Molecular diagnosis of a BRAF papillary thyroid carcinoma with multiple chromosome abnormalities and rare adrenal and hypothalamic metastases.
ABSTRACT Molecular characterization of thyroid tumors is rarely applied to patient management. Our aim was to demonstrate the application of molecular and cell biology to patient care.
Clinical and molecular case study.
A 57-year-old man with papillary thyroid carcinoma presented with adrenal and several other presumed metastases, pulmonary nodules, and mediastinal lymphadenopathy. Bronchial carcinoma was entertained for the pulmonary lesions because of a tobacco history. Mediastinal lymph node biopsy was nondiagnostic. Cells from the biopsy were grown in tissue culture and characterized by immunocytochemical (ICC), allele-specific polymerase chain reaction (PCR), reverse transcription (RT)-PCR, DNA sequencing, and cytogenetics. A panel of agents were tested the cells for tumoricidal activity. The cells expressed thyroid-specific markers [thyroid-stimulating hormone receptor (TSH-R), thyroglobulin (TG), sodium iodide symporter (NIS)] and markers [thyroid transcription factor-1 (TTF-1), cytokeratin-7, epidermal growth factor receptor (EGF-R)] present in the primary tumor and adrenal metastasis. The BRAF V600E mutation was detected. The karyotype was 44-48,XY, + der(1) t(1;9)(p13;p13),add(9)(p13),-17,-18, + 0-3mar[cp20]. Lovastatin, gefitinib, paclitaxel, depsipeptide, and 17-AAG inhibited the growth of the cultured cells. Combinations of two or three drugs produced additive or synergistic effects depending upon the combination.
Unusual metastases may be associated with multiple molecular and cytogenetic abnormalities. Thus, molecular and cell-biological studies can allow otherwise difficult thyroid tumor diagnosis and may be used for targeted, individualized selection of potential treatments.
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ABSTRACT: ABSTRACT Background: Papillary thyroid cancer is the most common type of thyroid malignancy and has an excellent prognosis. Distant organ metastasis is rare. Bilateral adrenal metastases with iodine uptake has not been described before. Patient Findings: A 47-year-old woman presented for evaluation because of severe right upper arm pain and weakness. Magnetic resonance imaging of the thoracic spine showed a compression fracture at the third thoracic vertebra associated with a soft tissue mass. Computed tomography (CT)-guided biopsy of the mass showed metastatic papillary thyroid carcinoma. Ultrasonography of the neck showed an enlarged right thyroid lobe with cervical lymphadenopathy. A high resolution CT scan of the chest showed multiple bilateral pulmonary nodules. Treatment included total thyroidectomy and lymph node dissection, external beam radiation to the thoracic spine, and 131 iodine therapy. Initial whole body 131 iodine scintigraphy showed faint uptake in the right upper abdomen, interpreted as a sign of physiologic bowel activity; however, repeat whole body 131 iodine scintigraphy showed increased uptake in both adrenal glands, consistent with metastatic disease. Serial abdominal CT scans showed progressively enlarging bilateral adrenal masses. Despite additional treatment with 131 iodine, the patient's disease deteriorated and progressed at all metastatic sites. Summary: This patient had bilateral adrenal metastases from advanced papillary thyroid cancer with distant metastasis to lung and bone at initial presentation and poor response to repeated 131 iodine therapy. Unilateral adrenal metastasis from thyroid cancer has been described previously in 6 cases; this is the first case report of bilateral adrenal metastases. Conclusions: Bilateral adrenal metastasis is rare in papillary thyroid cancer. Elevated abdominal uptake of 131 iodine in a high risk patient may be a sign of abdominal metastatic disease.Thyroid: official journal of the American Thyroid Association 05/2013; · 2.60 Impact Factor
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ABSTRACT: At present no successful treatment is available for advanced thyroid cancer, which comprises poorly differentiated, anaplastic, and metastatic or recurrent differentiated thyroid cancer not responding to radioiodine. In the last few years, biologically targeted therapies for advanced thyroid carcinomas have been proposed on the basis of the recognition of key oncogenic mutations. Although the results of several phase II trials look promising, none of the patients treated had a complete response, and only a minority of them had a partial response, suggesting that the treatment is, at best, effective in stabilizing patients with progressive disease. "Epigenetic" refers to the study of heritable changes in gene expression that occur without any alteration in the primary DNA sequence. The epigenetic processes establish and maintain the global and local chromatin states that determine gene expression. Epigenetic abnormalities are present in almost all cancers and, together with genetic changes, drive tumor progression. Various genes involved in the control of cell proliferation and invasion (p16INK4A, RASSF1A, PTEN, Rap1GAP, TIMP3, DAPK, RARβ2, E-cadherin, and CITED1) as well as genes specific of thyroid differentiation (Na+/I- symport, TSH receptor, pendrin, SL5A8, and TTF-1) present aberrant methylation in thyroid cancer. This review deals with the most frequent epigenetic alterations in thyroid cancer and focuses on epigenetic therapy, whose goal is to target the chromatin in rapidly dividing tumor cells and potentially restore normal cell functions. Experimental data and clinical trials, especially using deacetylase inhibitors and demethylating agents, are discussed.Frontiers in Endocrinology 01/2012; 3:40.
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ABSTRACT: Introduction: Epigenetic changes have been detected in thyroid cancer cells, and evidence indicates that they may contribute to altered differentiation and proliferation of these cells. Histone acetylation/deacetylation represents a major mechanism for modulating the expression of genes, including those involved in neoplastic transformation, and drugs that inhibit histone deacetylase (HDAC) activity have displayed promising anti-tumor activity in many pre-clinical studies. Areas covered: We provide a brief overview of the mechanisms underlying histone acetylation-mediated regulation of gene expression and the principal epigenetic alterations detected in thyroid cancer cells. The review then focuses on the results of pre-clinical and clinical studies (some still underway) in which HDAC inhibitors (HDACi) have been used to treat thyroid cancer. Expert opinion: HDACs are a potentially important target for thyroid cancer treatments. Inhibition of HDAC activity has produced encouraging results in terms of reducing proliferation rates and restoring the iodine-uptake capacity in transformed thyrocytes. HDACi, especially when combined with other molecularly targeted drugs, may represent an important option for those tumors that are unresponsive to the currently available treatments.Expert Opinion on Therapeutic Targets 12/2012; · 4.90 Impact Factor
Molecular Diagnosis of a BRAF Papillary Thyroid Carcinoma
with Multiple Chromosome Abnormalities and Rare Adrenal
and Hypothalamic Metastases
John A. Copland,1Laura A. Marlow,1Sandra F. Williams,2Stefan K. Grebe,3,4Michelle L. Gumz,1
William J. Maples,5Victor E. Silverman,6and Robert C. Smallridge2
Objective: Molecular characterization of thyroid tumors is rarely applied to patient management. Our aim was to
demonstrate the application of molecular and cell biology to patient care. Design: Clinical and molecular case study.
Main Outcomes: A 57-year-old man with papillary thyroid carcinoma presented with adrenal and several other
presumed metastases, pulmonary nodules, and mediastinal lymphadenopathy. Bronchial carcinoma was en-
tertained for the pulmonary lesions because of a tobacco history. Mediastinal lymph node biopsy was non-
diagnostic. Cells from the biopsy were grown in tissue culture and characterized by immunocytochemical (ICC),
allele-specific polymerasechain reaction (PCR),reverse transcription (RT)-PCR, DNA sequencing, and cytogenetics.
stimulating hormone receptor (TSH-R), thyroglobulin (TG), sodium iodide symporter (NIS)] and markers [thyroid
transcription factor-1 (TTF-1), cytokeratin-7, epidermal growth factor receptor (EGF-R)] present in the primary
tumor and adrenal metastasis. The BRAF V600E mutation was detected. The karyotype was 44-48,XY,+der(1)
t(1;9)(p13;p13),add(9)(p13),-17,-18,+0-3mar[cp20]. Lovastatin, gefitinib, paclitaxel, depsipeptide, and 17-AAG in-
hibited the growth of the cultured cells. Combinations of two or three drugs produced additive or synergistic effects
depending upon the combination. Conclusions: Unusual metastases may be associated with multiple molecular and
cytogenetic abnormalities. Thus, molecular and cell-biological studies can allow otherwise difficult thyroid tumor
diagnosis and may be used for targeted, individualized selection of potential treatments.
since it typically retains some features characteristic of the
normal thyroid follicular cell (2–4). Features postulated to
play a role in its pathogenesis include somatic genetic
changes that activate the RET=RAS=RAF=MEK=ERK path-
way (5). Rearrangements in RET are seen in up to 30% of
PTC, while other receptor tyrosine kinases, such as TRKA,
may also occasionally be altered. Downstream in the ERK
pathway, activating RAS mutations occur in about 10% of
PTC (5). Finally, the BRAF V600E activating mutation is the
most frequent genetic alteration in PTC, being found in 30–
40% of cases with very few occurrences described in other
thyroid tumor morphotypes (6).
APILLARY THYROID CARCINOMA (PTC) comprises up to 88%
of thyroid cancers (1) and is classified as differentiated
PTC tends to remain intrathyroidal or metastasizes pri-
marily to regional lymph nodes (7), accounting for its typi-
cally favorable prognosis. When distant metastases do occur,
they portend a dismal outcome. The most common sites of
metastases are lung (41–80%), bone (6–36%), and brain (1–
10%) (8),while adrenal (9–14) and pituitary (15,16) metastases
are rare. Typically, the ability to metastasize distantly signals
the acquisition of aggressive tumor behavior and correlates
with the loss of differentiated features. This can complicate
the morphological diagnosis of metastatic lesions, not only in
unusual locations, but also within the lungs of patients with
risk factors for primary broncho-pulmonary malignancy.
Thyroid cancer mortality is related to the inability to treat
progressive metastatic disease with conventional chemo-
therapy (2). The lack of an effective therapy for tumors that are
resistant to radioiodine and thyroid-stimulating hormone
1Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida.
2Division of Endocrinology, Mayo Clinic College of Medicine, Jacksonville, Florida.
3Division of Endocrinology, Department of Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota.
4Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, Minnesota.
5Division of Oncology, Mayo Clinic College of Medicine, Jacksonville, Florida.
6Center for Medicine Endocrinology and Diabetes, Atlanta, Georgia.
Volume 16, Number 12, 2006
ª Mary Ann Liebert, Inc.
(TSH)-suppressive therapy has led to ongoing research into
novel therapeutic modalities targeted to receptors, signaling
molecules, transcription factors, and de-differentiation factors.
These investigations allowed the elucidation over the past two
decades of the genetic events that underlie thyroid cancer
monstrates the diagnostic potential of targeted molecular, cell-
biological, and immunocytochemical (ICC) testing, as well as
based upon in vitro studies of patient-derived tumor cells.
Materials and Methods
THJ-2N cells derived from primary cultures of human
normal thyroid tissue and LAM1 cells derived from trans-
were established in our laboratory after informed consent and
the approval of Institutional Review Board. DRO 90-1 (DRO)
cells derived from primary cultures of human anaplastic
thyroid carcinoma were kindly provided by Dr. G.J.Juillard
(University of California-Los Angeles, Los Angeles, CA).
Thyroid cells (without TSH) were cultured in Roswell Park
Memorial Institute (RPMI) 1640 medium (Cellgro, Herndon,
VA) supplemented with 10% charcoal-strippedfetal bovine se-
rum (Hyclone, Logan, VT), non–essential amino acids, sodium
pyruvate, and penicillin–streptomycin–amphotericin B at 378C
in a humidified atmosphere with 5% carbon dioxide (CO2).
A549 lung cells (ATCC, Manassas, VA) were cultured in F-12K
supplemented with 10% fetal bovine serum and penicillin–
For cell proliferation inhibition studies, LAM1 cells (pas-
sages 20–30) were plated in 12-well culture plates in triplicate
for each condition at an initial concentration of 2?104cells per
well. After overnight incubation, cells were treated with lo-
vastatin (Mayo Clinic Pharmacy), paclitaxel (Sigma-Aldrich),
gefitinib (Iressa?Mayo Clinic Pharmacy), depsipeptide (NSC
630176, Gloucester Pharmaceticals, Cambridge, MA and Na-
tional Cancer Institute), or 17-Allylaminogeldanamycin (17-
AAG) (Sigma-Aldrich) diluted in dimethylsulfoxide (DMSO)
(Sigma-Aldrich, St. Louis, MO). All cells received identical
volumes of DMSO and were exposed to each drug for 6days
with medium and drug changed every 48hours. After 6days,
cells were washed with PBS (Cellgro), trypsinized, and coun-
ted by Beckman Coulter Counter.
RNA extraction and RT-PCR
Total cellular RNA was extracted from cells using RNA-
queous (Ambion, Austin, TX) according to the manufacturer’s
protocol. Total RNA from normal and papillary thyroid tissue
was extracted using TOTALLY RNA (Ambion) according to
subjected to DNAse digestion to eliminate potential genomic
DNA contamination. Following DNAse inactivation, cDNA
was synthesized from 1mg RNA using reverse transcriptase
(Biorad, Hercules, CA) followed by polymerase chain reaction
(PCR) using the ThermalACE kit (Invitrogen, Carlsbad, CA)
according to the manufacturer’s protocol. The primers used to
amplify thyroglobulin (TG), thyroid-stimulating hormone re-
ceptor (TSH-R), sodium iodide symporter (NIS), RET=PTC1,
RET=PTC2, RET=PTC3, and glyceraldehyde-3-phosphate-
dehydrogenase (GAPDH) as an internal control are listed in
Table 1. Reverse transcription (RT) conditions were 258C for
by the indicated number of cycles for PCR. PCR conditions
were denaturation at 958C for 3minutes, annealing at indi-
cated temperatures for 30seconds, primer extension at 748C
for 1minute, and then final extension for 10minutes. No RT
control experiments were included in any of the RT-PCR re-
actions. PCR products were electrophoresed in 2% agarose
gels, stained with ethidium bromide, and checked for the ex-
Table 1. RT-PCR Primers Used to Examine mRNA Levels for TG, TSH-R, NIS, RET=PTC1,
RET=PTC2, RET=PTC3, and GAPDH Are Shown
Sense (R1?) 50-AGGGAGCTTTGGAGAACTTG-30
Antisense (R12B) 50-CTTTCAGCATCTTCACGG-30
Sense (R1?) 50-AGGGAGCTTTGGAGAACTTG-30
Antisense (C2R) 50-TGCAGGCCCCATACAATTTG-30
RET=PTC2 568C 40 203
GAPDH 608C 35450
TG: thyroglobulin; TSH-R: thyroid-stimulating hormone receptor; NIS: sodium iodide symporter; and GAPDH: glyceraldehyde-3-
1294COPLAND ET AL.
BRAF mutational analysis
Cells were grown to confluence and genomic DNA was
purified using AQUAPURE Genomic DNA isolation kit
(Biorad) according to the manufacturer’s protocol. Wild-type
and possible V600E mutant BRAF DNA were amplified in
separate lightcycler real-time PCR reactions using allele-
primer-probe pairs. The wild-type-specific reaction used 50-
GATTTTGGTCATGC‘‘T’’ACAGT-30(‘‘T’’ denotes the position
of the LC-640 reporter dye) as a forward primer and 50-
CACTCCATCGAGATTTCACTG-fluorescein-30as a FRET do-
nor probe. For the V600E mutant detection reaction, we used
50-GATTTTGGTCATGC‘‘T’’ACAGA-30as a forward primer
and 50-CACTCCATCGAGATTTCTCTG-fluorescein-30as a
FRET donor probe. The reverse primer was identical in both
was PCR-amplified using 50-TCATAATGCTTGCT-CTGAT
as forward and reverse primers, respectively. Unincorporated
by Exo-SAP treatment. After heat inactivation of the exonu-
clease and the phosphatase, 1mL of the PCR product was se-
quenced in forward and reverse directions on an automated
DNA sequencer using BigDye dye terminator chemistry.
LAM1 cells were grown in Chang C=MEM alpha using
standard culture conditions and harvested with conventional
cytogenetic procedures with colcemid,hypotonicsolution,and
methanol–acetic acid fixative for chromosome analyses. Me-
taphases were stained by GTG- or QFQ-banding and analyzed
to determine the number and structure of chromosomes (18).
LAM1 cells were plated on coverslips at 1?105cells per well
and were allowed to adhere overnight. Cells were then fixed
with2% paraformaldehydefollowed by permeabilizationwith
ice-cold 100% methanol. Coverslipswere blocked with Diluent
(Dako, Carpinteria, CA) and probed with each primary anti-
TG (Dako), NIS (Chemicon, Temecula, CA), thyroid transcrip-
tion factor-1 (TTF-1) (Dako), cytokeratin-7 (Dako), epidermal
growth factor receptor (EGF-R) (Upstate, Charlottesville, VA),
and C-kit (Dako). Coverslips were washed with PBSþ0.05%
Tween followed by incubation of either goat anti-rabbit-
fluorescein isothiocyanate (FITC) or goat anti-mouse-FITC
(Jackson Labs, Westcove, PA) at 1:200 in Diluent. Coverslips
were again washed with PBSþ0.05% Tween and bound anti-
body was visualized using a FITC filter on a confocal micro-
Soft agar assays and xenograft studies
Soft agar assays for anchorage-independent growth were
performed on LAM1 cells. Each 60mm plate was first layered
with 0.75% agar diluted with 10% fetal bovine serum–
layer was then prepared as previously stated with 103cells per
plate. Plates were maintained at 378C under 5% CO2 for
3weeks. For xenograft studies, suspensions of 5?106=0.1mL
LAM1 cells in RPMI medium and Matrigel were injected sub-
mice (Harlan, Indianapolis, IN) for a total of 10 tumor sites.
Institutional Animal Care and Use Committee protocols were
Data are presented as mean?SD unless otherwise speci-
fied. Comparisons of treatment groups were analyzed by two-
tailed paired or unpaired Student’s t test, as appropriate based
upon F tests. A p<0.05 was considered statistically significant.
A 57-year-old man had developed a left neck mass with
biopsy positive for PTC. Total thyroidectomy and pathologi-
cal examination revealed a 1.2cm well-differentiated upper
left lobe PTC. Metastatic disease was noted in the 3.5cm
left cervical mass, in multiple foci of neck soft tissue and in 5
of 30 left cervical nodes. Postoperatively 130mCi I131was
administered, and a posttreatment total body scan revealed
intense uptake within the neck without uptake in any
other regions. At that time, the serum TSH concentration
was 43.5mIU=L with a concurrent serum TG of 14.5ng=mL.
TSH-suppressive levothyroxine therapy was initiated, but
area. Computed tomography (CT) of the neck demonstrated
left-sided nodularity, and chest CT showed left and right hilar
adenopathy. Increased uptake in the left neck, bihilar regions,
and left adrenal was noted on fludeoxyglucose-positron em-
mission tomography (FDG-PET) imaging. Additional dissec-
tissues. ICC stains were positive for TG, TTF-1, EGF-R, and
cytokeratin-7. CT-guided biopsy of the left adrenal gland
confirmed metastatic PTC with ICC staining positive for TG,
TTF-1, and cytokeratin-7 and negative for cytokeratin-20.
Pulmonary adenopathy was progressive and remained undi-
agnosed with attendant diagnostic dilemma given the pa-
tient’s significant past tobacco history.
Eight months following his initial diagnosis, the patient
presented to Mayo Clinic with recurrent neck nodularity and
associated pain, polyuria, and polydipsia. CT and magnetic
resonance imaging confirmed recurrent neck disease with
infiltrated cervical soft tissues and increased adenopathy in
the right posterior cervical triangle. Imaging also revealed
persistence of a 4cm left adrenal mass, bihilar and mediast-
inal adenopathy, new bilateral pulmonary nodules, and
thickening of the pituitary infundibulum and hypothalamus,
suspicious for metastatic disease (Fig. 1). Concurrent serum
TSH and TG were<0.1mIU=L and 71ng=mL, respectively,
in the absence of TG auto-antibodies. There was evidence of
secondary hypogonadism [testosterone¼195ng=mL (nor-
mal: 240–950), luteinizing hormone¼0.8IU=L (normal: 1–9),
leading to initiation of testosterone replacement.
The patient was started on empiric DDAVP therapy,
based on the clinical setting, radiographic findings, and un-
detectable plasma AVP (<0.5pg=mL), with resolution of
PTC METASTASIS MOLECULAR DIAGNOSIS1295
polyuric=polydipsic symptoms. External beam radiation,
5960cGy, was delivered to the thyroid region and adjacent
lymph nodes, and 3000cGy to the hypothalamus-pituitary
with resultant significant clinical improvement.
An attempt at diagnosis of pulmonary lesions with bron-
choscopy and transbronchial aspiration of left subcarinal
lymph nodes was nondiagnostic, but a sample from this as-
pirate was grown in tissue culture and subjected to ICC and
molecular analysis. One month later a second treatment dose
of I131(206mCi) was administered after withdrawal of le-
vothyroxine therapy. Posttreatment total body scan revealed
mild uptake in the right lower neck only. Re-evaluation
2months later revealed a new focal cortical lytic lesion
(2.3?1.3cm) in the left femur and progressive asymmetry in
the right choroid plexus with intense contrast enhancement.
Pulmonary adenopathy and pulmonary nodules were stable,
and infundibular thickening and enhancement were im-
proved. Lumbar puncture was unremarkable. Further ra-
diation, 3250cGy to the right choroid plexus and 1800cGy to
the left femur, was administered.
Cytogenetic analysis of patient-derived cell line
Cells obtained from the transbronchial biopsy specimen
were grown in tissue culture and named LAM1. There were
multiple numerical and structural chromosomal abnormal-
ities (Fig. 2). Each of the 20studied metaphases had monos-
1;9 translocation, add(9)(p13), and up to 3 marker chromo-
somes containing genetic material of uncertain origin. The
consensus karyotype was 44-48,XY,þder(1)t(1;9) (p13;p13),
add(9)(p13),-17,-18,þ0-3mar (Fig. 2).
Molecular analysis of LAM1 cells
Through ICC (Fig. 3) and RT-PCR (Fig. 4), these cells were
shown to possess thyroid-specific markers. LAM1 cells ex-
hibited NIS, TG, and TTF-1 expression and A549 lung carci-
noma cells were used to demonstrate specificity with no
expression of NIS and TG, while TTF-1 is expressed (Fig. 3A).
LAM1 cells are epithelial as demonstrated by cytokeratin-7
expression and are negative for c-Kit, but positive for EGF-R
(Fig. 3B). RT-PCR analyses confirmed mRNA expression of
thyroid-specific molecular markers (Fig. 4). LAM1 cells ex-
pressed moderate levels of NIS and low levels of both TSH-R
and TG (Fig. 4). Since it has been recently reported that his-
tone deacetylase (HDAC) inhibitors can induce differentia-
tion in thyroid carcinomas (19), LAM1 cells were treated with
depsipeptide and TSH-R mRNA levels are clearly induced
with moderate induction of NIS and none for TG (Fig. 4, last
column). These findings provide convincing evidence that the
pulmonary lesions represent metastatic PTC.
adrenal, (B) enlarged mediastinal lymph node, and (C) pulmonary nodule, and MRI of (D) thickened infundibulum.
Radiographic imaging of hypothalamic stalk, adrenal gland, and lung. CT imaging of (A) metastatic lesion to left
1296 COPLAND ET AL.
Mutational analysis of LAM1 cells
LAM1 cells were negative for RET=PTC1, 2, or 3 rear-
rangements (data not shown). Sequence-specific real-time PCR
for wild-type andV600E mutantBRAFshowed both wild-type
and mutant copies in LAM1 DNA indicated by an approx-
imate 1:1 ratio of BRAF wild-type and V600E mutant (Fig. 5A).
The presence of a heterozygote BRAF V600E mutation was
confirmed by independent repeat experiments and DNA se-
quencing (Fig. 5B).
Novel molecular targeted combinatorial therapies
for metastatic papillary carcinoma
Based upon the cytochemical and molecular findings ob-
tained upon testing and the medical literature in regard to
t(1;9)(p13;p13) (arrows) and monosomy of chromosomes 17 and 18. No marker chromosomes are visible in this particular
Representative karyotype of LAM1 cells. There is an additional derivative chromosome 1, containing an unbalanced
specific markers for thyroglobulin (TG), sodium iodide symporter (NIS), and thyroid transcription factor-1 (TTF-1) were used
to diagnose thyroid tumor in cells (LAM1) grown in culture isolated from the transbronchial biopsy. A549 lung carcinoma
cells, which express TTF-1, were used as a control. (B) Other markers were also used to verify previous findings of the
primary lesion and adrenal metastasis. These markers are c-Kit, epidermal growth factor receptor (EGF-R), and cytokeratin-7.
Immunocytochemical analyses of cells in culture isolated from transbronchial biopsy from patient. (A) Thyroid-
PTC METASTASIS MOLECULAR DIAGNOSIS 1297
the potential for antitumor activity with the use of novel new
agents, several drugs were tested for possible growth in-
hibition. Laboratory analyses revealed significant growth
inhibition when these cells were incubated with molecular-
targeted drugs from the following classes: taxane (paclitaxel),
HMG CoA reductase inhibitor (lovastatin), HDAC inhibitor
(depsipeptide), EGF-R antagonist (gefitinib), and Hsp-90
destabilizer (17-AAG) (Fig. 6A). Three of these drugs are
already FDA approved and have known antitumor activity
in experimental models for poorly differentiated thyroid
The concentration at which 25% of cell proliferation is in-
hibited (IC25) was calculated for each drug, and two or three
agents were then combined to determine whether they pos-
sessed additive, synergistic, or antagonist antiproliferative ac-
tivity. As shown in Figure 6B, the percent inhibitions of each
compound alone using IC25 concentrations were lovastatin
(P)¼24.0?.03%; and depsipeptide (D)¼29.7?0.22%. In
combinations of twos, gefitinibþpaclitaxel (GP), gefiti-
nib þlovastatin (GL), and paclitaxelþlovastatin (PL) demon-
strated additive to mild synergistic drug activity. For example,
PL had a growth inhibition of 61.8?2.0% compared to 42.2%
(24.0þ18.2%) using the fractional inhibition method (23).
When three compounds were combined, LGP, LDP, and
DGP also demonstrated additive to mild synergistic drug
activity. For example, DGP has a growth inhibition of 85.3?
1.9% compared to the addition of monotherapy (29.7þ
18.3þ24.0%¼72.0%). Depsipeptide in double drug combi-
nations produced additive effects and as a component of
triple drug combinations provided the greatest synergism for
two ofthethree combinatorial treatments(Fig.6B). Ourdesire
was to test the promising drug combinations in a nude mouse
cancer model. Unfortunately, LAM1 cells neither grew in
nude mice nor formed colonies on soft agar (data not shown).
On the other hand, antiproliferative activity of 17-AAG ap-
pears to possess antagonist activity when combined with
LAM1 cells. RT-PCR was performed as described in ‘‘Mate-
rials and Methods’’ for thyroid-stimulating hormone recep-
tor (TSH-R), sodium iodide symporter (NIS), thyroglobulin
(GAPDH) for LAM1?depsipeptide (HDAC inhibitor). The
positive control as indicated by ‘‘þ’’ is THJ-2N (passages 2–
3) and the negative control as indicated by ‘‘?’’ is A549 lung
cells. GAPDH levels are not changed indicating that all lanes
were loaded equally.
Thyroid-specific genes are expressed in cultured
primers (WT) and mutant primers (mut) that recognize the respective DNA sequences of wild-type and V600E mutant. Six
amplification curves are depicted. DRO cells were used as a positive control for the BRAF V600E mutation, showing a homo-
zygous V600E pattern with no amplification with wild-type primers (horizontal line [DRO WT] at the far right that does not rise)
and good amplification with themutant primer (second line[DRO Mut] from theright). LAM1 DNA amplifies with both mutant
(third line [LAM1 Mut] from the right) and wild-type primers (fourth line [LAM1 WT] from the right), as does an artificial
heterozygote control, consisting of a mixture of DRO DNA and normal control DNA (fifth line [pos Mut] from right for mutant
primers, and far left line for [pos WT] wild-type primers, respectively). (B) DNA sequence of LAM1 cell DNA (left panel) and
normal control DNA (right panel). A heterozygote A=T base position is present in the LAM1 cell DNA (arrow), changing the
amino acid code on one allele from V to E. The software basecaller has called an ‘‘A,’’ as this peak is slightly larger than the ‘‘T’’
visible beneath it. The control DNA on the right shows the wild-type ‘‘T’’ at the corresponding base position.
BRAFmutationinLAM1 cells. (A) Lightcycler real-timesequence-specific PCR analysis was performed using wild-type
1298 COPLAND ET AL.
lovastatin, paclitaxel, and gefitinib (Fig. 6C). In fact, when 17-
AAG is combined with either paclitaxel (17P) or gefitinib
(17G), it appears to significantly increase cell proliferation
We have shown that molecular diagnosis by thyroid-
specific ICC and tumor morphotype–specific RT-PCR and
PCR can allow unequivocal diagnosis of PTC in a situation
where conventional techniques have failed. Moreover, it al-
lowed targeted selection of possible therapies through in vitro
testing of the compounds as mono and combinatorial ther-
apies. In addition, we provide evidence that the tumor con-
tained the BRAF V600E mutation, as well as other cytogenetic
abnormalities, which may provide insights into this tumor’s
BRAF mutations have been linked to more aggressive
tumor behavior in PTC, compared to tumors with RET=PTC
rearrangements or RAS mutations. In addition, this tumor
displayed significant aneuploidy. Aneuploidy, as a marker
of genetic instability, has been linked to worse patient out-
comes in PTC (24). The underlying chromosomal changes in
this case encompass a wide range of abnormalities from
deletions to rearrangements and amplifications. While the
BRAF V600E mutation might have been the initial com-
mitted tumorigenic change, and may even have contributed
to subsequent genetic instability (25), several of the other
genomic changes observed are likely to have contributed to
the aggressive phenotype of this tumor. The monosomy 17 in
this tumor means that there is at least haplo-insufficiency of
several tumor suppressor genes that are known to be loca-
lized on chromosome 17. These include AXIN2, BRCA1, NF1,
PRKAR1A, TOC, and TP53. Of these, TP53 has been firmly
linked to progressive thyroid carcinoma (26) and PRKAR1A
is a known endocrine tumor suppressor gene. Its inactivation
underlies the chromosome 17 linked variant of Carney
complex (27). In addition, there is evidence that additional
AAG demonstrate a dose-responsive inhibition of cell proliferation at the indicated doses. The IC50 concentrations were
depsipeptide (130pM), paclitaxel (3.2nM), 17-AAG (56nM), lovastatin (1mM), and gefitinib (30mM). (B) Using IC25 values
calculated from Figure 6A, 750nM lovastatin, 10mM gefitinib, 90pM depsipeptide, and 300pM paclitaxel were combined to
identify combinations of drugs that inhibit LAM1 cell proliferation in an additive or synergistic fashion. The fractional
inhibition method was used to determine synergism or additive effects. Synergism is defined as i1,2>i1þi2 and additivity is
defined as i1,2¼i1þi2, where ‘‘i’’ is defined as the percent inhibition of compound 1 (i1) and compound 2 (i2) alone or in
combination (i1,2) (23). (C) 17-AAG (40nM) demonstrated antagonistic effects in combination with other drugs.
Different classes of drugs inhibit LAM1 cell proliferation. (A) Lovastatin, gefitinib, depsipeptide, paclitaxel, and 17-
PTC METASTASIS MOLECULAR DIAGNOSIS1299
thyroid tumor-specific tumor suppressor genes are located
on the short arm of chromosome 17 (28).
The partial trisomy of chromosome 1 [þder(1)], with the
associated unbalanced translocation between the derivative
chromosome 1 and chromosome 9 [t(1;9)(p13;p13)], also has
potential bearing on tumor behavior. A duplication of large
chromosomal regions, in this case an almost complete chro-
mosome 1, is often associated with oncogene amplification. In
addition, the chromosome 9 breakpoint region involved in the
t(1;9)(p13;p13) includes the PAX5gene. This gene codes for a
developmental transcription factor, normally restricted to ex-
pression in B-lymphocytes during their development and ma-
turation. Like many other members of the PAX gene family, its
inappropriate expression through translocation or amplifica-
tion has been linked to malignancies (29–32). Intriguingly, the
tumorigenic mechanism of aberrant PAX5 expression might
involve transcriptional repression of TP53 (33), which could be
particularly pertinent in this tumor with its monosomy 17.
Finally, chromosomal instability per se is an indicator of
increased genetic variability and potentially rapidly chan-
ging genetic make-up of evolving tumor subclones. This re-
sults in increased tumor somatic heterogeneity and increases
the likelihood that a tumor subclone will emerge that is re-
sistant to a given treatment regimen.
Small molecule kinase inhibitors may be effective in sev-
eral types of thyroid cancer. For example, EGF-R inhibitors
and antibodies have reduced cell growth in anaplastic (34)
and papillary (35) thyroid cancer cell lines. In this patient, the
adrenal metastasis (data not shown) and the cell line derived
from his mediastinal lymph node metastasis stained positive
for EGF-R (Fig. 3B). Furthermore, gefitinib (Iressa), an EGF-R
inhibitor, decreased cell proliferation in vitro (Fig. 6A) so the
patient was given a trial of gefitinib. The IC50 for gefitinib
was 30mM, compared to a median of *4mM in many tumors
(36). Furthermore, mutation of the EGF-R, not just over-
expression, may be required to inhibit tumor growth (37).
Either of these observations may account, at least in part, for
the patient’s lack of response.
Several other agents were tested in LAM1 cells including
Hsp-90 inhibitors, which decrease proliferation, increase
apoptosis, and down regulate EGF-R expression in a variety
of thyroid cancer cell lines (38). Geldanamycin (data not
shown) and 17-AAG (which is currently in clinical trials (39))
proved to be effective in vitro (Fig. 6A). Therefore, the patient
participated in a phase 2 clinical trial with 17-AAG, but
unfortunately had disease progression (unpublished data).
In fact, 17-AAG, while effective in vitro as monotherapy, was
detrimental in combination with lovastatin, paclitaxel, and
gefitinib due to antagonistic activity (Fig. 6C). These in vitro
data are provocative, but not conclusive, requiring further
testing of combinatorial therapy in multiple cell lines and in
animal models to confirm synergistic, additive, or antag-
onistic antitumor activity when used in combination.
Moreover, the patient’s lack of response to gefitinib and 17-
AAG monotherapy emphasizes the difficulty of extrapolating
favorable lab results to the clinic and suggests that combina-
torial therapy for attacking several genes, receptors, or sig-
naling pathways simultaneously may be more successful in
controlling aggressive cancers. The demonstrated genetic di-
versity of this tumor cell line (BRAF; chromosomal losses,
gains, and translocation; and general genetic instability) de-
monstrates that tumors either may not depend on a single
oncogenic signaling pathway or may not be homogeneous.
Either scenario would increase thetumor’s chance ofsurviving
any given monotherapeutic assault. Thus, combinatorial ther-
apy of gefitinib with paclitaxel, lovastatin, or depsipeptide
might be beneficial as seen with the in vitro study (Fig. 6B).
However, multiple drug therapy is not always advantageous.
For example, 17-AAG enhances the therapeutic response in
some cancers when combined with a second drug (40,41), but
antagonism with combination therapy also may occur (42).
This was verified by our 17-AAG studies in LAM1 where 17-
AAG was detrimental in combination with other drugs (Fig.
6C). Therefore, preclinical studies of drug combinations pro-
vide valuable insights into rational therapies.
In conclusion, we have shown the utility of ICC and mo-
lecular methods to more accurately identify the underlying
genetic abnormality and to better characterize metastatic le-
sions. Importantly, the establishment of a cell line enables
one to examine a patient’s tumor in the laboratory for tar-
geted therapies that demonstrate efficacy. The lack of a
clinical response to two agents used as monotherapy was
disappointing. One explanation is the possibility that cell
cultures may not truly represent the response in the primary
tumor or metastases in vivo. Another is that aggressive dis-
ease may respond better to multiple drugs simultaneously
(see Fig. 6). Nevertheless, the strategy of examining a pa-
tient’s tumor response in vitro is a step in the direction of a
truly individualized therapy.
The authors are indebted to Kendal W. Cradic, M.S., who
performed the work on BRAF genotyping and sequencing,
and Gordon W. Dewald, Ph.D., for the cytogenetic studies.
This work was funded in part from NIH grant P30CA15083
(Cancer Center Support Grant) to RCS and a grant from Ellis
and Donna Brunton to JAC.
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1302 COPLAND ET AL.