NEW YORKDNA sequencing is the gold standard for determining the
presence or absence of p53 mutations in breast tumors, said Jorge A.
Leon, PhD, corporate director for biotechnology research and
development, Corning Nichols Institute/Corning Clinical Laboratories.
Once considered complex and cumbersome, complementary DNA (cDNA)
sequencing is actually "very simple, accurate, and
reproducible," using new technology from Pharmacia Biotech
(Uppsala, Sweden), he said.
In fact, such testing can now be done in a matter of days at a cost
of approximately $200, Dr. Leon said at a roundtable on molecular
diagnostics in breast cancer sponsored by Pharmacia Biotech.
Furthermore, he said, sequencing now can be performed with as few as
2,500 tumor cells. "This is important," he said,
"because breast cancers are being diagnosed earlier and smaller,
and often only a small amount of biopsy tissue is available for study."
If validated in large clinical trials, cDNA sequencing could someday
be used routinely to identify patients with p53 mutations who have a
worse prognosis and require more aggressive treatment.
Dr. Leon and his colleagues at Georgetown University and the
University of Texas Health Science Center, San Antonio, are
conducting a retrospective study of p53 cDNA sequencing using
Pharmacia's automated sequence-based diagnosis (SBD) software and
Corning's large database of breast cancer samples.
The first phase of the study, comparing cDNA sequencing with
immunohistochemistry (IHC) staining, found inconsistencies in the IHC
technique, which detects p53 mutations indirectly by finding the
protein expressed by p53. If the protein is not found, the gene is
In the Corning study, p53 defects were found in 32% of breast cancer
patients using cDNA sequencing, but in only 20% with antibody
staining, and about 18% of defects found through staining proved to
be false-positives for genetic mutations. Overall, he said, about 30%
of the staining results conflicted with those of cDNA sequencing.
The second phase of the study, currently ongoing, is attempting to
validate p53 as a prognostic marker for use in clinical trials. The
researchers are looking for correlations between p53 mutations found
in the stored tissue samples and breast cancer outcomes. At present,
Corning is performing cDNA sequencing of the p53 gene only on a
research basis for patients in clinical trials. "We do, maybe,
25 to 50 a month," he said.
The company has encouraged researchers to incorporate p53 into
clinical trials, and trials are expected to begin next year "to
identify which mutations are important, as well as how those
mutations compare with other markers."
He also noted that companies are now actively developing gene therapy
agents to target p53. cDNA sequencing would be used to select
patients who could potentially respond to these therapies.