Immunohistochemical and molecular analysis of human melanomas for expression of the human cancer-testis antigens NY-ESO-1 and LAGE-1.
OBJECTIVE
NY-ESO-1 and LAGE-1 are homologous cancer-testis antigens, which are expressed in many different cancers. It is essential to type tumors accurately to assess patient suitability for clinical trials which target these. This study evaluates typing strategies used to distinguish these two homologous but distinct antigens and to characterize and quantitate expression of each in clinical samples.


METHODS
We typed 120 malignant melanomas for the expression of NY-ESO-1 and LAGE-1 with immunohistochemistry, reverse transcription-PCR (RT-PCR), and quantitative real-time (qRT-PCR), which was also used to explore the relationship between NY-ESO-1 and LAGE expression.


RESULTS
The two monoclonal antibodies ES121 and E978 had very similar immunohistochemistry reactivities. Both were specific for NY-ESO-1 because neither bound to homologous LAGE-1 peptides despite 84% overall amino acid homology. Of 120 melanomas tested by immunohistochemistry, NY-ESO-1 was expressed in >50% of cells in 23 melanomas (19%), between 11 and 50% cells in 15 (12.5%), <11% cells in 16 (13.5%), and negative in 66 (55%). Although specific for both antigens, the PCR methods did not provide this information about microheterogeneity. Polymorphisms in the LAGE-1 gene resulted in false negative LAGE-1 typing by qRT-PCR by inhibiting binding of oligonucleotide primers, thereby showing the exquisite specificity of qRT-PCR as a typing method.


CONCLUSIONS
For NY-ESO-1 typing, immunohistochemistry compared favorably with the RT-PCR, with the added advantage of being able to characterize heterogeneity of antigen expression. Because neither mAb bound LAGE and because there was no coordinate expression LAGE and NY-ESO-1, separate typing for each is required.
Vaughan
Hilary A
HA
Ludwig Institute for Cancer Research, Melbourne, Australia.
Svobodova
Suzanne
S
Macgregor
Duncan
D
Sturrock
Sue
S
Jungbluth
Achim A
AA
Browning
Judy
J
Davis
Ian D
ID
Parente
Philip
P
Chen
Yao-Tseng
YT
Stockert
Elisabeth
E
St Clair
Fiona
F
Old
Lloyd J
LJ
Cebon
Jonathan
J
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D000954:Antigens, Surface; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000556:secondary; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011110:Polymorphism, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000556:secondary; D013737:Testis / Q000276:immunology / Q000378:metabolism; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; C104948:CTAG1B protein, human; C510162:CTAG2 protein, human; D008565:Membrane Proteins