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Thyroid Cancer

Translational medicine is an expanding area of modern medicine. It embodies the


collaboration between basic science and clinical medicine, the purpose of which
is to deepen our
insight into disease pathogenesis and prognosis in order to advance and refine t
herapeutics. The
ultimate aim of translational medicine is to individualize care by tailoring the
rapy to best fit each
person and his or her specific disease. Much of this approach to medicine involv
es the use of molecular
techniques to study genetics, gene expression, and signaling pathways, while inc
orporating
innovations in modern technology from the computer industry. Therefore, this for
m of medicine
is also called molecular medicine. The management of thyroid cancer involves man
y challenges
and uncertainties relating to disease development, diagnosis, and management. In
this chapter, we
discuss some of the molecular biology techniques involved in the study of thyroi
d cancer and the
important role of translational medicine in revolutionizing thyroid cancer care.
1.1 INTRODUCTION
Translational medicine is the term applied to the integration of basic science a
nd clinical medicine.
The collaboration between basic science and clinical medicine, or bench to bedsid
e medicine, is
a process of clinical inquiry that inspires scientific research, leading to nove
l discoveries and new
insights into disease pathogenesis, diagnosis, and management that can then be i
ncorporated into
patient care. These discoveries result in the synergistic advancement of both cl
inical medicine and
basic science, as scientific research leads to new clinical knowledge, which spa
rks subsequent questions
for science to answer and the need for technological advancements to help answer
these questions.
Therefore, a perpetual flow of discovery circulates from the bench to the bedsid
e and back.
CONTENTS
1.1 Introduction................................................................
...............................................................1
1.2 Molecular Biology Methods...................................................
...................................................2
1.3 Molecular Biology and Thyroid Cancer........................................
............................................4
1.3.1 Chromosomal Translocations and Mutations..................................
..............................4
1.3.1.1 Gene Expression.........................................................
....................................6
1.3.1.2 MicroRNA................................................................
......................................6
1.3.1.3 Proteomics..............................................................
........................................7
1.3.2 Clinical Implications.....................................................
................................................7
1.4 Conclusion..................................................................
...............................................................7
References......................................................................
.....................................................................8
2 Thyroid Cancer: From Emergent Biotechnologies to Clinical Practice Guidelines

Much of this molecular medicine involves the study of genetics, gene expression, a
nd signaling
pathways, incorporating micro- and nanotechnologies from the computer industry.1
It is hoped that
translational medicine will allow medical care to be tailored to the individual,
based upon one s
genetic makeup or the genetics of one s disease. Genetic polymorphisms and mutatio
ns can make
an individual susceptible to certain illnesses, or more likely to respond to or
develop side effects to
certain treatments. Similarly, knowledge of the genetic mutations in cancer cell
s can predict their
aggressiveness and response to chemotherapy. With this knowledge, an individual
could avail of
targeted therapies and avoid potentially harmful treatments, in a new form of me
dicine called personalized
medicine.
While the concept of integrating basic science and clinical medicine is not new,
it is seen as an
essential part of the evolution of modern medicine, and the term translational m
edicine was adopted
to draw attention to and advance this strategy. Historically, cancers were diagn
osed by tissue and
histological subtype alone, with the choice of therapy determined on the same ba
sis. This approach
led to unpredictably varied responses to the same therapeutic agents, in individ
uals with apparently
the same tumor. In more recent years, the genetic and biological basis of many c
ancers has
been determined, which is allowing for great advances in the development of targ
eted therapies.2,3
Based on this genetic and biological knowledge, a clinician could predict a pati
ent s prognosis and
likelihood of response to treatment, thereby distinguishing those patients who m
ay be cured with
surgery alone, so avoiding unnecessary and potentially toxic medication, from th
ose with tumors
that are likely to recur or metastasize, and therefore would benefit from adjuva
nt therapy. The care
of patients with thyroid cancer involves many challenges, for example, diagnosin
g follicular thyroid
carcinoma (FTC) with fine-needle aspiration biopsy (FNAB), predicting which pati
ents with papillary
thyroid cancer (PTC) are likely to develop recurrent or metastatic disease, how
to treat patients
who do not respond to radioactive iodine (RAI), and how to alter the poor progno
sis associated
with anaplastic thyroid cancer (ATC). Genetic breakthroughs since the 1980s and
ongoing breakthroughs
in molecular biology are confronting these challenges.
1.2 MOLECULAR BIOLOGY METHODS
Observational studies of humans and animals in the early part of the twentieth c
entury described
inherited forms of cancer and evoked theories on the presence of genetic mutatio
ns inciting cancer
development in these families. But scientific evidence was wanting, and these th
eories remained
unproven for some time.4,5 Advances in basic science followed, with the descript
ion of the DNA
double helix in 1953, and subsequent depiction of transcription and translation,

succeeded by major
advances in molecular biology techniques through the latter part of the twentiet
h century, allowing
for the completion of the Human Genome Project in 2003.6,7 While DNA contains th
e entire
genome, it consists of both coding and noncoding regions. Of the coding regions,
certain genes are
only expressed at certain times in certain tissues. Studying RNA allows us to id
entify the genes that
are expressed, upregulated, downregulated, or silenced in a particular cell, giv
ing a truer reflection
of the biological activity within that cell. RNA undergoes posttranscription mod
ifications that result
in splice variants and variations in the amino acid sequence produced during pro
tein translation.
The recently discovered microRNAs (miRNAs) add another dimension to the regulati
on of gene
expression, by binding to mRNA transcripts, leading to their degeneration or pre
venting their translation.
8 Proteins also undergo conformational changes after translation, forming differ
ent isoforms
and potentially lipoproteins or glycoproteins. Proteomics is an evolving field o
f translational medicine
that studies these proteins and their contributions to cell activity.
Some of the techniques frequently used in molecular biology and their applicatio
ns are outlined
in Table 1.1. Notable breakthroughs in molecular biology include the development
of recombinant
DNA techniques, as described in 1972.10 Recombinant DNA involves the integration
of a DNA
sequence of interest (target DNA) into the self-replicating DNA of a host, which
may be a virus
(bacteriophage), a bacterial plasmid, a bacterial artificial chromosome (BAC), o
r a yeast artificial chromosome (YAC). The choice of vector is made based on the
size of the target DNA sequence.
Recombinant DNA technology is a cornerstone of modern molecular biology. It faci
litates DNA
cloning, the study of transfected cell lines, and the generation of transgenic m
ouse models of disease.
11 13 Another major advance was the development of polymerase chain reaction (PCR)
to clone
DNA.14 PCR allows the rapid generation of many copies of a target DNA sequence.
PCR has been
adapted to clone RNA in the form of complementary DNA (cDNA) by reverse transcri
ptase PCR
(RT-PCR). It has also been modified to sequence DNA by nucleic acid hybridizatio
n and to quantify
RNA (real-time PCR, TaqMan). Most recently, microarray technology has revolutioni
zed DNA
hybridization and sequencing through integrating molecular biology techniques wi
th nanotechnology
from the computer industry. Microarrays were employed in the sequencing of DNA f
or
the Human Genome Project. They are also being used widely to study gene expressi
on in certain
cancers.9,15
1.3 MOLECULAR BIOLOGY AND THYROID CANCER
1.3.1 Chromosomal Translocations and Mutations
From the 1900s, chromosomal abnormalities were suspected to be associated with t

umor development.
It took until the latter part of the twentieth century to prove this with certai
nty. The first chromosomal
translocation was identified in the 1970s. This was the reciprocal translocation
between
chromosomes 9 and 22 (t(9;22)), named the Philadelphia chromosome, which was fou
nd to be a consistent
abnormality in chronic myelogenous leukemia (CML). Subsequently, many chromosoma
l
translocations were identified throughout the different hematological malignanci
es.16,17
Chromosomal translocations can alter gene expression in two ways: an activating
gene may
become situated proximal to a proto-oncogene, leading to activation of the oncog
ene; alternatively,
a break in the gene can create a new fusion gene and result in a novel protein.
While these chromosomal
abnormalities are salient in hematological malignancies, relatively few have bee
n identified
in solid tumors. PTC was the first solid tumor in which a chromosomal translocat
ion was detected.
The initial abnormality detected was the fusion of the RET gene with the H4 gene
, by a paracentric
inversion of the long arm of chromosome 10, known as the RET/PTC1 rearrangement.
18 The RET
gene encodes a transmembrane tyrosine kinase that is found in the parafollicular
C cells, but is not
present in normal thyroid follicular cells. RET/PTC rearrangements result in a c
onstitutively activated
form of the tyrosine kinase due to the fusion of a promoter region to the RET on
cogene. Many
forms of RET/PTC rearrangements have been identified in thyroid cancer cells, re
sulting from paracentric
inversions of the long arm of chromosome 10 (RET/PTC1, RET/PTC3) and others from
reciprocal translocations, such as the t(10;17) involved in RET/PTC2. These rear
rangements are
particularly common in individuals with thyroid cancer and a history of radiatio
n exposure.19 21
FTC cells have been found to have the chromosomal translocation t(2;3) in approx
imately oneto
two-thirds of cases.22,23 The result of this translocation is the fusion of tran
scription factor gene
PAX8 with the gene for peroxisome proliferator-activated receptor ? (PPAR?), and
leads to upregulation
of the PPAR? transcription factors.22 FTCs with PAX8/PPAR? translocation appear
to have a
more aggressive phenotype, although some adenomas also carry this translocation.
23
Initial karyotyping identified chromosomes by their distinct banding pattern (Gbanding) and
could detect gross chromosomal abnormalities, such as the Philadelphia chromosom
e.16 However
hybridization has allowed for more subtle anomalies to be detected.24 Fluorescen
t in situ hybridization
(FISH) uses large DNA probes to locate specific DNA sequences on chromosomes, an
d comparative
genomic hybridization (CGH) compares the binding pattern on the DNA of interest
to normal
DNA.11 FISH revealed the positions of chromosomes during interphase, demonstrati
ng how a single

radiation beam could create simultaneous breaks in the RET/PTC genes, allowing f
or the observed
rearrangements seen in individuals with thyroid cancer after radiation exposure.
25 Multiplex FISH
(M-FISH) and spectral karyotyping (SKY) use the same principles, but use multipl
e probes for each chromosome. The pattern of probe hybridization undergoes compu
ter analysis so that the origin
of each chromosome involved in a rearrangement can be distinguished. ATC cells h
ave complex
chromosomal rearrangements that have been demonstrated with SKY.24,26,27 These t
echniques also
allowed for the identification of the multiple different RET/PTC fusion genes in
PTC.20
Germline point mutations in the RET oncogene are present in familial medullary t
hyroid carcinoma
(fMTC), the multiple endocrine neoplasia (MEN) 2A and MEN2B syndromes, and are
inherited in an autosomal dominant manner. These mutations were found in MEN2A t
o be a single
base pair change that most commonly altered the amino acid produced by codon 380
.28 Screening
different tumor types for point mutations led to the discovery of mutations in t
he BRAF gene in
melanoma, colorectal cancer, and ovarian cancer.29 The RAF proteins are serine/t
hreonine protein
kinases that affect cell growth, differentiation, and apoptosis through the mito
gen-activated protein
kinase (MAPK) pathway signaling. These RET and BRAF point mutations were detecte
d by DNA
sequencing using nucleotide hybridization.28,29 Heteroduplex analysis was also u
sed to detect the
BRAF mutations. This process involves the use of PCR to replicate DNA from two s
ources, normal
DNA and DNA with a suspected mutation. The PCR products from the normal and abno
rmal
DNA templates recombine to form heteroduplexes, but the point mutation results i
n unsuccessful
base pairing at that nucleic acid. This leads to a bubble in the DNA double heli
x and allows it to be
separated on gel electrophoresis from the normal double-stranded DNA. The abnorm
al nucleic acid
can then be identified by sequencing.29 Forty to seventy percent of PTC has been
shown to carry
the BRAF mutation, but it is uncommon in follicular variants of papillary thyroi
d cancer (FVPTCs)
and FTC.29,30 BRAF mutations translate into single amino acid substitutions that
alter the activity of
BRAF kinase and activate MAPK signaling.29
FTC, FVPTCs, ATC, as well as some follicular adenomas have been found to harbor
point
mutations in the RAS gene.21,23,31 The RAS proteins are signal transducing prote
ins involved in cell
growth and differentiation and are associated with the same signaling pathway as
PPAR?. As the
RAS mutations and PAX8/PPAR? translocations are rarely found in the same tumor,
it has been suggested
that these two mutations may represent two distinct tumor types.23 Transgenic mo
use models
of thyroid cancer have been produced to study these genetic mutations in vivo. T
he use of transgenic
mouse models allows the introduction of these known mutations of the RAS and BRA

F genes to
see if thyroid cancer can be attributed to these mutations, rather than being me
rely coincidental
anomalies. Studies of BRAF mutations in transgenic mice confirmed that these mut
ations induce
thyroid tumors with histological findings similar to those of human PTC, and RAS
mutations have
been shown to lead to the development of aggressive FTC.32,33
Since the discovery of germline mutations in RET, the screening of relatives of
affected individuals
with MTC and MEN2 involves DNA testing. Previously, stimulated calcitonin (CTN)
levels
were monitored in all family members. CTN monitoring had suboptimal sensitivity,
and there was
unnecessary testing and follow-up of unaffected relatives. Since MTC is the majo
r cause of mortality
among individuals with MEN2A, a positive DNA test for the mutation should lead t
o prophylactic
thyroidectomy in childhood, as this appears to be curative. Therefore, DNA testi
ng for MEN and
fMTC has transformed the care of these patients.34,35
The presence of the BRAF mutation in PTC is associated with increased disease re
currence and
metastases, so testing thyroid FNAB specimens for this mutation has been propose
d preoperatively to
guide the extent of surgical resection.36,37 Also, PTC with this mutation has a
less avid response to RAI,
and therefore may benefit from molecular targeted therapies such as inhibitors o
f tyrosine kinases.38
As mentioned above, the identification of different translocations and mutations
within thyroid
cancers, such the PAX8/PPAR? translocation and RAS mutation within FTC, may prov
ide highly
useful information regarding tumor behavior. This information may ultimately lea
d to a reclassification
of thyroid cancers, based not on their histological subtype, but on their geneti
cs. However, even
though these mutations may be present in the majority of thyroid cancers, noncan
cerous tissues have
also been found to harbor some of these genetic anomalies. Therefore, detecting
these mutations is
not diagnostic ; they are still unable to completely distinguish benign from malign
ant disease.
Hence, further clinical insights can be gained by studying additional molecular
biological mechanisms,
such as gene expression and patterns of protein synthesis and interactions.
1.3.1.1 Gene Expression
Gene expression is studied by analyzing the RNA produced by gene transcription.
Although mutations
and translocations affecting DNA can lead to thyroid tumors, the disease phenoty
pe can vary
due to differences in gene expression. For example, BRAF mutations lead to more
aggressive forms
of PTC than RET/PTC translocations, even though they both act through the same s
ignaling pathway.
Microarray and real-time PCR are the two methods most commonly used to analyze R
NA.
Microarrays have been used to analyze gene expression in many cancers, but they
have been best

studied in breast cancer, where the expression profiles of genes regulating cell
cycle, metastases,
and angiogenesis can be used to predict tumor behavior and guide therapy. Gene e
xpression has also
been used in colorectal cancer to establish whether tumors will respond to certa
in chemotherapeutic
agents.9 Microarray allows for the detection of known sequences of RNA or its cD
NA produced by
RT-PCR. The microarray has predefined sequences fixed to the glass chip. Labeled
cDNA hybridizes
to the complementary sequences on the chip in a process called reverse hybridiza
tion. The gene
expression is demonstrated by the signal intensity, which is related to the stre
ngth of hybridization
when compared to the normal tissue.40 In real-time PCR, the quantity of RNA is d
etected by the
quantification of fluorescent molecules that are bound to the PCR product. The T
aqMan probe
contains a fluorescent molecule and a quencher that masks the fluorescence. It i
s used as a third
primer that binds downstream to one of the conventional DNA primers during PCR.
As the DNA is
elongating, DNA polymerase encounters the quencher and releases it, allowing the
DNA strand to
fluoresce. The intensity of the fluorescence increases with greater quantity of
the PCR product.41
In PTC, gene expression analysis has shown different expression patterns in BRAF
- and RET/
PTC-associated tumors. The BRAF mutation has been shown to promote methylation a
nd silencing
of a number of tumor suppressor genes and upregulation of the angiogenic vascula
r endothelial
growth factor (VEGF), matrix metalloproteinases (MMPs) that promote destruction
of the interstitial
matrix and promote angiogenesis, and c-Met, a cell surface tyrosine kinase that
stimulates mitogenesis
and is associated with a number of tumors. Iodide metabolizing genes are downreg
ulated
in BRAF mutations, perhaps accounting for the poor response to iodide treatment
in patients with
metastatic PTC.37 Comparing the gene expression profiles of RET/PTC, BRAF- and R
AS-associated
PTC have shown a defining gene expression profile associated with each mutation,
involving genes
associated with signal transduction and the immune response.19 Microarrays could
potentially be
used in thyroid cancer to determine therapy based on these different gene expres
sion profiles.
1.3.1.2 MicroRNA
In 1993, miRNAs first appeared in the genomics cast. They were initially discove
red in the nematode
Caenorhabditis elegans, but were later recognized in many eukaryotes, including
humans.8,42
In oncology, they were first noted to be involved in chronic lymphocytic leukemi
a (CLL), where
a deletion on chromosome 13 was found to cause downregulation of microRNA 15 (mi
R15) and
miR16, prompting further research into these small molecules and their role in c
ancer.43 miRNAs
are transcribed from nonprotein coding regions of DNA, to form the precursor pri

-miRNA. PrimiRNA
is then enzymatically cut to form pre-miRNA in the nucleus and is transported to
the
cytoplasm as a double strand. One strand forms the mature miRNA that enters a pr
otein complex,
mi-ribonucleoprotein (mi-RNP). The second strand was thought to be degraded, but
may also form
functional mature miRNA.44 The miRNA binds to the 3 untranslated region (3 UTR) of
mRNA,
that is, the region of mRNA between the 3 end of the translated mRNA and the poly
adenylated
tail. How precisely the miRNA binds to the 3 UTR region determines whether it lead
s to mRNA
degradation or inhibition.8,45
Different miRNAs may be upregulated or downregulated in different tumor types. I
n thyroid
cancer, there appears to be distinct patterns of miRNA upregulation, linked to t
he different genemutations and translocations.46 In addition, single nucleotide
polymorphisms (SNPs) in the genome
have been associated with altered regulation of miRNAs and an increased risk of
PTC.47 It appears
that the potential for tumorigenesis is related to the mRNA affected by the miRN
A. In PTC, miRNA
also affects expression of the protein KIT, a tyrosine kinase receptor, whereas
in B cell leukemia,
miRNAs activate the oncogene Myc.8 It has been suggested that these miRNAs also
mediate drug
resistance. Therefore, miRNAs may be useful for diagnostics and guiding therapeu
tics. It is hoped
that expression profiles of miRNAs may assist in FNAB diagnoses. Additionally, p
harmacological
research continues to study the forms of anti-miRNAs that can bind to and inhibi
t the action of
miRNAs.45
1.3.1.3 Proteomics
The product of gene transcription and translation and the ultimate effector of a
ny alterations in DNA or
RNA is protein. Proteomics studies these molecular products of genetic mutations
. Proteins can be studied
by 2D electrophoresis and mass spectrometry. Mass spectrometry has a greater sen
sitivity for protein
detection than electrophoresis. Two common types of mass spectrometry are matrix
-assisted laser
desorption ionization (MALDI) and electrospray ionization (ESI). Analysis of who
le tissue sections is
possible using MALDI, and reveals which proteins are present and where in the ti
ssue they are functioning.
In thyroid cancer, mutations frequently lead to continuous activation of kinases
, increased signaling
through the (MAPK) pathway, and angiogenesis resulting from the production of VE
GF.23,48 Therefore,
inhibitors of tyrosine kinase, VEGF and PPAR? agonists are potential therapeutic
agents.49,50
As well as identifying the alterations in signaling pathways that result from ge
netic mutations or
altered gene expression, proteomics can identify biomarkers for cancer that migh
t predict response
to therapies and monitor response to treatment.9 Protein profiling of thyroid FN
AB specimens is an
area under investigation, with the hope that markers will be identified that can

define the malignant


potential of the thyroid tissue and its likely response to treatment.51
1.3.2 Clinical Implications
Ultimately, the aim of translational medicine is to apply scientific knowledge t
o clinical medicine
and thereby improve patient care. As mentioned, DNA testing in patients with MEN
syndromes and
fMTC has allowed for curative surgery in those who carry this germline mutation,
and no need for
ongoing screening of unaffected relatives. In thyroid cancers of follicular cell
origin, insights into
the associated mutations have allowed a new understanding of cancer development
and its progression,
and may redefine how we think of this disease. The identification of potential t
reatment targets
through gene expression has led to pharmacological trials of novel treatments, s
uch as tyrosine
kinase inhibitors in advanced cancers that were previously untreatable.
Eventually, gene expression analysis of mRNA, miRNA, or biomarker detection coul
d be performed
on FNAB samples, and the results could determine the malignant potential of the
lesion and
the surgical intervention required. In addition, a patient s likelihood of disease
progression could be
determined, as well as the optimal treatment strategy for that individual. New t
herapeutic targets
are being investigated, with new molecular targets, such as miRNAs. Perhaps with
the development
of proteomics, sensitive and specific biomarkers will be available that can dete
ct tumors before they
are clinically apparent and allow for intervention before the disease progresses
.