| Lung cancer
is the leading cause of cancer death in the United
States and Western Europe. Adenocarcinoma, the
histological subtype most frequently seen in never
smokers and former smokers, is now the most common
type of lung cancer in men and women in the United
States. The increasing incidence of lung adenocarcinoma
and its lethal nature underline the importance
of understanding the development and progression
of this disease, and the need for the development
of accurate tools for early diagnosis. Atypical
adenomatous hyperplasia (AAH) and bronchioalveolar
carcinoma (BAC), defined as non-invasive lesions,
are thought to be sequential precursors along
the path of progression to lung adenocarcinoma.
Elucidation of the molecular changes underlying
the development and progression of lung adenocarcinoma
is of great importance for devising targeted drugs
and methods of early detection.
DNA hypermethylation at promoter CpG islands
is now recognized as a key mechanism for tumor
suppressor gene inactivation in cancer. Identification
of abnormal methylation changes could provide
important insights into the mechanism of cancer
development, and in addition could yield powerful
biomarkers for cancer. At USC, we have developed
a sensitive, quantitatively accurate, automated
DNA methylation analysis technique based on real
time polymerase chain reaction (PCR) with methylation-specific
primers (MethyLight®). Using this high-throughput
assay we have recently identified fifteen loci
that show highly significant differences in methylation
between lung adenocarcinoma and adjacent non-tumor
tissue from the same patient (P<<0.0001
for 13/15 markers, Wilcoxon signed rank test).
The identified loci represent genes with functions
in cell cycle, differentiation, cell-cell contacts,
signaling, and apoptosis. To determine the role
of the methylation of these genes during the progression
from AAH to BAC and to invasive disease, we have
initiated a collaboration with expert lung pathologist
Dr. Keith Kerr, one of the world’s top authorities
in pre-invasive lesions preceding adenocarcinoma.
Dr. Kerr will provide a unique collection of normal
adjacent lung, pre-invasive lesions (AAH and BAC),
mixed adenocarcinoma samples with a BAC component,
and adenocarcinoma, representing adenocarcinoma
in its different developmental stages. This collection
will form the basis for the proposed study. We
will use MethyLight to determine the methylation
status of the previously identified markers (and
any new ones we uncover during our ongoing studies)
in these different samples. In a subset of representative
samples, we will analyze the methylation of the
loci in more detail through bisulfite genomic
sequencing. This allows the examination of methylation
along single DNA strands, and may provide information
on the mechanism by which methylation spreads
to new loci. The identification of sequential
epigenetic alterations during progression to lung
adenocarcinoma will broaden our molecular understanding
of the disease, providing insights that may be
applicable to the development of targeted drugs.
Equally important, such DNA methylation changes
will be powerful markers for early detection and
patient classification. We envision that the data
from this proposal will support a future R01 grant
application that will be aimed at a detailed elucidation
of the mechanistic role of DNA methylation in
adenocarcinoma development and progression.
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