Mutations within the kinase domain and truncations of the epidermal growth factor receptor are rare events in bladder cancer: implications for therapy.
OBJECTIVE
It has previously been reported that the patient response to gefitinib depends on the presence of mutations within the kinase domain of epidermal growth factor receptor (EGFR) or the expression of its truncated form, EGFR variant III (EGFRvIII). The focus of this study was to determine if these alterations are present within the tyrosine kinase and ligand-binding domain of EGFR in urothelial carcinoma.


METHODS
The kinase domain found within exons 18 to 21 of the EGFR from 11 bladder cancer cell lines and 75 patient tumors were subjected to automated sequencing. EGFRvIII expression was determined by immunohistochemistry using a urothelial carcinoma tissue microarray, and its expression was subsequently verified by reverse transcription PCR, real-time PCR, and Western blot analysis, using an EGFRvIII-transfected glioblastoma cell line and glioblastoma tumors as positive controls.


RESULTS
Our analysis failed to detect mutations within the tyrosine kinase domain of EGFR in the 11 cell lines and 75 patients tested. The initial analysis of EGFRvIII expression by immunohistochemistry revealed that at least 50% of the patient tumors expressed EGFRvIII in a urothelial carcinoma tissue microarray. Conflicting reports exist, however, regarding the extent of EGFRvIII expression in tissues owing to the specificity of the antibodies and the methodologies used. Therefore, we sought to validate this observation by reverse transcription PCR, real-time PCR, and Western blot analysis. In these assays, none of the samples were positive for EGFRvIII except for control transfectants and glioblastomas.


CONCLUSIONS
When our results are taken together, we conclude that alterations within the tyrosine kinase domain and expression of EGFRvIII are rare events in bladder cancer. The present study has clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.
Blehm
Kelly N
KN
Department of Cancer Biology, Brain Tumor Center, The University of Texas M.D. Anderson Cancer Center, Houston, 77030, USA.
Spiess
Philippe E
PE
Bondaruk
Jolanta E
JE
Dujka
Melanie E
ME
Villares
Gabriel J
GJ
Zhao
Yi-Jue
YJ
Bogler
Oliver
O
Aldape
Kenneth D
KD
Grossman
H Barton
HB
Adam
Liana
L
McConkey
David J
DJ
Czerniak
Bogdan A
BA
Dinney
Colin P
CP
Bar-Eli
Menashe
M
D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015331:Cohort Studies; D004252:DNA Mutational Analysis / Q000379:methods; D004354:Drug Screening Assays, Antitumor; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D017354:Point Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D046888:Tissue Array Analysis / Q000379:methods; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology
D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases