and androgens. The interest of several protocols is to collect a sufficient number of oocytes (and thus of embryos to be transferred), making it possible to obtain reasonable rates of pregnancy. However, it arises that the rates of pregnancy observed among these women depend not only on their ovarian reserve and their age, but are also function of the type of infertility, of the cycle number and the uterus.

[Management of an ovarian stimulation in case of a Kallmann-De Morsier syndrome. The role of LH]. / Prise en charge en induction de l'ovulation d'un cas de syndrome de Kallmann-De Morsier. Réflexions sur le rôle de la LH.

We report a case of ovarian stimulation in a woman with a Kallmann-De Morsier syndrome, which resulted in a triple pregnancy and childbirth by caesarean section at 36 weeks of amenorrhea of three girls weighing from 1,950 to 2,300 g. Starting from a literature review of Kallmann-De Morsier syndrome, we discuss the role of LH during the follicular phase and the monitoring of ovarian stimulation.

A new MAGE gene with ubiquitous expression does not code for known MAGE antigens recognized by T cells.

A number of genes of the MAGE family have been shown to code for antigens that are recognized on many human tumors by autologous CTLs. These antigens should be strictly tumor specific because the encoding MAGE genes are not expressed in normal adult cells, except for male germ-line cells, which lack HLA expression. Here, we report that a distant relative of the previously identified MAGE genes is expressed in many, if not all, normal tissues. This gene, which was named MAGE-D, is located in Xp11. Its exon-intron structure is completely different from that of the other MAGE genes. None of the 20 MAGE antigenic peptides presently known to be recognized by T lymphocytes is encoded by the new MAGE gene. It appears, therefore, that this new finding leaves intact the tumor specificity of the antigens encoded by the MAGE genes that are expressed only in tumor and germ-line cells.

Characterization of the GAGE genes that are expressed in various human cancers and in normal testis.

The GAGE-1 gene was identified previously as a gene that codes for an antigenic peptide, YRPRPRRY, which was presented on a human melanoma by HLA-Cw6 molecules and recognized by a clone of CTLs derived from the patient bearing the tumor. By screening a cDNA library from this melanoma, we identified five additional, closely related genes named GAGE-2-6. We report here that further screening of this library led to the identification of two more genes, GAGE-7B and -8. GAGE-1, -2, and -8 code for peptide YRPRPRRY. Using another antitumor CTL clone isolated from the same melanoma patient, we identified antigenic peptide, YYWPRPRRY, which is encoded by GAGE-3, -4, -5, -6, and -7B and which is presented by HLA-A29 molecules. Genomic cloning of GAGE-7B showed that it is composed of five exons. Sequence alignment showed that an additional exon, which is present only in the mRNA of GAGE-1, has been disrupted in gene GAGE-7B by the insertion of a long interspersed repeated element retroposon. These GAGE genes are located in the p11.2-p11.4 region of chromosome X. They are not expressed in normal tissues, except in testis, but a large proportion of tumors of various histological origins express at least one of these genes. Treatment of normal and tumor cultured cells with a demethylating agent, azadeoxycytidine, resulted in the transcriptional activation of GAGE genes, suggesting that their expression in tumors results from a demethylation process.

MAGE-A genes are not expressed in human leukemias.

Immunotherapy is promising to improve the prognosis of human leukemias, at least as adjuvant treatment. Tumor-associated antigens such as antigens encoded by MAGE-A1, -A2, -A3, -A4, -A6 and -A12 genes might provide tools in this field. We demonstrated recently that the presentation peptides encoded by MAGE-A genes might make leukemic blasts suitable targets to cytolytic T lymphocytes. We reported previously negative data of MAGE-A1 gene expression in hematological malignancies, but in further studies positive results of MAGE-A gene expression were published in some subtypes of hematological malignancies such as T leukemia, myeloma and Hodgkin's disease. This led us to enlarge the screening of MAGE-A gene expression in human leukemias. In the RT-PCR screening of a large panel including 154 patients, only weak signal were detected in a few samples. We conclude that MAGE-A genes are not expressed in human leukemias.

Expression of MAGE and GAGE in high-grade brain tumors: a potential target for specific immunotherapy and diagnostic markers.

The mRNA expression of the tumor-associated antigens MAGE and GAGE was examined in 60 high-grade brain tumors. This analysis was performed by using reverse transcription-PCR, Southern blotting, and sequencing. It was demonstrated that, of the eight GAGE genes, GAGE-2 and -7 were expressed in five of seven normal brains. Four groups of tumors--adult glioblastoma multiforme (n = 20), pediatric glioblastoma multiforme (n = 9), medulloblastomas (n = 15), and ependymomas (n = 14)--were analyzed for mRNA expression. The following frequencies were observed: MAGE-1, 0, 0, 13, and 0%, respectively; MAGE-2, 5, 11, 60, and 57%; MAGE-3 & -6, 0, 0, 13, and 0%; GAGE-1, 65, 11, 13, and 43%; and GAGE-3-6 and -8: 75, 78, 47, and 93%, respectively. Two unclassified tumors expressed GAGE-3-6 and -8 only. The absence of GAGE-1 expression in normal brain, its relatively high frequency of expression in high-grade brain tumors, and its unique 3' sequence, suggest it may represent a useful target for specific immunotherapy. The detection method of reverse transcription-PCR and Southern blotting may also be useful for rapid screening of biopsy specimens both for diagnostic purposes and to determine a patient's eligibility for specific immunotherapy.

Expression of prostate-specific membrane antigen in transitional cell carcinoma of the bladder: prognostic value?

The expression of Prostate-specific membrane antigen (PSMA) mRNA was assessed in the normal bladder urothelium (n = 9), transitional cell carcinoma (TCC) specimens (n = 52), TCC-derived cell lines (n = 3), and preoperative blood samples from TCC patients (n = 27). Specific PSMA mRNA was found in 100% of normal and malignant tissues and two cell lines. PSMA protein was detected in normal (n = 3) and malignant tissues (n = 4). Using a PSMA-specific substrate, PSMA enzymatic activity was found in two bladder cell lines and correlated with immunostaining. Seven of the 27 TCC preoperative blood samples were positive by reverse transcription-PCR. These preliminary results, obtained on a nonrandomized cohort of patients, correlated with tumor invasion (positive RT-PCR: 0% for pT < or = 2 versus 41% for pT > or = 3) and 2-year survival rate (81% in the PSMA-negative group versus 29% in the PSMA-positive group). Although the clinical usefulness of this assay requires confirmation in larger prospective randomized trials, current preliminary results suggest that a blood-borne PSMA mRNA PCR assay may be a useful tool to predict a poor outcome in TCC patients.

Use of GNRH antagonists in reproductive medicine.

Gonadotrophin-releasing hormone (GnRH) plays a key role in the secretion of gonadotrophins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which regulate steroidogenesis and folliculogenesis. Two GnRH antagonists, Cetrorelix and Ganirelix, deprived of histaminergic side-effects, have been introduced into ovarian stimulation protocols to prevent premature LH surges and proved their safety in clinical trials. At present, most of the published studies have not found significant differences in follicular recruitment, oocyte quality, and so on, except for a decrease in pregnancy and implantation rates in in vitro fertilization and embryo transfer (IVF-ET) cycles when the GnRH antagonist rather than the agonist was used. This decrease in pregnancy rates was in relation with a necessary learning curve of the physicians. Another possibility is the impact of the GnRH antagonist on endometrium through its GnRH receptor; this effect was cancelled after cryopreserved embryo transfers because the pregnancy rates were similar between GnRH antagonist and agonist in this case. GnRH antagonists were also interesting in poor responders and polycystic ovarian syndrome, where the agonists have not permitted to obtain the better results in IVF-ET cycles. Similarly, the GnRH antagonists could prevent the LH surge in the intrauterine insemination cycles.

Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: a quality-assurance initiative of the EORTC Melanoma Cooperative Group.

Reverse transcription-polymerase chain reaction (RT-PCR)-based assays detecting occult neoplastic cells are increasingly being used for the study of tumour dissemination and minimal residual disease. However, different methods are employed by various research groups and the results are heterogenous. We prospectively assessed the results from nine laboratories performing tyrosinase RT-PCR assays for the detection of melanoma cells on a series of blind samples. After complete analysis, the results were compared for sensitivity and specificity. All laboratories reported correct results for cDNA standards. Five laboratories attained acceptable specificity and a sensitivity detecting 10 cells in 10 ml of whole blood. Four laboratories had unacceptable specificity and/or sensitivity. This blind study highlights the difficulty of RT-PCR data interpretation and the need for quality assurance between laboratories. Measures to increase the reliability of RT-PCR assays are proposed, which have to be prospectively evaluated in future studies.

Correlation between size and external temperature in four rat tumours after treatment with cytostatic agents.

The reduction in size of four experimental tumours (ISIS 130 and ISIS 208 immunocytomas, S 437 mammary adenocarcinoma, S 447 colon adenocarcinoma) was investigated in LOU rats under the influence of cytostatic agents belonging to different classes (5-fluorouracil, methotrexate, vinblastine, cisplatin, doxorubicin, cyclophosphamide). External tumour and rectal temperatures were measured at the same time, twice daily, during the whole experiment. With the rectal temperature of the rats kept constant, the reduction in tumour dimensions following chemotherapy correlated via a linear relationship with the duration and degree of tumour hypothermia for the three tumours S 437, ISIS 208, ISIS 130. However, for the same reduction in tumour volume following chemotherapy, the duration and degree of transient tumour hypothermia varied according to the type of tumour and cytostatic agent studied. There was not correlation between the decrease in size of S 447 and external tumour hypothermia. Even when the reduction in tumour size was statistically significant, the hypothermic tumour phase after drug administration was not sufficient to be significant, except for vinblastine. However, the temperature of this slowly growing tumour before chemotherapy was particularly low. The measurement of the degree and duration of external tumour hypothermia of tumours following chemotherapy would represent a new physiological technique for measuring the efficacy and duration of action of cytostatic agents.

Tissue distribution of doxorubicin associated with polyisohexylcyanoacrylate nanoparticles.

The body distribution of i.v. doxorubicin depends mainly on the physicochemical characteristics of the molecule. However, entrapment of that cytostatic drug inside polyalkylcyanoacrylate nanoparticles has been shown to modify its distribution profoundly in the mouse. Polysiohexylcyanoacrylate nanoparticles loaded with [14C]-doxorubicin were studied in comparison with free drug, with emphasis on their distribution pattern in mouse tissue after i.v. administration. An autoradiographic study showed that most of the radioactivity was found in the reticuloendothelial system as soon as a few minutes after i.v. administration of the doxorubicin-loaded nanoparticles. Quantitative determinations by liquid scintillation counting in fresh tissue (spleen, heart, kidneys, liver, lungs, bone marrow) and blood samples confirmed these observations. When the drug was linked to nanoparticles, doxorubicin blood clearance was reduced during the first few minutes after administration, whereas heart and kidney concentrations were substantially decreased. Assays of doxorubicin and doxorubicinol by a specific HPLC analytical method gave results very similar to those obtained by scintillation counting.

MAGE-1 expression threshold for the lysis of melanoma cell lines by a specific cytotoxic T lymphocyte.

Human gene MAGE-1 codes for an antigen that is recognized on a melanoma by an autologous cytotoxic T lymphocyte (CTL). Because MAGE-1 is expressed on a significant proportion of tumours of various histological types and not on normal tissues, the encoded antigen may serve as a target for cancer immunotherapy. Evaluation of the expression of the gene by reverse transcription polymerase chain reaction (RT-PCR) in various tumour samples and tumour cell lines has suggested great variability in the level of expression. It was therefore important to evaluate the minimal level of expression required for lysis by CTL. We tested a number of melanoma cell lines by a quantitative RT-PCR assay to correlate their level of MAGE-1 expression and recognition by the relevant CTL clone. We found that only cell lines expressing more than 10% of the MAGE-1 messenger RNA (mRNA) level of reference cell line MZ2-MEL.3.0 (i.e. more than three mRNA molecules per cell) were lysed by the CTL or induced significant tumour necrosis factor release.

Expression of MAGE genes in esophageal squamous-cell carcinoma.

The genes MAGE-1, -2, -3 and -4 are expressed in tumors of different histological types, but not in normal tissues, with the exception of testis and placenta. Short peptides derived from MAGE-1 and MAGE-3 gene products are recognized by cytolytic T lymphocytes when presented by HLA-class-I molecules, and represent potential targets for specific immunotherapy. We have determined whether esophageal carcinoma patients should be eligible for MAGE-peptide-based vaccine therapies. The expression of genes MAGE-1, -2, -3 and -4 in tumor samples was assessed by reverse-transcription and polymerase-chain-reaction amplification. Out of the 49 esophageal squa-mous-cell carcinomas studied, 53% expressed MAGE-1, 49% MAGE-2, 47% MAGE-3 and 71% MAGE-4. Eighty-four percent of the tumors expressed one or more of the four MAGE genes. Owing to the high incidence of MAGE gene expression in esophageal squamous-cell carcinoma, a large proportion of patients could be suitable candidates for immune therapies involving tumor-specific antigens encoded by MAGE genes.