LUNG TISSUE–BASED MOLECULAR ASSAYS

Of course, the gold standard for lung cancer detection is examination
of lung tissue itself, either by conventional means
(microscopy, immunohistochemistry, and the like), or by newer
molecular means. Among the newer means, fluorescence in situ
hybridization (FISH) for larger-region genomic deletions or
duplications (see Chapter 49), microsatellite examinations (see
Chapter 7), DNA methylation patterns (see Chapter 7), microarray
messenger RNA (mRNA) expression signatures, candidate
mRNA expression signatures, microRNA microarrays, candidate
microRNA expression signatures, proteomic and antibody arrays,
and metabolomic approaches are all in use in experimental
and/or beta-testing for early clinical application. The National
Cancer Institute (NCI) empowered, in the last decade, the Early
Detection Research Network (EDRN) to create a consortium
of tissue repositories and technology platforms for this purpose,
and the technologies presented at the most recent 2008 meeting
are truly astonishing. As this chapter emphasizes evolving
early detection modalities, largely by molecular assays on noninvasively
obtained tissues from individuals without symptoms,
those molecular techniques requiring robust, large surgical level
tissue specimens are covered elsewhere in this volume.
For noninvasively obtained tissues used in early detection
studies on asymptomatic populations, such as exfoliated cells
in sputum, or the circulating macromolecules of blood, polymerase
chain reaction (PCR)-based assays of DNA and RNA are
most often employed in the translational research setting. Their
sensitivity is extraordinary, allowing analysis of these lung-surrogate
tissues with greater ease. It must be acknowledged that
although conferring great sensitivity, these nucleic acid end
points may, even for cancer syndromes, often be considered
more biologically proximate, or twice-removed end points, as
opposed to direct measurement of the full-dimensional cancer
biological phenotype (uncontrolled growth, invasion, metastasis),
which is generally not feasible. Even other proximate
markers (metabolites or proteins/enzymes generating those
metabolites) that are the direct underpinnings of these cancer
phenotypes are often beyond the level of sensitivity of current
assays, although proteomic, and antibody microarray technologies
are advancing rapidly as are informatic approaches to
these multidimensional data sets.
DNA-based genome-wide search for aberrant copy number
in tumors have revealed copy number aberrations using single
nucleotide polymorphism (SNP) arrays or array comparative
genomic hybridization (CGH) and reveal that many of the
copy number alterations found to cluster in lung cancer involve
unknown genes. 16
DNA-based methylation markers in lung tissue, by example :
cytosine-phosphodiester-guanine (CpG) methylation of gene promoter
DNA is a major correlate of biologic pathway regulation
and deregulation. 17–19 In many ways, progressive DNA methylation
of tumor suppressor genes has been revealed over the last 10
to 15 years to be a strong correlate of lung carcinogenesis.
As a recent example of a comprehensive methylation
biomarker search, a concerted genome-wide effort to assay in
vitro genes likely to be methylated, and responsive to a methylation
inhibitor, was checked for consistency with expression
and methylation profiles in human lung. Those genes whose
expression was most discriminatory for lung tumor from
adjacent nontumor tissue (for lung, specifically) by methylation-
specific PCR (MSP) sampling of one or a few (CpG)
methylation sites in each gene locus, included BNC1 , MSX1 ,
CCNA1 , RASSF1A , p16 , ALDH1A13 , LOX , and CTSZ . This
is one of the first such comprehensive genome-wide searches
for methylation-silenced genes in lung cancer 20 and has been
followed up by others. 21
In another study, a panel of candidate gene DNA
methylation markers, using methylation-specific PCR sampling
was constructed, and using the top four markers
( CDKN2A EX2 , CDX2 , HOXA1 , OPCML ) could distinguish
lung tumors from normal tissue with 94% sensitivity and 90%
specificity. 22
A workshop was convened in 2005 on the use of methylated
DNA sequences as cancer biomarkers, in risk assessment
and disease detection, enjoining the NCI’s EDRN and
the National Institute of Standards and Technology. The
summary 23 has emphasized the importance of specimen choice
and handling, bisulfite modification strategy, choice of assay
based on scale, sensitivity and cost, tissue specificity, and other
factors. These issues remain at play for current lung cancer
screening biomarkers (see Chapter 7).