2012 EDUCATIONAL BOOK - American Society of Clinical Oncology

histologic section–based molecular tests (e.g., FISH and
IHC).
On the other hand, our growing understanding of the
cancer biology of NSCLC, particularly the molecular evolution
of tumors during local progression and metastasis and
the identification of molecular abnormalities contributing to
resistance to TK inhibitor therapies, emphasizes the importance
of characterizing the molecular abnormalities of the
disease at every stage of its evolution. For molecular testing
of advanced metastatic NSCLC, it is important to sample
and analyze the tumors’ sample at each time point of clinical
decision making.
Relevant Molecular Pathologic Analysis Methods
Currently, most of the predictive molecular markers available
for therapy selection in NSCLC are oncogene mutations
and amplifications. However, other molecular and genetic
changes modulate the sensitivity of tumor to targeted therapies,
including protein overexpression, gene methylation,
and gene expression abnormalities. The need for analysis of
multiple molecular and genetic changes in small, clinically
relevant biopsy and cytology specimens has driven the
scientific community and the molecular pathology laboratories
to develop multiplex approaches for molecular testing
of small tumors samples, particularly for gene mutation
analysis.
Mutation Analysis
Currently, direct nucleic acid sequencing with previous
polymerase chain reaction (PCR) amplification of extracted
DNA is the most commonly used technique for gene mutation
analysis of tumor biopsy and cytology samples to detect
mutations of clinically relevant genes. Several sequencing
methods are available for mutation analysis of DNA extracted
from tumor tissue and cell specimens, especially
for formalin-fixed paraffin-embedded (FFPE) samples. The
current PCR-based sequencing mutation analysis methods
can be divided into uniplex (e.g., Sanger sequencing and
pyrosequencing) and multiplex (e.g., matrix-assisted laser
desorption ionization time-of-light mass spectrometry and
primer extension assay) methods. In the uniplex method,
one hotspot sequence is examined at a time, while the
multiplex technique multiple hotspot mutations are examined
simultaneously. The multiplex approaches are clearly
the preferred methods since they allow more efficient mutation
analysis of small amounts of DNA for multiple hotspots
(approximately 100 to 200) from a panel of genes (approximately
10 to 30).
Sanger Sequencing
In solid tumors, including lung, Sanger sequencing is the
most commonly used sequencing method to detect hotspot
mutations of oncogenes (e.g., EGFR, KRAS, BRAF). This
type of sequencing can detect essentially all base substitutions,
small insertions, and deletions. Its main disadvantage
is the relatively low sensitivity (approximately 20%) for the
detection of mutant alleles in the DNA sample extracted
from tumor specimens. These DNA samples are usually a
mixture of mutant and wild-type alleles as a result of the
presence of malignant and nonmalignant (from adjacent
normal or tumor stroma) cells in the tumor tissue specimens.
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Pyrosequencing
Pyrosequencing uses sequencing by a synthesis method
to sequence nucleic acids and relies on the detection of
pyrophosphate release on nucleotide incorporation. Pyrosequencing
is considered to be more sensitive than Sanger
sequencing and detects approximately 5% of mutant compared
with wild-type alleles. 26
Matrix-Assisted Laser Desorption Ionization Time-of-Light
Mass Spectrometry
Matrix-assisted laser desorption ionization time-of-light
mass spectrometry (Sequenom) utilizes a high-throughput
PCR-based sequencing assay to detect multiple hotspot
mutations simultaneously using small amounts of DNA
obtained from biopsy and cytology specimens. 27 It has a high
level of sensitivity (approximately 5% of the mutant alleles)
and allows quantification of the percentage of mutant
DNA. 27 The Sequenom method is also useful to assess gene
amplification.
Primer Extension Assay
Primer extension assay is a primer extension–based
method that allows simultaneous analysis of up to 10 different
mutations. 28 It is a sensitive, low-cost, and rapid method
to screen for mutations and to analyze methylation. This
assay uses the SNaPshot Multiplex Kit (Applied Biosystems
Inc), which contains a reaction mix of four differentially
fluorescently labeled ddNTPs, allowing the interrogation of
each base at a mutation site. 28
Translocation and Gene Copy Number Analyses
FISH is a cytogenetic technique that uses fluorescentlabeled
probes to hybridize specific sequences of DNA on
chromosomes. 29 It is applied to visualize chromosome deletions,
amplification, and structural rearrangements. The
main advantage of this technique is that it allows in situ localization
of the specific sequences and the simultaneous
detection of multiple sites by using hybridization probes
labeled with different fluorophores. The main disadvantage
of FISH is the need of additional equipment for analysis,
such as dark-field microscopy and multiband fluorescent
filters. Chromogenic in situ hybridization is a variant of the
in situ hybridization technique that visualizes the specific
DNA sequence by a peroxidase reaction and allows the
visualization in a light microscope. The main disadvantage
of this method is that it limits the use of different label
probes to target multiple sites; however, recent advances in
methodologies permit the use of dual colors to target two
sequencing sites and enable the visualization of tissue
architecture and cytomorphologic analysis. Although there
are other methods available for gene copy number and
fusion genes analyses, such as DNA quantitative PCR assay
for copy number assessment and messenger RNA quantitative
PCR assay for gene fusion analysis, FISH continues to
be the preferred method for gene copy number (e.g., MET,
FGFR1) and gene fusion (e.g., EML4-ALK) in lung cancer.
Protein Expression Analysis
IGNACIO I. WISTUBA
IHC is a widely used technique in pathology laboratories
to detect the presence and levels of expression of a specific
protein in FFPE tumor cytology and cytology specimens. In
lung cancer, the use of IHC is currently used for histopatho-