A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma.

We have identified an antigen recognized on a human melanoma by autologous cytolytic T lymphocytes. It is encoded by a gene that is expressed in many normal tissues. Remarkably, the sequence coding for the antigenic peptide is located across an exon-intron junction. A point mutation is present in the intron that generates an amino acid change that is essential for the recognition of the peptide by the anti-tumor cytotoxic T lymphocytes. This observation suggests that the T-cell-mediated surveillance of the integrity of the genome may extend to some intronic regions

A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma.

We have identified an antigen recognized on a human melanoma by autologous cytolytic T lymphocytes. It is encoded by a gene that is expressed in many normal tissues. Remarkably, the sequence coding for the antigenic peptide is located across an exon-intron junction. A point mutation is present in the intron that generates an amino acid change that is essential for the recognition of the peptide by the anti-tumor cytotoxic T lymphocytes. This observation suggests that the T-cell-mediated surveillance of the integrity of the genome may extend to some intronic regions

Quantitative evaluation of the expression of MAGE genes in tumors by limiting dilution of cDNA libraries.

The MAGE-A genes are expressed in tumor cells but not in healthy tissues, except in male germ line cells and in placenta. They encode tumor-specific antigens recognized by autologous cytolytic T lymphocytes (CTLs). On the basis of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, 6 of the 12 members of the MAGE-A family, including MAGE-A1, were previously reported to have a high level of expression in tumors, whereas 5 other members, including MAGE-A10, were expressed at a much lower level, deemed to be insufficient for CTL recognition. However, analysis with antibodies has shown that some melanoma cell lines contain equivalent amounts of MAGE-A1 and MAGE-A10 proteins. This discrepancy appeared to be due to the low efficacy of the primers that had been used for the previous MAGE-A10 RT-PCR assays. This led us to develop a method that is independent of the efficacy of the PCR primers to evaluate MAGE-A gene expression. cDNA libraries from tumor cell lines were introduced into bacteria, of which 200 pools of about 500 bacteria were maintained in microcultures. The frequencies of the MAGE-A cDNA clones in each library were evaluated by performing PCR assays on each of these pools. The abundance of MAGE-A10 cDNAs was found to be similar to that of MAGE-A1 in 3 of the libraries that were analyzed, including 2 with high expression (1/6,400), confirming that MAGE-A10 is expressed at a high level. MAGE-A2, A3, A4, A6 and A12 cDNAs were also confirmed often to be present at a frequency of more than 1/10,000, a level of expression that should suffice for recognition of antigenic peptides encoded by these genes by cytolytic T cells. The remaining MAGE genes are either not expressed in tumors or are expressed at a very low level, with the exception of MAGE-A8 and 11, which show high expression in a very small number of tumors. This method also allowed us to isolate 5 MAGE-A cDNAs that we had not obtained previously, enabling us to delineate the exons in the sequences of genes MAGE-A5, A8, A9, A10 and A11

The majority of autologous cytolytic T-lymphocyte clones derived from peripheral blood lymphocytes of a melanoma patient recognize an antigenic peptide derived from gene Pmel17/gp100.

Anti-melanoma cytolytic T-lymphocyte (CTL) clones were derived from peripheral blood lymphocytes of HLA-A2 melanoma patient LB265 after stimulation with the autologous tumor cell line LB265-MEL, which showed high expression of melanocyte-lineage specific genes. Of 55 CTL clones, 46 recognized HLA-A2-restricted antigens. These 46 CTL clones were studied for their ability to specifically release tumor necrosis factor in the presence of COS cells cotransfected with the HLA-A2 gene and the cDNA of either tyrosinase, Melan-A/MART1, Pmel17/gpl00, gp75/TRP1, or MSH receptor. Six CTL clones recognized the Melan-A/MART1 antigen, whereas the remaining 40 CTL clones recognized a Pmel17/gp100 antigen. These 40 anti-PmelI7/gpl00 CTL clones were all able to lyse T2 cells pulsed with the antigenic peptide YLEPGPVTA, as previously reported. The T-cell receptor beta chain hypervariable region was sequenced and found to be identical in the 15 CTL clones analyzed. Taken together, these data show a high frequency of Pmell7/gp100-specific T cells in autologous antitumor CTL clones derived from peripheral blood of a melanoma patient

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

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

Frequent expression of HAGE in presentation chronic myeloid leukaemias

Cancer testis (CT) antigens provide attractive targets for cancer-specific immunotherapy. Although CT genes are expressed in some normal tissues, such as the testis and in some cases placenta, these immunologically protected sites lack MHC I expression and as such, do not present 'self' antigens to T cells. To date, CT genes have been shown to be expressed in a range of solid tumours, but rarely in haematological malignancies. We have extended previous studies to investigate the expression of a comprehensive range of CT genes (MAGE-A1, -A3, -A6, -A12, BAGE, GAGE, HAGE,LAGE-1, NY-ESO-1 and RAGE) for their expression in a cohort of acute and chronic myeloid leukaemia patient samples. CT expression was not detected in 20 normal bone marrow or peripheral blood stem cell samples. In acute myeloid leukaemia (AML) nine of the 26 (35%) samples analysed expressed one or more of the CT genes with six of the samples (23%) expressing HAGE. In chronic myeloid leukaemia (CML) 24 of 42 (57%) presentation chronic myeloid leukaemia (CML) patient samples expressed one or more CT antigen with 23 expressing HAGE. We have shown that HAGE is frequently expressed in CML, and to a lesser extent in AML patient samples. This is the first demonstration of HAGE gene expression in myeloid leukaemia patients and the frequent expression of HAGE at disease presentation opens up the possibility of early immunotherapeutic treatments