Dirk Usener

Tumor-specific antigens in cutaneous T-cell lymphoma: Expression and sero-reactivity

Cutaneous T‐cell lymphoma (CTCL) is a heterogeneous group of extra‐nodal non‐Hodgkin lymphomas with primary manifestation in the skin with poor treatment options in the advanced stages. As basis for future immune‐therapeutic strategies we have investigated the possible expression of tumor‐specific targets in CTCL focusing mainly on so‐called cancer‐germline genes. cDNAs derived from 20 CTCL tissues and 4 CTCL cell lines were tested with 15 gene‐specific and 4 gene family‐specific primers by RT‐PCR and confirmative Northern blotting. The most frequently detected mRNAs were LAGE‐1 (55% with only partial coexpression of the splicing variants), cTAGE‐1 (35%), MAGE‐A9 (27%) and the GAGE‐3–7 group (35%). Furthermore, we could detect NY‐ESO‐1 (21%) and a MAGE‐A subgroup (15%), whereas sub‐specification of the latter proved absence of MAGE‐A1, ‐A2, ‐A3, ‐A6 and ‐A12. SCP‐1 was found in only one specimen and a several antigens could not been detected in any tumor tissue or cell line (MAGE‐B, GAGE‐1,2,8 and all 4 RAGE genes). 90% of all CTCL samples were positive for at least 1 of the frequent mRNAs in RT‐PCR (LAGE‐1, NY‐ESO‐1, cTAGE‐1, MAGE‐A9, or GAGE‐3to7). Using a secondary SEREX approach we could detect sero‐reactivity in sera of CTCL patients against recombinant cTAGE‐1 (10/29), GAGE (3/19), MAGE‐A1 (1/18), ‐A3 (1/18), ‐A6 (2/18) and ‐A9 (4/18) protein, but not against LAGE‐1a, MAGE‐A4b or MAGE‐A12 protein (n = 19). We conclude, that certain cancer‐germline genes can be detected frequently in CTCL and are able to elicit a systemic immune response. These candidate genes might therefore be promising targets for immunotherapeutic interventions in CTCL.

mRNA expression of tumor‐associated antigens in melanoma tissues and cell lines

Abstract: Tumor‐associated antigens (TAA) are increasingly used as specific targets for immune therapy of malignant melanoma. The aim of the present study was to provide a basis for selecting the most suitable TAA by analyzing the mRNA expression of a large panel of TAA by RT‐PCR and Northern blotting. We have chosen primers differentiating four groups of TAA (MAGE‐A, MAGE‐B, and two groups of GAGE‐genes) and 13 individual TAA (MAGE‐A2 and ‐A3, RAGE‐1, ‐2, ‐3, and ‐4, LAGE‐1a and ‐1b, NY‐ESO‐1, GAGE‐1, SSX‐2, SCP‐1, and cTAGE‐1) based on most recent sequence data. In addition, the RAGE‐gene family has been separated into its four members by a novel designed nested PCR, which was confirmed by Northern analysis. Furthermore, the chromosomal organization and relationship between the RAGE‐family and MOK was analyzed. RAGE‐4 mRNA could be shown for the first time to be present in testis tissue. The most frequently expressed TAA were the MAGE‐A and the GAGE‐3,‐4,‐5,‐6,‐8 group, whereas among individual TAA MAGE‐A2, ‐A3, RAGE‐1, ‐3, and LAGE‐1a/b were found within most specimens and are thus promising candidates for immune therapy. In comparison, melanoma metastatic specimens and cell lines show similar profiles of TAA expression, but individual TAA differ notably between both types of samples indicating that results from cell lines are not always applicable to tumor specimen.

Tumor-associated Antigens as Possible Targets for Immune Therapy in Head and Neck Cancer: Comparative mRNA Expression Analysis of RAGE and GAGE Genes

Specific immune therapy targeting residual areas of cancer cells may emerge as a powerful treatment strategy for head and neck squamous cell carcinoma (HNSCC). In order to define possible targets for immune therapy, we evaluated the frequency of two groups of tumor antigens - the RAGE and GAGE families - by means of reverse transcriptase polymerase chain reaction using primary HNSCCs ( n =28), mucosa specimens as normal controls ( n =10) and HNSCC cell lines ( n =6). By means of specific primer selection we could differentiate between RAGE-1, -2, -3 and-4, as well as between two groups of GAGE genes (GAGE-1,2,7 vs GAGE-3,4,5,6,8). While all mucosa tissues (from smokers and non-smokers) were negative for both antigen families, 24/28 investigated tumors were positive for up to 5 tumor antigens. Among the RAGE genes, RAGE-1-positive tumors were the most abundant (8/28), followed by RAGE-2 (7/28) and RAGE-4 (6/28). Differences in the locations of HNSCCs were reflected by different RAGE family members being expressed most frequently: larynx, RAGE-1; oropharynx, RAGE-2; and hypopharynx, RAGE-4. Primers against GAGE-1,2,7 and GAGE-3,4,5,6,8 revealed 6/27 and 16/27 positive tumors, respectively. This report suggests that RAGE genes and GAGE-3,4,5,6,8 may be promising candidates for specific immune therapy in HNSCC.

Seroreactivity against MAGE-A and LAGE-1 proteins in melanoma patients

Summary  Background Cancer‐testis antigens exemplify a growing number of tumour antigens which are expressed in a variety of malignancies, but not in normal tissues other than germ cells, primarily those of the testis.