Rapid and sensitive p53 alteration analysis in biopsies from lung cancer patients using a functional assay and a universal oligonucleotide array: a prospective study.
OBJECTIVE
Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA.


METHODS
p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay.


RESULTS
Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells.


CONCLUSIONS
The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.
Fouquet
Coralie
C
EA3493, UPMC-IC of [corrected] Laboratoire de génotoxicologie des tumeurs, Paris, France.
Antoine
Martine
M
Tisserand
Pascaline
P
Favis
Reyna
R
Wislez
Marie
M
Commo
Fréderic
F
Rabbe
Nathalie
N
Carette
Marie France
MF
Milleron
Bernard
B
Barany
Francis
F
Cadranel
Jacques
J
Zalcman
Gérard
G
Soussi
Thierry
T
D000368:Aged; D000483:Alleles; D001706:Biopsy; D015331:Cohort Studies; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D016158:Genes, p53; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009696:Nucleic Acids / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D009696:Nucleic Acids; D009841:Oligonucleotides; D016159:Tumor Suppressor Protein p53; D012313:RNA; D004247:DNA