Molecular genetic study comparing follicular variant versus
classic papillary thyroid carcinomas: association of N-ras
mutation in codon 61 with follicular variantB
Julie Di Cristofaro PhDa, Myriam Marcy MDa,b, Vasily Vasko MD, PhDa,b,
Fre ´de ´ric Sebag MDc, Nicolas Fakhry MDc, David Wynford-Thomas MD, PhDd,
Catherine De Micco MD, PhDa,b,*
aFaculte ´ de Me ´decine, Institut National de la Sante ´ et de la Recherche Me ´dicale (U555), 13385 Marseille Cedex 5, France
bLaboratory of Pathology, School of Medicine, 13916 Marseille Cedex 20, France
cDepartment of General and Endocrine Surgery, University Hospital La Timone, 13005 Marseille, France
Received 20 May 2005; accepted 24 January 2006
Summary Although the follicular variant of papillary thyroid carcinoma (FVPTC) has been classified as a
papillary cancer based on nuclear features, its follicular growth pattern and potential for hematogenous
spread are more characteristic of follicular carcinoma. To gain insight into the biologic nature of FVPTC,
we compared genetic alterations characteristic of papillary and follicular thyroid carcinomas in 24
FVPTCs and 26 classic PTC (CPTCs). In FVPTCs, we observed ras mutation in 6 of 24 cases (25%),
BRAF mutation in 1 of 13 cases (7.6%), and ret rearrangement in 5 of 12 cases (41.7%). In CPTCs, we
found ras mutation in no case, BRAF mutation in 3 of 10 cases (30%), and ret rearrangement in 5 of
11 cases (45%). One FVPTC exhibited simultaneous ras mutation and ret/PTC1 rearrangement, and one
CPTC harbored simultaneous BRAF mutation and ret/PTC3 rearrangement. Based on these findings, we
concluded that ras mutation correlates with follicular differentiation of thyroid tumors whereas ret
activation is associated with papillary nuclei but not with papillary architecture. ret activation is not
exclusive of ras or BRAF mutation, whereas ras and BRAF mutations are mutually exclusive. The
implications of these results for follicular and papillary carcinogenesis are discussed.
D 2006 Elsevier Inc. All rights reserved.
International classifications divide differentiated thyroid
carcinomas into 2 categories—papillary thyroid carcinomas
(PTCs) and follicular thyroid carcinomas (FTCs)—based on
nuclear appearance and invasive propensity . Tumors
showing a combination of follicular architecture and PTC
nuclear characteristics have been classified in a subgroup
0046-8177/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
BThis work was funded by a clinical research contract from the
Marseille Public Hospital System.
* Corresponding author. Faculte ´ de Me ´decine, Institut National de la
Sante ´ et de la Recherche Me ´dicale (U555), 27 Bd Jean Moulin, 13385
Marseille Cedex 5, France.
E-mail address: email@example.com (C. De Micco).
Human Pathology (2006) 37, 824–830
called follicular variant of PTCs (FVPTCs) . However,
FVPTCs exhibit a number of differences from classic PTCs
(CPTCs). Papillary nuclei are less typical in FVPTCs than in
CPTCs and may be intermediate between follicular and
papillary cancers on fine needle aspiration [3-5]. Several
recent reports have underlined the special challenge of
distinguishing FVPTCs from encapsulated nodules that are
commonly observed either isolated or in goiters  and
present a follicular architecture and an incomplete PTC
nuclear feature. Because differential diagnosis between
adenoma and carcinoma relies solely on the appreciation
of nuclear morphology, discordance in these cases may
exceed 60% between pathologists having large experience
in the diagnosis of thyroid tumors . Cytogenetic and
comparative genomic hybridization analyses have demon-
strated that the number and profile of chromosomal
aberrations are completely different in FVPTC and CPTC
and that FVPTC is closer to FTC . Recently, gene
expression profiling differentiated conventional papillary
carcinoma from both FVPTC and FTC . From a clinical
standpoint, FVPTC resembles FTC insofar as it produces
fewer node metastases and more lung and bone metastases
than CPTC [10,11].
The patterns of ras oncogene–activating mutations are
different in follicular and papillary thyroid tumors. Muta-
tions in the first exon of K-ras have been observed in PTCs
whereas mutations in codon 61 of H-ras and N-ras have
been found in atypical and malignant follicular tumors .
Mutations in codon 61 of H-ras and N-ras have occasion-
ally been reported in PTCs, but the histologic subtypes of
these tumors have rarely been specified. Studies have found
that the ras mutation pattern of FVPTC was more similar to
that of FTC than to that of PTC, suggesting a correlation
between ras mutation and follicular differentiation of
thyroid tumors [13,14].
Oncogenic rearrangements of the ret tyrosine-kinase
receptor gene called ret/PTC are thought to be specific to
PTCs . More than 10 ret/PTCs have been described,
but ret/PTC1 and ret/PTC3 are the most frequent and
account for more than 90% of cases. The prevalence of
these rearrangements varies from 5% to more than 80%,
depending on clinical and environmental factors [16-19].
Evidence correlating ret/PTC1 with CPTC and occult PTC
and ret/PTC3 with solid and tall-cell variants have been
reported, but the correlation between ret/PTC rearrange-
ments and follicular differentiation of PTC has received
less attention and current evidence is contradictory [19-22].
Until now, the most commonly observed oncogenic event
in PTC has been the somatic activating mutation of BRAF
at residue 600 (V600E) [23-25]. This mutation occurs in
29% to 69% of PTCs, almost exclusively in classic and
tall-cell subtypes [21,26].
In this study, we compared the prevalence of ret/PTC1
and ret/PTC3 rearrangements and that of H-ras, K-ras,
N-ras, and BRAF mutations in FVPTC and CPTC to
determine whether the molecular profile of FVPTC is
closer to the follicular or to the papillary subtype of
2. Materials and methods
2.1. Patients and tumors
The study was performed on archival paraffin-embed-
ded material from 26 CPTCs and 24 FVPTCs. Ten of the
24 FVPTCs were analyzed to detect ras mutations as
described by Vasko et al . Histologic diagnoses were
reviewed by 2 pathologists with extensive experience in
thyroid diseases (V. V., C. D. M.). Cases liable to
discordant diagnosis were excluded. Based on standard
criteria, differentiated tumors showing more than 99% of
follicles without well-developed papillary formation were
classified as FVPTCs . More than 50% of cells in these
tumors exhibited characteristic PTC nuclear features: clear
overlapping nuclei, marginated chromatin, irregular shape,
grooves, and pseudoinclusions. The CPTC group included
well-differentiated tumors with a predominant papillary
architecture. The clinicopathologic features of these tumors
are listed in Table 1.
Samples of normal thyroid tissue adjacent to benign
nodules from 10 patients were used as negative controls.
The study protocol was approved by the Clinical
Research Board of the Marseille Public Hospital System’s
2.2. Nucleic acid preparation
2.2.1. DNA extraction
After dewaxing, paraffin sections were digested for
2 days at 558C with proteinase K (20 mg/mL) and heated
at 958C for 10 minutes to inactivate the enzyme. DNA was
extracted by phenol/chloroform, precipitated by ethanol,
FVPTCs and CPTCs studied
Comparative clinicopathologic features of the
(n = 24)
(n = 26)
Age (y; mean F SD)
(mm; mean F SD)
Vascular invasion [n (%)]
Cervical lymph node
metastasis [n (%)]
Distant metastasis [n (%)]
Other nodules [n (%)]
38.5 F 13
21.5 F 8.5 25.7 F 11.3 NS
43.3 F 17.3 NS
21 (87)10 (38)
15 (62)5 (19)
Abbreviation: NS, nonsignificant.
Molecular genetic study comparing FVPTCs versus CPTCs 825
dissolved in TE buffer, and quantified by fluorimetry
(BioRad Versafluor, BioRad Laboratories, Marnes La
Coquette, France). The concentration of DNA was checked
by UV spectrophotometry (OD260 nm).
2.2.2. RNA extraction and reverse transcription
After dewaxing, paraffin sections were digested overnight
at 378C using a solution of guanidine thiocyanate (1 mol/L,
Gibco, Invitrogen SARL, Cergy Pontoise, France), Sarcosyl
(0.5%, Sigma-Aldrich Chimie SARL, Lyon, France), Tris-
HCl (20 mmol/L, pH 7.5), b-mercaptoethanol (25 mmol/L,
Sigma), and proteinase K (10 mg/mL, Roche Diagnostics
Meylan, France). The liquid phase was recovered by
centrifugation at 13000 rpm and treated using 200 lL of
phenol/chloroform (70:30) and 200 lL of phenol/chloro-
form/isoamylic acid (25:24:1). RNA pellets were precipitat-
ed overnight at ?208C in 400 lL of ethanol and 20 lL of
sodium acetate 3 mol/L, pH 5.2. After centrifugation, pellets
were resuspended in 20 lL of DEPC-treated water and
stored at ?808C. The concentration of RNAwas checked by
UV spectrophotometry (OD260 nm).
Reverse transcription (RT) was carried out using an RNA
amplification kit on 7.5 lL of RNA (Applied Biosystem,
Applera France SA, Courtaboeuf, France) in a final volume
of 50 lL containing 5 mmol/L of MgCl2, buffer 1?, l U/lL
of RNase inhibitor, 1 mmol/L of each dNTP, 2.5 lmol/L of
random primers, and 2.5 U/lL of MuLV transcriptase.
Quality of cDNA was checked by amplification of a
sequence of the GAPDH gene using a forward primer
overlapping exons 2 and 3 to prevent genomic DNA
amplification (Table 4). Pending use, cDNAs were stored
2.3. Detection of BRAF mutations
DNA sequences in exons 11 and 15 of BRAF were ampli-
fied using the primer pair sets shown in Table 2. Set A was
used first; set B, which was designed to amplify a shorter re-
chain reaction (PCR) products. The same set of primers was
used for subsequent sequencing (one for each direction).
Amplification was performed using up to 200 ng of
extracted genomic DNA in a standard 50-lL PCR reaction
mixture containing 1.5 mmol/L of MgCl2, 40 pmol of each
primer, and 200 lmol/L of each dNTP together with 1.5 U
of Taq polymerase. Initial denaturation at 948C for 5
minutes was followed by 40 cycles comprising 1 minute
at 948C, 1 minute at 558C, 1 minute at 728C, and a final 10-
minute extension at 728C. After being checked by agarose
gel electrophoresis, PCR products were purified using
Qiagen spin columns (Qiagen SA, Courtaboeuf, France) to
remove unincorporated primers and then subjected to cycle
sequencing using the appropriate forward or reverse
primers. Sequencing was performed with an ABI Big Dye
kit (Applied Biosystems, Applera France, Courtaboeuf,
France) according to the manufacturer’s instructions applied
to an ABI 3100 capillary sequencer. Forward and reverse
sequences on electropherograms were analyzed visually to
minimize the risk of overlooking a mutation caused by
coexisting wild-type signal.
2.4. Detection of ras mutations
DNA sequences of H-ras, K-ras, and N-ras in exons 1
and 2 were amplified using the primer pairs listed in Table 3.
Direct sequencing was performed in an Applied Biosystem
373 XL sequencer according to the manufacturer’s instruc-
tions on PCR products purified using a Qiagen gel
extraction kit. In samples exhibiting mutations, both sense
and antisense strands were sequenced for confirmation.
2.5. Detection of ret/PTC rearrangements
The methods and primers that we used to amplify ret/
PTC1 and ret/PTC3 were similar to those used by Santoro
et al . To improve the sensitivity and specificity of
detection, we amplified ret rearrangements using nested
primers (Table 4). Polymerase chain reaction was carried out
with an RNA amplification kit (Applied Biosystem, Applera
of 25 lL of 5 mmol/L of MgCl2, buffer 1?, 4 ng/lL of each
primer, and 0.25 U/lL of Taq polymerase. Nested PCR was
performed with 2 lL of the first PCR products. Amplifica-
5minutes,amplificationby 35 cycles of denaturation at 948C
for 30 seconds, hybridization at Tm primers for 1 minute,
elongation at 728C for 1 minute, and elongation at 728C for
7 minutes. Polymerase chain reaction products were electro-
phoresed in a 2% agarose gel and blotted to a nylon
membrane (Hybond N+, Amersham Biosciences [GE
Primer pairs used to amplify BRAF DNA sequences
J. Di Cristofaro et al. 826
Healthcare], Orsay, France) by NaCl sodium citrate buffer
10? capillary transfer. Nylon membranes were hybridized
with either a radiolabeled ret probe covering the tyrosine
kinase domain or a GAPDH probe (Table 4).
RNA from TPC1 cells and RNA from a PTC harboring
ret/PTC3 were used as positive controls for ret/PTC1 and
ret/PTC3, respectively. They were kindly provided by Dr A.
Fusco (University Federico II, Naples, Italy). RNA extracted
from 10 samples of normal thyroid tissue was used as
negative control in addition to usual no-template controls.
2.6. Statistical analysis
Statistical analysis was done using v2or Fisher tests. A
P value less than .05 was considered significant.
As shown in Table 1, the clinicopathologic features that
correlated significantly with the follicular differentiation of
papillary carcinomas were encapsulation of the tumor, lower
frequency of lymph node metastasis, and more frequent
association with benign nodules or multinodular disease in
the rest of the thyroid. Nodules were found in 15 FVPTCs as
4 nodules associated with FVPTC revealed limited nuclear
features of PTCs in a varying number of cells. Such features
were not found in nodules associated with CPTC.
As shown in Table 5, ras mutations were found in 6
of the 50 papillary carcinomas analyzed (12%). H-ras
mutation GGC Y GTC at codon 12 was found in 1 of
the 26 CPTCs (3.8%) and in none of the FVPTCs
(P = nonsignificant). N-ras mutation CAA Y CGA at
codon 61 was found in 5 of the 24 FVPTCs (20.8%) and
in none of the CPTCs. The 2 groups were statistically dif-
ferent with regard to N-ras mutation at codon 61 (P b.05).
Sufficient cDNA for study of ret/PTC rearrangements
was obtained from 23 tumors and 10 normal thyroid tissue
samples. A ret/PTC rearrangement was found in 10 papillary
cancers (43.5%) and in none of the normal thyroid tissue
samples. Five ret/PTC1 rearrangements were observed in
12 FVPTCs (41.7%) as compared with 4 ret/PTC1 and
1 ret/PTC3 rearrangements in 11 CPTCs (45%). The
difference between FVPTC and CPTC was not significant
for any rearrangement. One FVPTC presented both N-ras
mutation at codon 61 and ret/PTC1 rearrangement.
Amplification and sequencing of BRAF were successful
in 23 tumors. Mutation in exon 15 at codon 600 (V600E)
was found in 1 of 13 FVPTCs (7.7%) and in 3 of 10 CPTCs
(30%) (P N .05; nonsignificant). No mutation was found in
exon 11. One case of CPTC exhibited both a ret/PTC3
rearrangement and a BRAF mutation.
Primers and probes used to detect ret/PTC rearrangements and GAPDH
Forward: 5V-AGT CAA CGG ATT TGG TCG-3V
Reverse: 5V-GCA AAT TCC ATG GCA CCG-3V
Forward: 5V-ATT GTC ATC TCG CCG TTC-3V
Reverse: 5V-CTT TCA GCA TCT TCA CGG-3V
Forward: 5V-AAG CAA ACC TGC CAG TGG-3V
Reverse: 5V-CTT TCA GCA TCT TCA CGG-3V
Forward: 5V-GCA AAG CCA GCG TTA CC-3V
Reverse: 5V-TTC GCC TTC TCC TAG AGT-3V
Forward: 5V-CCC CAG GAC TGG CTT ACC-3V
Reverse: 5V-TTC GCC TTC TCC TAG AGT-3V
5V-CCT GGT GAC CAG GCG CCC AAT-3V
ret/PTC15V-GAG GAT CCA AAG TGG GAA TTC CCT CGG-3V
ret/PTC35V-GAG GAT CCA AAG TGG GAA TTC CCT CGG-3V
ret/PTC1 nested5V-GAG GAT CCA AAG TGG GAA TTC CCT CGG-3V
ret/PTC3 nested5V-GAG GAT CCA AAG TGG GAA TTC CCT CGG-3V
Primer pairs used to amplify H-ras, K-ras, and N-ras
Molecular genetic study comparing FVPTCs versus CPTCs827
None of the molecular alterations of ras, BRAF, or ret
genes was statistically correlated with any of the clinico-
In the present study, we compared FVPTCs and CPTCs
with regard to the prevalence of the 3 most common
molecular alterations in PTC and FTC (ie, BRAF muta-
tions, ret/PTC rearrangements, and ras mutations). The
prevalence of BRAF mutations was low in CPTC but
remained in the range usually reported for this gene .
This finding may be explained by the fact that 20 of the
27 patients were younger than 40 years because the
prevalence of BRAF mutations has been shown to correlate
positively with age [26,29]. In agreement with those of
several recent studies, our results showed that BRAF
mutations mainly affected PTCs with a papillary architec-
ture and are rarely observed in FVPTCs [21,26]. This
finding confirms that BRAF mutation is specifically
correlated with the papillary phenotype of PTC.
The incidence of ret/PTC rearrangements varied from
40% to 45% in both CPTC and FVPTC and, as is consistent
with the sporadic nature of the tumors, most were of the
PTC1 type. Most studies on ret/PTC have also reported that
the incidence of rearrangements was similar in CPTC and
FVPTC and thus is independent of follicular or papillary
architecture [19,22,30]. However, the ret/PTC rearrange-
ments have been correlated with PTC nuclei, both exper-
imentally and in human tumors [27,31]. Thyroid adenomas
possessing incomplete nuclear features of PTC have been
shown to express the RET protein and to harbor ret/PTC
rearrangements, as well as tumors possessing PTC nuclei in
focal areas only [27,32,33]. In these tumors, the distribution
of ret/PTC is heterogeneous and clusters of positive and
negative cells are admixed . This suggests that ret/PTC
is a secondary event occurring within a preexisting nodule
and that another event is responsible for tumor initiation. In
our study, the significant association of FVPTC with
multinodular disease and nodules possessing incomplete
nuclear features of PTC is in accordance to these observa-
tions. In the context of heterogeneous tumors, the RT-PCR
method is not sensitive enough to detect subclones of
ret/PTC–containing cells. The method used in this study
was initially designed to detect small numbers of ret/PTC
rearrangements and has been shown to be highly sensitive
[17,28]. Specificity was ascertained based on the negativity
of normal tissue and controls and of the low rate of ret/
Our data confirm earlier results showing a similar
prevalence (20.8%) of N-ras mutation at codon 61 in
FVPTC, atypical adenoma, and follicular carcinoma [12-14].
These findings suggest a common biologic process in the
mutated subset of these follicular proliferations. Conversely,
mutation of N-ras at codon 61 was not observed in CPTC.
Based on these findings, we conclude that this mutation is
significantly correlated with the follicular differentiation of
thyroid tumors. A study indicated that N-ras mutation is an
early event in thyroid follicular carcinogenesis  and is
significantly correlated with malignant progression of
follicular tumors as observed in malignant follicular tumors
and tumors of uncertain malignant potential but not in
common adenomas . It is thus reasonable to postulate
that N-ras mutation is responsible for tumor initiation in
affected FVPTCs. However, considering that only a small
proportion of these tumors possess the N-ras mutation,
there must be another yet unknown factor with similar
repercussions in unaffected cases. In a study on follicular
proliferations by image analysis, we demonstrated that the
N-ras mutation at codon 61 was independent of nuclear
morphology . This suggests that the occurrence of
papillary nuclei within N-ras–mutated follicular prolifer-
ations is a secondary event. It could be correlated with the
expression of the wild-type RET protein that was found by
RT-PCR and immunohistochemistry in approximately 50%
of FVPTCs [27,35].
This is the first time that the 3 most common molecular
alterations of thyroid carcinomas (ie, N-ras mutation, BRAF
mutation, and ret/PTC rearrangements) have been analyzed
simultaneously in FVPTC and CPTC. As expected from
earlier data [23,25], our results confirm that N-ras and
BRAF mutations are mutually exclusive because they were
never found in the same tumor. However, in contradiction
with most studies [14,22,23,25], we found minimal over-
lapping between N-ras or BRAF mutation and ret/PTC
rearrangements. An N-ras-2 mutation was associated with a
ret/PTC1 rearrangement in one FVPTC and a BRAF
mutation was associated with a ret/PTC3 rearrangement in
one CPTC. It should be underlined that some studies
showing combined ret/PTC rearrangement and ras or BRAF
mutation have already suggested that these alterations were
not necessarily mutually exclusive [36,37]. Some experi-
mental findings also support the idea that these molecular
alterations, which exhibit a low oncogenic potential when
present individually, may act synergistically to generate
more aggressive malignant phenotypes . In this regard,
it should be noted that the most invasive tumor in this series
was the CPTC, presenting both BRAF mutation and ret/
PTC3 rearrangement. This isolated 2.5-cm encapsulated
Molecular alterations in the FVPTCs and CPTCs
Molecular eventFVPTCs CPTCs
H-ras-1 mutation [n (%)]
N-ras-2 mutation [n (%)]
K-ras-1 mutation [n (%)]
K-ras-2 mutation [n (%)]
ret/PTC1 rearrangement [n (%)]
ret/PTC3 rearrangement [n (%)]
BRAF mutation [n (%)]
J. Di Cristofaro et al.828
tumor in a 31-year-old man thoroughly invaded the capsule
as well as the lymphatic channels within the same thyroid
lobe, 23 lymph nodes, and the extranodal fat tissue. Despite
2 successive courses of radioactive iodine, several node
metastases persisted after 1 year of follow-up. We believe
that such association is not fortuitous and that the stepwise
accumulation of genetic defects could explain not only the
development of PTC within preexisting thyroid nodules 
but also the existence of aggressive tumors with complex
morphology associating features of both papillary and
Experimental testing using multiple gene transfer in vitro
and/or in vivo transgenic approaches should be developed to
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