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Basic Concepts of Molecular Pathology

Timothy Craig Allen, MD, JD; Philip T. Cagle, MD; Helmut H. Popper, MD

T his month’s issue of the Archives of Pathology & Labora-

tory Medicine includes a unique special section on
‘‘Molecular Signatures of Lung and Pleural Tumors’’ that
building blocks. Nucleotides, made up of a sugar-phos-
phate backbone with a nitrogenous base, are either pu-
rines—adenine (A) and guanine (G) in DNA and RNA—
represents the proceedings of a special Joint Symposium or pyrimidines—thymine (T) and cytosine (C) in DNA
of the European Working Groups for Molecular Pathology (uracil [U] replaces T in RNA). The nucleotides that make
and Pulmonary Pathology at the 21st European Congress up the genes are arranged in a double-stranded right-
of Pathology in Istanbul, Turkey, in September 2007. This handed helix. Nucleotides in DNA are arranged sequen-
symposium and the subsequent special section were or- tially so that a gene will code for a matching protein.
ganized by Dr Helmut Popper of the Institute of Pathology Within a double helix pattern, A, a purine, always binds
at the Medical University of Graz, Graz, Austria, president with T, a pyrimidine, and G always binds with C, giving
elect of the Austrian Society of Pathologists and immediate a nucleotide sequence for which 1 strand is a ‘‘mirror im-
past president of the European Working Group for Pul- age’’ of the other strand.1–6
monary Pathology. In this brief review, we have provided There are 46 chromosomes (23 pairs) in a human dip-
some definitions of terms and concepts used in the pro- loid cell, on which all genes are located. Chromosomes are
ceedings of the ‘‘Molecular Signatures of Lung and Pleu- paired, and as such a gene is found on a locus on each of
ral Tumors’’ special section for those readers who are not the 2 paired chromosomes, giving 2 copies, or alleles, of
already familiar with molecular pathology. genes. Gametes are haploid rather than diploid and there-
Molecular pathology may have once been a specialized fore contain only 1 allele for each gene. Diploid status is
component of the research laboratory or the clinical lab- reestablished when the nuclear material from an egg and
oratory, but today molecular diagnostic and prognostic sperm combine during fertilization.1–6
techniques are in common use within the anatomic pa- Transcription, the synthesis of messenger RNA (mRNA)
thology laboratory, especially in the realm of infectious from a DNA strand, is a key step in the formation of pro-
organism diagnosis and cancer diagnosis. Molecular test- tein coded by DNA. During transcription, enzymes called
ing continues to expand as more easily obtainable archival topoisomerases break a DNA strand and allow the DNA
paraffin-embedded tissue replaces fresh and frozen tis- double-helix to uncoil, giving 2 DNA strands, 1 of which
sues as the source of DNA and RNA needed for molecular is the template for mRNA, called the DNA template. Base
analysis, and as newer technologies allow for more pairs are matched with the DNA template to produce a
streamlined methods of testing. This review addresses the mirror image of the DNA template, except with the sub-
increased application of molecular testing in lung pathol- stitution of U for T, forming a strand of mRNA. A series
ogy, specifically how the current state of molecular pa- of 3 base pairs in a gene, called a codon, code for a specific
thology may be applied to practical, everyday lung pa- amino acid, so that a series of codons code for a particular
thology diagnosis. sequence of amino acids resulting in the synthesis of a
GENES specific protein. Translation, the assembly of the protein
molecule from the mRNA template, occurs with the ad-
Genes, made up of nucleic acids, contain the informa- dition of amino acids in a particular sequence based on
tion necessary for the construction of proteins from amino the specificity of the mRNA. Transfer RNA assists in
acids within a cell. Genes code for proteins required for translation.1–6
metabolic reactions and cellular structure. DNA makes up After translation, modifications to the newly formed
genes, and RNA transcribes the genetic code held within protein occur in order for it to function, to move within
the DNA into proteins. The genetic code within the genes the cell, or to fold properly. Methylation, acetylation, phos-
is composed of nucleic acids, for which nucleotides are the phorylation, glycosylation, posttranslational cleavage, and
the addition of lipid groups are examples of posttransla-
Accepted for publication June 17, 2008. tional modifications. End regions of chromosomes are
From the Department of Pathology, The University of Texas Health made up of telomeres, hundreds of repeats of the nucle-
Science Center at Tyler (Dr Allen); the Department of Pulmonary Pa- otide sequence TTAGGG. Some of these telomere sequenc-
thology, The Methodist Hospital, Houston, Tex (Dr Cagle); and the Insti- es are lost each time a cell divides, until they are lost and
tute of Pathology, Medical University of Graz, Graz, Austria (Dr Popper). the cell can no longer divide. This process is called senes-
The authors have no relevant financial interest in the products or
companies described in this article. cence. A polymerase called telomerase is able to replace
Reprints: Timothy Craig Allen, MD, JD, Department of Pathology, the DNA sequences at the end regions, allowing for con-
University of Texas Health Science Center at Tyler, 11937 US Hwy 271, tinuing cell division—a significant feature in some can-
Tyler, TX 75708-3154 (e-mail: cers.1–6
Arch Pathol Lab Med—Vol 132, October 2008 Basic Concepts of Molecular Pathology—Allen et al 1551
The polypeptide chain formed by the specific amino probe that can be seen as a chromogenic reaction under
acid sequence causes the newly formed protein to fold into light microscopy, whereas FISH is available as probe sets
a tertiary arrangement giving it a 3-dimensional structure. and multiprobe FISH cocktails. Peripheral blood, urine,
Often, the newly formed protein is inert until made func- sputum, endoscopic brushings and washings, and paraf-
tional by a posttranslational modification such as phos- fin-embedded, formalin-fixed tissue are all suitable for
phorylation or proteolytic cleavage. Phosphorylation, the FISH. However, fixation of tissue with formalin for longer
addition to the protein of a phosphate group catalyzed by than 48 hours may yield poorer FISH results.23
enzymes called kinases, may cause, for example, translo- DNA and RNA probes hybridize to a specific target se-
cation of the protein from the cytosol into the nucleus. quence that is of interest, for example, genes implicated in
Dephosphorylation is the removal of a phosphate group a specific type of cancer or an inherited disease, or to a
catalyzed by enzymes called phosphatases. Phosphoryla- certain microorganism.16–22 Probes are made using DNA
tion and dephosphorylation of proteins are often impor- fragments cloned from yeast or bacterial artificial chro-
tant in the activation and deactivation of cell cycle pro- mosomes and, with FISH, are directly or indirectly fluo-
teins, signaling pathway proteins, and transcription factor rophore labeled, most often using Texas Red, fluorescing
proteins.1–6 red, and fluorescein isothiocyanate, fluorescing green.24
Protein degradation is necessary to remove damaged Directly labeled probes have a fluorophore-labeled nucle-
proteins and limit signaling proteins such as those in- otide inserted into the probe, so binding of the probe to
volved in cell survival and cell death. This degradation its target in 1 hybridization step is all that is required to
often needs to proceed quickly. Reversible cross-linkage visualize the probe. Indirectly labeled probes have a re-
to a polypeptide, termed ubiquitin, leads to the rapid deg- porter molecule such as biotin or digoxigenin attached co-
radation of proteins and is called ubiquinylation or poly- valently, requiring the additional step of the application of
ubiquinylation.7 a fluorophore-labeled avidin or fluorophore-labeled anti-
The control of gene expression is for the most part con- digoxigenin. The additional step required with indirectly
trolled by regulation of transcription initiation. Proteins labeled probes is a disadvantage; however, indirectly la-
called transcription factors, also termed transactivators or beled probes generally allow for stronger signals because
trans-acting factors, bind to DNA and regulate RNA poly- of greater signal amplification.24 There are 4 general types
merase activity, affecting gene expression either by induc- of probes: chromosome enumeration probes, locus-specific
ing or activating the gene or by inhibiting the gene by indicator probes, telomeric probes, and chromosome
reducing transcription levels.8–13 Transcription factors are paints. Chromosome enumeration probes hybridize to re-
necessary for RNA polymerase to initiate transcription, petitive DNA sequences located near chromosome centro-
and transcription factors called transcriptional activators meres, and because the loss of a centromere is generally
stimulate transcription of an RNA molecule from its DNA indicative of the loss of an entire chromosome, they are
template.14,15 used to enumerate the number of copies of a certain chro-
mosome within a cell.25,26 Locus-specific probes are probes
THE TOOLS OF MOLECULAR PATHOLOGY to unique sequences and are most frequently used to de-
A variety of techniques exist for molecular pathology termine whether specific genes are amplified, translocat-
diagnosis, including nucleic acid extraction, Southern blot- ed, or deleted. Telomeric probes hybridize to unique DNA
ting, restriction fragment length polymorphism, sequenc- sequences located very close to telomeres. The probes do
ing, liquid bead microarrays, mass spectrometry, and not hybridize to telomeric sequences. Chromosomal paints
comparative genomic hybridization, among others. Nu- are a mix of probes that probe to the entire length of 1 or
cleic acid extraction historically has used organic tech- more chromosomes.
niques using chloroform and phenol; however, automated A FISH specimen must undergo prehybridization to al-
nucleic acid extraction exists today and is frequently used low a probe to efficiently hybridize to cellular DNA tar-
to purify nucleic acids for their use with other molecular geted by the probe while protecting the cell from mor-
methods. For practical laboratory-based molecular diag- phologic disruption. Following prehybridization, the
nosis of lung disease, polymerase chain reaction (PCR) probe and cellular DNA are denatured so that the probe
and fluorescence in situ hybridization (FISH) are 2 of the can hybridize to the cellular DNA it is targeting. Hybrid-
most important techniques commonly used. ization usually takes between 4 and 12 hours. A type of
DNA called Cot DNA is added during hybridization to
hybridize highly repetitive DNA sequences that are locat-
Fluorescence In Situ Hybridization ed within the genome so that the probe DNA does not
In situ hybridization uses DNA or RNA probes to eval- nonspecifically bind these repetitive sequences and yield
uate intact cells for genetic changes. Probes visualized a multitude of nonspecific signals rather than the appro-
with a chromogen that produces a colored chemical at the priately specific signal.24,25,27,28 The nonbound probe is then
reaction site is called chromogenic in situ hybridization removed by washing, a nuclear counterstain that weakly
and probes using fluorescent labels are called FISH. Eval- fluoresces is added so that the nucleus can be identified,
uation of genetic alterations within intact cells allows for and an antifade is added to retard fluorophore photo-
the detection of genetic alterations occurring in a specific bleaching. The signal produced by the FISH fluorophore
group of cells or within a small number of examined cells is then examined with fluorescence microscopy.
and is a major benefit of the use of in situ hybridization
in anatomic pathology. It is commonly used with cytologic Polymerase Chain Reaction
and surgical specimens to detect tumor cells and certain Since its introduction in 1985,29 PCR has been refined
microorganisms, and with surgical specimens of tumors to be an efficient and sensitive method of studying the
for its prognostic utility and to determine treatment re- molecular pathology of primary and metastatic neo-
sponse.16–22 Chromogenic in situ hybridization uses a plasms, inflammatory mechanisms, and infectious diseas-
1552 Arch Pathol Lab Med—Vol 132, October 2008 Basic Concepts of Molecular Pathology—Allen et al
es.30–37 Today, automated instruments designed for the lab- PCR has become popular for detecting and quantifying
oratory tabletop are available. Polymerase chain reaction RNA targets in a variety of cancers. It requires no post-
amplifies DNA via a repeated 3-step process of denatur- PCR analysis and is efficient and automated. Several stud-
ation, annealing, and extension. Double-stranded DNA ies have used real-time quantitative RT-PCR to evaluate
that is the target is denatured at high temperature to yield lymph nodes for micrometastases, including for non–small
2 intact single strands of complementary DNA. Then at a cell lung carcinoma (NSCLC), and to examine peripheral
lower temperature, specially designed single-stranded blood for potential dissemination of lung cancer cells dur-
DNA primers anneal or bind to a specific targeted area of ing lobectomy.49–52
the single-stranded DNA. Because there are very large
numbers of DNA primers relative to the full-length com- LOSS OF HETEROZYGOSITY
plementary DNA strand, the target DNA anneals with the
primer DNA much more frequently than with the com- Normal human DNA contains 2 alleles for every genetic
plementary DNA when cooling occurs. In the third step, locus, the majority of which are identical, or homozygous,
extension, Taq polymerase identifies the now partially and the loss of 1 allele results in no pathologic change.
double-stranded DNA and extends the primers by poly- Some genetic loci have 2 differing copies of alleles and are
merization, to yield, at the end of the first cycle, 2 double- heterozygous. The majority of these heterozygous alleles
stranded copies of a portion of the target DNA generated allow for normal variance and do not result in pathologic
from 1 copy. Because the DNA primers only recognize the changes; however, some of these heterozygous loci have
DNA for which they have been specifically designed, only the potential to cause pathologic genetic variations, with
that specific segment of DNA, making up a small part of resultant disease. If there is a loss of the normal, or ‘‘wild-
the entire DNA present in the original DNA, is preferen- type,’’ allele at a locus, with resultant ‘‘loss of heterozy-
tially amplified. Subsequent cycles produce numerous gosity,’’ the remaining aberrant allele can cause cellular
shorter double-stranded DNA PCR products, so that more
damage. At homozygous loci, the loss of an allele can oc-
than a billion copies of the original double-stranded DNA
cur and be followed by gene silencing or point mutation,
are produced after 30 cycles, and more than a trillion are
produced after 40 cycles. Following amplification, post- causing loss of tumor suppressor genes.53,54 Detection of
PCR analysis of the markedly increased amount of tar- loss of heterozygosity using older methods such as South-
geted DNA sequence product can be performed. The tar- ern blot analysis and restriction fragment length poly-
get sequence can be shown to be present in a specimen morphism analysis are low-throughput, tedious, and in-
by the use of amplicons run on polyacrylamide or agarose efficient; however, newer, high-throughput single nucleo-
electrophoresis, or via Southern blotting with probe hy- tide polymorphism arrays have allowed for more efficient
bridization, comparing the lengths of the targeted DNA examination of loss of heterozygosity.55–57
sequence with DNA ‘‘ladder’’ markers. DNA sequencing Cancers, including lung cancers, commonly exhibit loss
can be performed on the amplicons, or studies to identify of heterozygosity, causing the inactivation or silencing of
mutations may also be performed.38,39 Because PCR enor- genes critical for growth regulation and homeostasis. Cig-
mously amplifies DNA, great caution must be used in per- arette smoking has been associated with loss of hetero-
forming PCR to avoid cross-contamination of a specimen geneity of sites on chromosome 3, and the association
with even very small amounts of DNA. is greater in patients who began smoking at a young
Real-time PCR is becoming a more and more popular age.58–61 More than 90% of small cell carcinomas and more
method of molecular pathology research and diagnosis than 70% of NSCLCs contain loss of heterozygosity.57,62,63
that eliminates the need for post-PCR analysis and allows Among NSCLCs, squamous cell carcinomas exhibit loss of
for relatively quick detection of DNA targets, including heterozygosity in more than 90% of cases, compared with
specific mutations.40–44 adenocarcinomas, showing loss of heterozygosity in ap-
The best way to examine specific genes present in a proximately 70% of cases. In NSCLC, loss of heterozygos-
certain cell type, such as in tumor cells, is to examine those ity generally involves genetic foci on chromosomes 1p, 3p,
cells’ mRNA. As RNA is not stable enough to work with 8p, 9p, 13q, 17p, 19p, Xp, and Xq. In small cell carcinomas,
easily in a laboratory, reverse transcription can be used to loss of heterozygosity generally involves chromosomes 3p,
convert mRNA into its complementary DNA. With reverse 4q, 5q, 4q, 10q, 13q, 15q, and 17p.57,62–66 Losses found in
transcription, mRNA is the template used for the produc- both small cell carcinomas and NSCLCs, involving chro-
tion of a strand of DNA, opposite or reverse of typical
mosomes 3p, 13q, and 17p, are probably related to inac-
cellular transcription. The complementary DNA can be
tivation of critical tumor suppressor genes including reti-
used as the template for PCR in a process called reverse
noblastoma, p53, and fragile histidine triad (FHIT).57,62,63
transcriptase–PCR (RT-PCR). Reverse transcriptase–PCR
can be used to examine genes that are expressed, over- Loss of heterozygosity in premalignant conditions and
expressed, underexpressed, or not expressed in a specific malignant diseases of the lung represents both early- and
cell type by the isolation of specific mRNA.44–47 Altered late-stage changes in the progression of disease; however,
gene expression is a characteristic of malignant transfor- the continuum of losses makes it hard to evaluate the spe-
mation, and those alterations allow for the identification cific contribution of each loss. Loss of heterozygosity has
of the presence of cancer cells via the detection of mRNA also been identified in some benign lung diseases, includ-
transcripts specific to those tumor cells. Tumor markers ing asthma and chronic obstructive pulmonary disease,
have been identified that are specific to solid organ can- probably reflecting the genetic predisposition identified in
cers, and RT-PCR is highly sensitive in detecting differ- these diseases.67–70 Loss of heterozygosity has also been
entially expressed tumor-related mRNAs. Some studies identified in cases of usual interstitial pneumonia (idio-
have indicated that RT-PCR can detect as few as 1 cancer pathic pulmonary fibrosis) and suggests premalignant po-
cell in a million normal cells.44,48 Real-time quantitative RT- tential in those cases.71
Arch Pathol Lab Med—Vol 132, October 2008 Basic Concepts of Molecular Pathology—Allen et al 1553
SIGNAL TRANSDUCTION AND SIGNALING PATHWAYS pathway repairs small isolated foci of DNA damage in-
cluding reduced or oxidized single bases or fragments
Extracellular messenger molecules such as hormones,
and small, nonbulky adducts. The nucleotide excision re-
inflammatory cytokines, and growth factors, called li-
pair pathway repairs DNA damage involving both
gands, bind to specific cell surface receptors and activate
strands. The defects cannot be simply replaced because no
messengers within the cytosol leading eventually to acti-
matrix for the DNA segment is available. These defects can
vation of nuclear transcription factors that, due to the ex-
cause DNA helical structure deformity, such as cross-links,
tracellular message, direct the transcription of a specific
bulky chemical adducts, and pyrimidine dimers. The
gene product, such as the transcription of a protein in-
DNA damage response pathway, also termed the double-
volved in cell growth. This cascade of events is termed
strand break repair pathway, involves a cascade of events and
signal transduction, and the series of steps within the cas-
repairs damage to double-stranded breaks. The direct
cade is termed signal transduction pathway or signaling path-
damage reversal pathway repairs DNA damage caused by
way. Growth factor receptors are a common cell surface
alkylating compounds in cigarette smoke.94–102
receptor, on which polypeptide growth factors such as epi-
dermal growth factor are ligands attaching to those recep- CELL CYCLE
tor protein-tyrosine kinases, activating the receptor and
The cell cycle is a sequential series of very tightly reg-
causing it to bind with intracellular proteins, which in
ulated events governing cell proliferation, including entry
turn continue the signaling pathway. Epidermal growth
into DNA replication, replication, entry into cell division,
factor receptor is a member of the type I growth factor
cell division, and cell rest. The cell cycle is divided into 5
receptor tyrosine kinase family. Epidermal growth factor
phases: G0 (cell at rest), G1 (preparation for DNA synthe-
receptor has other ligands that bind to it other than epi-
sis), S (DNA synthesis or replication), G2 (integrity check
dermal growth factor, including transforming growth fac-
of replicated DNA), and M (mitosis with nuclear and cel-
tor ␣, and these ligands, receptors, and signaling path-
lular division), which provide orderly control of DNA rep-
ways play a central role in many lung cancers as well as
lication and cell division in response to external and in-
some nonneoplastic pulmonary diseases.72–77
ternal stimuli. Cyclin-dependent kinases form complexes
The extracellular ligands’ ‘‘messages’’ are transmitted
with proteins called cyclins that tightly regulate the pro-
via a signaling pathway. Cell differentiation and prolifer-
gression of the series of steps in the cell cycle by activating
ation, cell survival, and cell death and apoptosis are reg-
and inactivating proteins by phosphorylation, including
ulated by signaling pathways, the majority of which
proteins that act as ‘‘brakes’’ on cell cycle progression and
‘‘cross-talk’’ with other signaling pathways in a complex
cell proliferation. The cell cycle is controlled by many in-
manner. Several important signaling pathways have been
teracting pathways and positive and negative feedback
well studied. For example, the Wnt/B/catenin pathway,
loops, and it may be stimulated appropriately or inappro-
termed the canonical Wnt signaling pathway, involves Wnt
priately in various inflammatory diseases. Cell cycle reg-
binding to Frizzled cell surface receptors, which in turn
ulation loss is a very important step in uncontrolled cell
activate Disheveled, causing the inhibition of protein ki-
proliferation during carcinogenesis.
nase glycogen synthase kinase 3, which in turn releases
Cell cycle checkpoints prevent damaged DNA to be
dephosphorylated ␤-catenin from the adenomatous pol-
passed on to daughter cells by temporarily arresting the
yposis coli–axin complex. ␤-Catenin associates with T-cell
cell cycle at specific steps. It allows damaged DNA to be
factor/lymphoid enhancer–binding factor transcription
repaired, or, if the damage is too severe, to send cells into
factors causing the induction of Myc.78–84 Other important
apoptosis (programmed cell death). The primary check-
signaling pathways include the JAK/STAT pathway, in-
point in the cell cycle is the restriction point in G1 where
volving signal transducers and activators of transcription
‘‘commitment’’ to the cell cycle occurs. Other checkpoints
(STAT) proteins and Janus kinase (JAK) nonreceptor pro-
include an S-phase checkpoint and a G2-M checkpoint.103–106
tein tyrosine kinases; the Ras/Raf-1/MAPK pathway, a
A complex of damage sensor proteins, the Rad9-Rad1-
significant pathway in carcinogenesis, including epithelial
Hus1 heterotrimer complex and the Rad17-RFC complex,
cell proliferation; the nuclear factor-␬B transcription factor
detect DNA damage at these checkpoints. After DNA
and nuclear factor-␬B signaling pathways that regulate
damage is repaired, the DNA damage checkpoint is si-
immune system proteins, cell survival and proliferation
lenced and the cell cycle restarts.
proteins, and apoptosis proteins; and the PI3K/Akt/
Growth factor signaling initiates the cell cycle and
mTOR pathway important in regulating cell survival;
maintains the transition through the G1 phase; however,
among many others.85–93
once the cell passes through the cell cycle’s restriction
point, it no longer requires growth factor signaling, and
the cell is ‘‘committed’’ to the cell cycle. The retinoblas-
Errors in replication, extracellular influences such as UV toma (Rb) gene product, pRb, governs progression past
light, radiation, and chemicals, and endogenous influences the restriction point of the cell cycle and governs the ex-
such as oxygen radicals routinely cause DNA damage, pression of genes involved in DNA synthesis. Progression
generally depurination, deamination, nonenzymatic meth- of the cell cycle also depends on activation of cyclin D.107,108
ylation, and hydrolysis, sometimes with the attachment of One of the main functions of p53, the TP53 gene product,
a chemical group to DNA, the combination of which is is to ‘‘protect’’ the DNA through the arrest of the cell cycle
termed an adduct. Several DNA repair pathways exist to at checkpoints in response to DNA damage or to promote
excise the damaged DNA and replace it with newly syn- the induction of apoptosis when damage is too severe.109,110
thesized DNA based on its undamaged complementary As such, p53 has been referred to as the guardian of the
DNA strand. These DNA repair pathways are important genome. Both Rb and p53 have critical roles in the man-
in an individual’s susceptibility to lung cancer and re- agement of the cell cycle, and abnormalities of Rb and p53
sponse to lung cancer therapy. The base excision repair are the most common abnormalities associated with the
1554 Arch Pathol Lab Med—Vol 132, October 2008 Basic Concepts of Molecular Pathology—Allen et al
cell cycle dysregulation of malignancy; however, due to genetics: Protocols and Applications. Vol 204. Totowa, NJ: Humana Press; 2002:
the large number of redundancies, feedback loops, and 25. Van Stedum S, King W. Basic FISH techniques and trouble-shooting. In:
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