Molecular and Cellular Endocrinology 322 (2010) 8–28

Contents lists available at ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review

Molecular prognostic markers in papillary and follicular thyroid cancer: Current

status and future directions
Daria Handkiewicz-Junak a , Agnieszka Czarniecka b , Barbara Jarzab
˛ a,∗
a
Department of Nuclear Medicine and Endocrine Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej
14, 44-100 Gliwice, Poland
b
Department of Oncologic Surgery, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 14, 44-100 Gliwice, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Gene expression profiling shows that, by gene signature, the difference between BRAF-positive and BRAF-
Received 7 July 2009 negative PTC is so distinct that BRAF-positive cancer may be regarded as a molecular subtype of papillary
Received in revised form thyroid cancer (PTC). Since much enthusiasm surrounds the BRAF-oncogene as a molecular prognostic
20 December 2009
factor, a central focus of our consideration is to weigh the current arguments for and against applying BRAF
Accepted 9 January 2010
mutation status of the tumor in clinical practice. The frequency of BRAF mutation in PTC is high—45% on
average, with values over 70–80% in some populations. This will mean that implementing BRAF mutation
Keywords:
as a factor of poor prognosis will shift many PTC patients, considered up to now as low risk ones, to the
Papillary thyroid cancer
Follicular thyroid cancer
more extensive treatment. We estimate that 31% of all PTC patients and 39% of those diagnosed with
Prognosis stage I–II disease will face the risk of overtreatment if the decision will be based on the BRAF-positivity
BRAF of their tumors. Also, the risk of undertreatment in the young patients with BRAF-negative tumors is
Molecular markers evaluated with 26%. We think that, as of now, the evidence-based support for such consequences is
still weak. Thus, there is urgent need to look for genes or gene signatures which will be helpful in the
stratification of BRAF-positive tumors to specify these with poor prognosis with higher accuracy, needed
for clinical decisions. Considering this, in the review we summarize the present status of knowledge on
other prognosis-related gene expression changes in papillary and follicular cancer and relate them to he
tumor’s biology.
© 2010 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2. Pathological and clinical basis for evaluating prognosis in papillary and follicular thyroid cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.1. Histopathological and clinical characteristics of DTCs and their influence on disease prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2. Clinicopathological staging of DTC and its limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3. Molecular mechanisms of neoplastic transformation in papillary and follicular thyroid cancer: prognosis-related aspects . . . . . . . . . . . . . . 11
2.3.1. Activation of the RAS–RAF–MEK–ERK1/2 pathway and the PI3K/AKT pathway in thyroid cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3. Prognostic impact of the inititiating molecular events in papillary and follicular thyroid cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1. The role of BRAF proto-oncogene in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1.1. BRAF mutation is the most frequent initiating molecular event in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1.2. BRAF mutations are associated with poorer prognosis of PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.2. Is it the time to apply BRAF as clinically relevant prognostic/predictive factor in PTC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.3. Other molecular initiating events specific for PTC—do they have prognostic potential? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3.1. RET/PTC rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.3.2. NTRK rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.3.3. Gene rearrangements are frequent in thyroid cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.4. Is there some multiclonality in papillary thyroid cancer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.5. Gene mutations important for development of both papillary and follicular thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.5.1. RAS mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

∗ Corresponding author. Tel.: +48 322789339; fax: +48 322789325.

˛
E-mail addresses: dhandkiewicz@io.gliwice.pl (D. Handkiewicz-Junak), aczarniecka@io.gliwice.pl (A. Czarniecka), bjarzab@io.gliwice, bjarzab@io.gliwice.pl (B. Jarzab).

0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2010.01.007
 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28 9

3.6. Follicular thyroid cancer-specific gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.6.1. PAX8/PPAR rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.6.2. RAS homolog 1 (ARH1) silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.7. Mutations of other genes and their importance for thyroid cancer and its outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.7.1. PIK3CA mutations and amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.7.2. PTEN mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.7.3. ␤-Catenin mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.7.4. TP53 mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4. Prognostic aspects of other gene expression changes in papillary and follicular thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.1. Single genes expression changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.1.1. MET proto-oncogene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.1.2. Epithelial growth factor receptor (EGFR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.1.3. Mitogen-inducible gene-6 (MIG-6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.4. Vascular epithelial growth factor (VEGF) and VEGF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.5. E-cadherin and catenins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.6. Cyclin D1 and other cell cycle regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.7. Mucin 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.8. S100A4 calcium-binding protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.9. Osteopontin and CD44v6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.10. Immunity-related genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.2. Prognosis-related gene signatures in thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.2.1. Gene expression profiling and prognosis of thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.2.2. Metastasis-associated genes in thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.2.3. Epithelial-to-mesenchymal transition in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.3. Genetic germline background and the prognosis of papillary and follicular thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5. Final considerations on prognostic impact of genetic factors in papillary and follicular thyroid cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.1. Relation between BRAF status and gene expression profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.1.1. Is BRAF-positive DTC a distinct molecular subtype? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.1.2. BRAF-mutated PTCs exhibit fewer thyroid-specific gene expression features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5.1.3. BRAF mutation is associated with the more aggressive tumor phenotype in PTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5.2. Major questions in the therapeutic strategy in DTC which can be solved by molecular diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5.3. Conclusions from the transcriptome-wide analyses of DTCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

1. Introduction sion into two molecular subtypes – luminal and basal – is slowly
gaining influence in formulating disease management strategies
Papillary thyroid cancer (PTC) and follicular thyroid cancer (Sorlie et al., 2001; van’t Veer et al., 2005).
(FTC) are both described clinically as differentiated thyroid cancers In the present review, we highlight that the molecular diag-
(DTCs) due to their tumor cells structurally and functionally resem- nosis of thyroid cancer is already possible (Eszlinger et al., 2008;
bling normal follicular cells and due to their sharing a relatively Giordano, 2008). We therefore examine whether, to improve prog-
indolent natural history and good responsiveness to surgery and nostic/predictive ability, molecular diagnosis should now supplant
radioiodine in most – but far from all – cases. DTC constitutes the or at least supplement the clinicohistopathological evaluation of
commonest endocrine malignancy, and among neoplastic diseases, DTC. This question is increasingly pertinent, as in recent years,
possesses the most favorable overall prognoses. However, over 10% both research on single, cancer-specific mutations and genome-
of patients eventually die of DTC and an even greater proportion wide DNA microarray-based approaches have added greatly to our
faces the morbidity of recurrences (Eustatia-Rutten et al., 2006; understanding of DTC biology and clinical behavior. In our discus-
Mazzaferri and Kloos, 2001; Sherman, 2003). In these respects, lit- sion of the prognostic relevance of molecular studies, we focus on
tle to nothing has improved in DTC management in the last twenty data regarding gene mutations, essential for neoplastic transforma-
years (Clark and Duh, 1991). tion of follicular cells and, then, for gene expression changes, judged
As in other cancers, numerous studies in DTC have concentrated both by RNA and protein levels. We present details of microarray-
on relatively simple clinicopathological variables, e.g., primary based comparative genome hybridisation (CGH) studies on gene
tumor size, extent of disease, pathological histotype, patient’s age amplifications/deletions and of studies on epigenomic changes
at diagnosis or sex, to formulate risk group stratification or stag- insofar as this information is germane to the current role and
ing systems. The goal has been to select patients who will do well future investigation of molecular prognostic factors in DTC. Since
without further therapy (i.e., to identify prognostic factors) or who the BRAF-oncogene (v-raf murine sarcoma viral oncogene homolog
will do well with some treatments but not others (i.e., to identify B1) is the major genetic factor in PTC (Puxeddu and Moretti, 2007;
predictive factors). Xing, 2007), an important focus of the present review is to weigh
In recent years, the impact of molecular diagnosis is becoming the current arguments for and against applying BRAF mutation sta-
even more substantial in cancer care. This trend is already evident in tus in clinical practice. As we discuss in more detail below, the data
many hematological malignancies, where the presence of particu- on the correlation of BRAF status with DTC clinicopathological fac-
lar chromosomal rearrangement influences the natural history and tors are substantial and even show an impact on survival. However,
the choice of treatment, i.e., is not only of prognostic significance from the clinical point of view, a factor present in 50% of cases is of
but also of predictive significance (Haferlach et al., 2003; Jabbour limited use in managing a disease which has a poor outcome in no
et al., 2008; Meijerink et al., 2009). A similar phenomenon is also more than 10–15% patients, thus, a more detailed stratification is
occurring with solid tumors such as breast cancer, where the divi- necessary and we look for this possibility.
10 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
Our review begins by summarizing the current understanding
of histopathological and clinical prognostic factors in DTC. Next,
we describe BRAF mutations and other molecular events occurring
early in the thyroid neoplastic transformation in the context of their
relationship to thyroid cancer prognosis. Subsequently, we discuss
the expression changes in other single genes other than BRAF that
are known to have prognostic utility in DTC. Lastly, we present the
status of relevant genome-wide analyses and provide some closing
remarks where we consider not only the prognostic value of genetic
markers but also comment briefly on their predictive contribution.
2. Pathological and clinical basis for evaluating prognosis
in papillary and follicular thyroid cancers
2.1. Histopathological and clinical characteristics of DTCs and
their influence on disease prognosis
The follicular cells of the thyroid gland give rise to a variety
of tumors that differ markedly in their morphology and biologi-
cal and clinical behavior. Based on a number of diagnostic criteria
and morphological hallmarks, these tumors may be divided into
five major types: papillary, follicular, oncocytic, poorly differenti-
ated, and undifferentiated (anaplastic), the first two of which in
turn may be subdivided into a number of variants (LiVolsi and Asa,
1994; LiVolsi and Baloch, 2004). By far the most common is PTC,
representing up to 80% of all thyroid malignancies. This prevalence
is distantly followed by that of FTC, ranging from less than 10% or
15% to not more than 25%, depending in the series, while oncocytic
histotype (either variant of PTC or FTC) constitutes not more than 2%
of thyroid cancers (Kushchayeva et al., 2008). Several histopatho-
logical features of DTC have been claimed to influence its prognosis.
In PTC, the tall cell variant has been shown to have a worse prog-
nosis than does the classical type (Ito et al., 2008). Concomitant
thyroiditis, diffuse lymphocytic infiltration, or both may favorably
influence the course of PTC as well as that of FTC (Kashima et al.,
1998; Loh et al., 1999; Modi et al., 2003).
FTC, especially if widely invasive or oncocytic, was claimed to
have a worse prognosis than does PTC (Gulcelik et al., 2007; Passler
et al., 2004). However, a recent study of more than 1000 patients
found no difference in cancer-specific survival between PTC and
FTC when one accounted for clinical prognostic factors such as age
at diagnosis, primary tumor size and the presence of extrathyroidal
invasion or of metastases (Verburg et al., 2009). In FTC, the prog-
nosis for the minimally invasive subtype is excellent (Thompson
et al., 2001). Fig. 1 compares a variety of commonly used clin-
ical prognostic factors with respect to the associated significant
increases in the risks of death or DTC recurrence that were found
in a recent large, long-term study of DTC patients conducted at our
center (Czarniecka, 2004). As seen in that figure, the presence of
distant metastases is the strongest poor prognostic factor for over-
all survival in DTC (Elisei et al., 2008; Mazzaferri and Jhiang, 1994;
Mazzaferri and Kloos, 2001). However, there are exceptions to this
rule, as distant metastases in young patients are not regarded as a
poor prognostic factor. In fact, younger patients with distant metas-
tases have a better prognosis than do older patients diagnosed with
locally invasive tumors, lymph node metastases, or both (Jarzab et
al., 2005a).
The prognostic impact of lymph-node involvement in DTC has
long been a matter of controversy, especially regarding survival.
Several studies failed to demonstrate an influence on mortality
rates (DeGroot et al., 1990; Mazzaferri and Young, 1981) while oth-
ers have identified an association between lymph node metastases
and an increased risk of cancer-related death, especially when the
involved lymph nodes are large, bilateral or located in the medi-
astinum (Czarniecka, 2004; Mazzaferri and Kloos, 2001; Podnos
et al., 2005; Tubiana et al., 1985; Zaydfudim et al., 2008). In fact,
in the past, lymph-node involvement was even considered to be
a favorable prognostic sign, until the effect of age was recog-
nized: children and young adults with PTC have both far more
frequent lymph-node involvement and a far better outcome than
do older patients. However, almost all studies unequivocally agree
that lymph-node involvement is associated with a higher risk of
locoregional recurrences (Beasley et al., 2002; Handkiewicz-Junak
et al., 2007; Harwood et al., 1978; Sakorafas et al., 2009).
2.2. Clinicopathological staging of DTC and its limitations
At least 18 staging systems for PTC, FTC (Akslen, 1993; Cady
and Rossi, 1988; DeGroot et al., 1990; Greene et al., 2002; Hay et
al., 1987, 1993; Mazzaferri and Jhiang, 1994; Pasieka et al., 1992;
Shaha et al., 1994; Sherman et al., 1998) or both, have been formu-
lated based on retrospective patient outcome evaluations (Lang et
al., 2007a,b). In Europe, the UICC/TNM system is the most widely
adopted. It considers three main factors:
• T status—primary tumor size and extrathyroidal extension,
• N status—presence or absence of lymph node metastases,
• M status—presence or absence of distant metastases.
The UICC/TNM system also considers patient age at diagnosis,
a factor not commonly used in other non-TNM-based DTC staging
systems. All patients below 45 years of age who are diagnosed with-
out distant metastases, are considered to have stage I disease, i.e.,
belong to the “low-risk” group.
The clinicopathological factors upon which the majority of
staging systems are based are summarized in Table 1. These vari-
ables frequently overlap, e.g., older or very young patients tend
to more often have lymph node or distant metastases or both;
young patients with lymph node metastases more often suffer
from distant metastases (Jarzab et al., 2005a). Such overlap is usu-
ally addressed with multivariate analysis, which can help select
independent variables while rejecting factors that are only surro-
gates for those variables. Nonetheless, even with that statistical
approach, results are sometimes contradictory, as in the evaluation
of tumor histopathology (Gulcelik et al., 2007; Verburg et al., 2009)
or of lymph node metastases (Bardet et al., 2008; McConahey et al.,
1986). In the case of histopathology, the contradictory results may
be attributable to the somewhat subjective, not fully standardized
method of specimen evaluation. The rate of disagreement during
DTC evaluation can be high, especially in FTC where it can reach up
to 50% of cases (Franc et al., 2003; Lange et al., 2006).
Table 2 provides the survival by disease stage or risk group
according to the most popular staging systems. As seen in Table 2,
the systems show marked survival differences, especially for “high-
risk” groups/advanced stages, in which, according to most systems,
more than 50% of patients eventually succumb to disease (Durante
et al., 2006; Sampson et al., 2007; Schlumberger et al., 1996). It
is however, not established which patients in less advanced sub-
groups do or do not require more intensive treatment to cure their
cancer. This results in a very wide “gray zone” of DTC prognosis,
which leads to a significant risk of over- or undertreatment in a sub-
stantial number of patients (Gulcelik et al., 2007; Tuttle et al., 2008).
The MACIS and TNM staging systems have been demonstrated to be
the most accurate prognostic systems in some (Lang et al., 2007a,b;
Passler et al., 2003) but not all studies (Brierley et al., 1997). Never-
theless, even if evaluated with the best score, they explained barely
about 20% of the observed variation in survival time (Lang et al.,
2007a,b) and only a few studies yielded better results (D’Avanzo et
al., 2004).
Exacerbating this problem, the extent of treatment (e.g., rad-
icalness of surgery or use or activity of radioiodine) that is
 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
Fig. 1. Clinical and pathological risk factors of overall and recurrence-free survival in thyroid cancer (multivariate regression analysis of 1141 DTC patients, followed-up by
5 years) (Czarniecka, 2004).
recommended in guidelines is not consistent between “high-risk”
or “low-risk” groups according to different staging systems. It is, of
course, well known that the extent of treatment can influence dis-
ease outcome (Czarniecka, 2004; Gulcelik et al., 2007; Lang et al.,
2007a,b; Passler et al., 2003). In addition, since almost all current
clinicohistopathological prognostic factors are based on postsur-
gical tumor and patient evaluation, none of these staging systems
can be applied pre-operatively, and hence help tailor the extent of
surgery.
In summary, then, the utility of the current clinicohistopatho-
logical staging systems for DTC is limited by the abundance
of systems, their frequent overlap and sometimes contradictory
results, their far from perfect match with short- or long-term prog-
nosis, and their inapplicability pre-operatively.
2.3. Molecular mechanisms of neoplastic transformation in
papillary and follicular thyroid cancer: prognosis-related aspects
From the molecular point of view, papillary and follicular thy-
roid cancers are regarded as different diseases. This opinion is
supported by the disparate molecular initiating events leading to
neoplastic transformation. Also supporting this perspective are the
differences in DNA ploidy level (PTCs are generally diploid, FTCs
aneuploid) and the differences in localization and in the number
of gains and losses seen in comparative genomic hybridisation
(CGH)-based analysis. In PTC, aneuploidy, although observed in
minority of tumors (Rodrigues et al., 2007), is significantly associ-
ated with cancer-specific death (Sturgis et al., 1999). The profound
differences in gene expression will be described below. On the
other hand, many subsequent down-stream molecular mecha-
nisms are very similar between PTC and FTC, which explains the
11
commonalities in the clinical course of both cancers and cre-
ates a rationale for similar therapeutic approaches to both of
them.
2.3.1. Activation of the RAS–RAF–MEK–ERK1/2 pathway and the
PI3K/AKT pathway in thyroid cancers
DTC harbors several highly prevalent genetic alterations, some
of which are seen only in this cancer or are characteristic of one of its
histotypes. In PTC, the most important oncogenic mechanism, seen
in about 80% of tumors, is the activation of the mitogen-activated
protein kinase (MAPK) pathway (Fagin, 2004; Fagin and Mitsiades,
2008), which once constitutively activated, leads to tumorigene-
sis (Hilger et al., 2002; Peyssonnaux and Eychene, 2001). Three
most important initiating events, RET/PTC (rearranged during trans-
fection/papillary thyroid cancer), RAS (resistance to audiogenic
seizures) and BRAF mutation, are regarded as mutually exclusive,
alternative triggers for the activation of the pathway (Fagin, 2004).
The mutual exclusivity between BRAF and the RAS mutation is char-
acteristic not only of thyroid cancer but also of several other human
cancers (Xing, 2005). The activating BRAF mutation has only been
recognized relatively recently (Davies et al., 2002), but in fact is the
most frequent trigger in PTC.
A single oncogenic alteration along the receptor tyrosine
kinase–RAS–RAF–MEK–ERK1/2 pathway is likely sufficient to drive
thyroid cell neoplastic transformation, although further supportive
molecular events are necessary. BRAF mutation and RET/PTC (and
NTRK1—neurotrophic tyrosine kinase, receptor, type 1) rearrange-
ments differ to some extent in their effects on the shared oncogenic
pathway, respectively, resulting more frequently in the classic vari-
ant or the solid variant of PTC, while RAS mutations and RASSF1A
(Ras association (RalGDS/AF-6) domain family member 1) methy-
12 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28

Table 1
Clinical prognostic factors used in different staging systems for DTC.

Factor Positive prognostic indicator Negative prognostic indicator

Age Younger (usually below 40–45 years of age) Older

Primary tumor—diameter Small (the best prognosis for tumors ≤1 cm) Large
Primary tumor—extension beyond thyroid capsule No Yes
Lymph node metastasis Absent Present
Distant metastases Absent Present
Surgical resection Total thyroidectomy and appropriate extent of Incomplete removal of cancer tissues and/or
lymphadenectomy ensuring complete removal lack of total thyroidectomy and of appropriate
of cancer tissues extent of lymphadenectomy

Table 2
Survival by disease stage or risk group in selected DTC staging systems.

Staging system DTC type (number of patients) Primary outcome end-point Disease stage or risk group

TNM stage (Greene et al., 2002) PTC (6590) 5-Year survival I II 93% III IV
97% 84% 39%
FTC (1129) 5-Year survival 95% 90% 69% 41%

MACIS (Hay et al., 1993) PTC (1779) 20-Cause-specific survival rates I II 89% III IV
99% 56% 24%
Ohio State University Staging PTC and FTC (1355) Cancer-related deaths I II 6% III IV
system (Mazzaferri and 0% 15% 65%
Jhiang, 1994)
AMES (Cady and Rossi, 1988) PTC and FTC (821) Cancer-related deaths Low High
1.8% 46%
NTCTCS system (Sherman et PTC and FTC (1607) 5-Year CSS I II 100% III IV
al., 1998) 99.8% 91.9% 48.9%
University of Chicago (DeGroot PTC (269) 10-Year CSS I II 100% III IV
et al., 1990) 100% 87% 35%
GAMES Risk Groups (Shaha et PTC and FTC (1038) 10-Year survival Low Intermediate85% High
al., 1994) 99% 57%
AGES (Hay et al., 1987) PTC (860) 25-Year CSS Low High
98% 65%
DAMES (Pasieka et al., 1992) PTC (74) Cancer-related death or recurrence Low Intermediate45% High
8% 100%
SAG (Akslen, 1993) PTC (173) 10-Year CSS I II 88% III
98.3% 39%

CSS, cancer-specific survival; DTC, differentiated thyroid cancer; FTC, follicular thyroid cancer; PTC, papillary thyroid cancer.

lation are more prone to induce the follicular variant of PTC (Xing,
2005).
MEK–ERK1/2 pathway activation also plays a significant role
in FTC tumorigenesis (Liu et al., 2008). However, in FTC, neither
RET/PTC nor the BRAF mutation can be found, while RAS mutations
are quite frequent, seen not only in follicular cancers, but also in
follicular adenomas (FA). This pattern is repeated in the case of
PAX8/PPAR rearrangements, which however do not directly lead
to MEK–ERK activation and cause different down-stream effects
(Giordano et al., 2006).
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway also
plays a fundamental role in the regulation both of normal cell
growth, mitosis and survival and, when disarranged, of tumorige-
nesis. PI3K/AKT alterations are found in every histotype of thyroid
cancer, frequently in FTC and, even more distinctly, in ATC. In FTC,
phosphorylation of AKT, the key player in this pathway, was far
more frequent than that of ERK (MAPK kinase effector) (Liu et al.,
2008).
3. Prognostic impact of the inititiating molecular events in
papillary and follicular thyroid cancers
3.1. The role of BRAF proto-oncogene in PTC
BRAF is a serine–threonine kinase, expressed in a tissue-specific
manner and abundant in thyroid follicular cells, which among the
three known RAF kinases, most potently activates the MAPK path-
way (Peyssonnaux and Eychene, 2001) (for reviews see Puxeddu
and Moretti, 2007; Xing, 2005). Activating BRAF mutations are not
thyroid cancer-specific. To the contrary, BRAF belongs to the com-
monest oncogenes, mutated in 7–9% of all malignant solid tumors,
with the highest prevalence (about 60%) in melanomas (DeLuca
et al., 2008), and a frequent presence in ovarian, colorectal, and
lung cancers and even some low-grade astrocytomas (Pfister et al.,
2008). Interestingly, although the activation of the MAPK signaling
pathway is regarded as frequent in human cancer, the evidence for
this is most compelling in thyroid cancer and melanoma (Johansson
et al., 2007; Knauf and Fagin, 2009).
3.1.1. BRAF mutation is the most frequent initiating molecular
event in PTC
After melanoma, PTC is the second human malignancy where
BRAF mutations are especially frequent, found in 29–87% (with a
mean frequency of about 45%) of cases (Kim et al., 2009; Kimura
et al., 2003; Puxeddu et al., 2004; Xing, 2007). BRAF mutations are
not found exclusively in PTC but also, albeit less often (not more
than 20–26% of cases, mainly those originating from PTC) in ATC
(Nikiforova et al., 2003a; Smallridge et al., 2009; Takano et al.,
2007).
Although more than 40 mutations have been identified in the
BRAF gene, the most significant hot spot mutation, accounting for
over 90% of all BRAF mutations, is a thymidine to adenine transver-
sion at nucleotide 1799 (T1799A) in exon 15. This substitution
leads to a valine-to-glutamate transversion at residue 600 near
the catalytic center of the protein (BRAFV600E) and is believed to
produce a constitutively active kinase by disrupting hydrophobic
interactions between residues in the activation loop and residues
in the adenosine triphosphate (ATP) binding site (Wan et al., 2004).
 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
BRAFV600E potentiates the catalytic activity of the enzyme by more
than 500 times (Kim et al., 2005).
BRAF mutation is an early event in PTC tumorigenesis, which
can be found even in minute PTCs, less than 4 mm in diameter
(Ugolini et al., 2007). The constitutively active kinase subsequently
leads to tumorigenesis through aberrant activation of the MAPK
pathway (Davies et al., 2002; Garnett and Marais, 2004). Such acti-
vation includes hyperphosphorylation of retinoblastoma protein
(RB), which releases inhibition of E2F-dependent transcription fac-
tors, allowing the cell to pass from G1 into S phase, increasing
growth and promoting survival (for a review of this process see
Knauf and Fagin, 2009). At the same time, apoptosis is inhibited,
probably due to the action of NF␬B transcription factor (Palona
et al., 2006). Transgenic mouse studies clearly demonstrated the
driving force of the BRAF mutation in promoting malignant trans-
formation of thyrocytes and extrathyroidal invasion of PTC (Knauf
et al., 2005). One important molecular mechanism involved in this
process is BRAF mutation-promoted methylation and hence silenc-
ing of the tissue inhibitor of metalloproteinase-3 (TIMP-3; Hu et al.,
2004) and resultant overexpression of metalloproteinases (Melillo
et al., 2005; Mesa et al., 2006; Palona et al., 2006).
The BRAF-oncogene induces chromosomal instability
(Mitsutake et al., 2005), and is it not only an initiator of PTC
but is necessary for the maintenance of PTC proliferation and
tumorigenicity (Liu et al., 2007b).
3.1.2. BRAF mutations are associated with poorer prognosis of
PTC
Based on molecular results, numerous studies have investigated
the clinical significance of BRAF mutation in PTC. The general ten-
dency of the studies to show the association of this mutation with
factors related to poor prognosis is rather convincing (Table 3 ).
However, a careful overview of the literature indicates that the clin-
ical data on the link between BRAF mutation and DTC outcome are
not very consistent. The first data were summed up in a large meta-
analysis of 1168 patients and showed that BRAF mutation correlated
with histologic subtype, presence of extrathyroidal extension, and
advanced clinical stages, but not with age, sex, race, or tumor size of
PTC patients (Lee et al., 2007). More recent studies addressed even
more carefully the correlation between BRAF status and PTC clini-
copathological factors: two such studies (Frasca et al., 2008; Wang
et al., 2008a), which altogether included more than 400 patients,
found in multivariate analysis a positive correlation between BRAF
mutation and extrathyroidal extension, while two other such stud-
ies totaling about twice as many patients did not (Kebebew et al.,
2007; Lupi et al., 2007). In the analysis of Lupi et al., encompassing
500 consecutive PTC patients at the University of Pisa, the one-way
univariate correlations of BRAF mutation positivity with extrathy-
roidal extension and tumor stage-related data were very distinct.
However, the multivariate analysis discarded all the factors except
lack of a tumor capsule (whether the tumor was single or multifo-
cal) (P = 0.0005 in multivariate analysis). In the Lupi study, lack of
encapsulation remained statistically significant in separate multi-
variate analyses for micro-PTC, larger tumors and follicular variant
PTC but not for classical variant PTC. Presence of tumor capsule,
although relatively rare in PTC (present in 21.7% of the Lupi series),
is a strong predictor of an especially good disease-free prognosis,
seen very distinctly in follicular variant PTC (Kakudo et al., 2004).
Summing up the analysis of the relationship of BRAF muta-
tions with clinicopathological factors, there is only one factor
shown by the majority of studies to correlate with BRAF
mutations—extrathyroidal tumor extension, which is however
questioned if tumor encapsulation is also included in the analy-
sis. For one perusing these studies, questions arise on the origins of
the discrepancies between the study results. Many studies on the
correlation of BRAF with clinicopathological data (Costa et al., 2008;
13
Elisei et al., 2008; Guan et al., 2009; Kebebew et al., 2007; Lee et al.,
2009; Rosenbaum et al., 2005) were based on univariate analyses
that could not distinguish between overlapping significant factors.
In that case what seems to be BRAF related in univariate analysis can
result from other unknown factors affecting studied outcome (e.g.,
disease stage or recurrences) and be correlated with BRAF status.
For example, BRAF mutations are relatively rare in PTC cases with
concommitant autoimmune thyroiditis (Nikiforova et al., 2002),
and here the immune response may influence the clinical course
more than do the molecular consequences of initiating mutation
itself. Although the multivariate analysis may be somewhat help-
ful in making the distinction between diverse confounding factors,
it may only solve some but not all the problems, as only a limited
number of factors can be included and other, theoretically deci-
sive factors in the outcome of the disease can be unintentionally
omitted. Nevertheless at present, multivariate analysis seems to be
the best way to obtain the least biased results. A good example is
the already discussed study by Lupi et al., where BRAF mutation
positively correlated with extrathyroidal extension in univariate
analysis, but was abolished by the status of thyroid capsule in mul-
tivariate approach.
A second issue in interpreting these studies is how precisely
clinicopathological factors are defined. For example, both mini-
mal invasion beyond the thyroid capsule and gross invasion of
muscles and other neck tissue can be regarded as extrathyroidal
disease extension, but their clinical significance differs substan-
tially: tumors with gross invasion into surrounding tissues have a
worse prognosis and a higher risk for recurrence. Differences in the
methodology of BRAF detection can also cause conflicting results
among studies.
Surprisingly, distant metastases, the most significant factor of
poor PTC outcome, were not associated with BRAFV600E mutation
as a single factor. However, when that mutation was associated
with other genetic events, the conjunction of events appeared as
an independent predictor of distant metastasis (Costa et al., 2008).
A more definitive assessment of the impact of BRAF status on
PTC prognosis can be derived solely from studies evaluating the
long-term outcome of the disease. Only recently have reports on
disease-free survival or overall survival in BRAF-positive versus
BRAF-negative PTC patients been published (Table 4). Ito et al.
(2009) evaluated disease-free survival and distant metastases-free
survival with respect to BRAF mutation status in more than 600
patients followed for a mean 83 ± 35 months. The BRAFV600E muta-
tion was detected in 38% of their series, but these investigators
did not search for other far less frequent BRAF point mutations or
rearrangements. The only significant difference among patient sub-
groups was the frequency of BRAF mutations in micro-PTC (28%)
versus tumors larger than 1 cm (41%) (P = 0.0175), however, BRAF
mutation prevalence did not successively increase with size in
patients having tumors larger than 1 cm. In multivariate analysis,
BRAF mutation was not linked to patient gender, disease stage, mas-
sive extrathyroidal extension, or lymph node/distant metastases at
diagnosis. Importantly, the authors also did not find any difference
in 5- or 10-year disease-free survival or metastases-free survival,
which were about 90% in both BRAF-positive or BRAF-negative
cases. This observation confirmed the findings of a previous mul-
ticenter Italian report involving PTC of all types (Fugazzola et al.,
2004) and a Korean study (Kim et al., 2006b) involving the clas-
sic PTC variant. Contrary conclusions, supporting the correlation of
BRAF status with disease-free survival, were reported by Kebebew
et al. (2007) (mean follow-up of 6 years) who in multivariate
analysis, noted a significant association of BRAF mutation with
recurrence risk.
The recent study by Elisei et al. (2008), who analyzed 102
patients with a median follow-up of >10 years, is the only published
study until now to address the relationship of BRAF mutation status
 14
Table 3
Correlation of BRAF mutation and clinicopathological prognostic factors in papillary thyroid carcinoma: data from recent studies.

D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28

Author Number of Type of statistical Number of Tumor size Gender Age Extrathyroidal Tumor Lymph node Distant Cancer stage
patients analysis BRAF-positive cases extension capsule metastases metastases
(%)
Rosenbaum et al. (2005) 85 Univariate 54 (64%) – – 0.0001 – – – – –

Kim et al. (2006a) 103 Univariate 34 (33%) 0.0001 ns 0.01 – – 0.0001 – 0.0001
Multivariate ns – ns – – 0.0001 – ns

Kim et al. (2006b) 203 Univariate 149 (73%) 0.006 0.006 ns 0.06 – ns – ns
Multivariate 0.03 0.04 0.005 (gross invasion) – 0.02 – –

Kebebew et al. (2007) 274 Univariate 133 (49%) ns ns 0.03 ns ns ns ns 0.04

Lupi et al. (2007) 500 Univariate 217 (44%) – ns ns 0.0001 <0.0001 0.0009 – 0.00001
Multivariate – – – ns 0.0005 ns – ns

Lee et al. (2007) (meta-analysis) 1168 Univariate 570 (49%) ns ns ns 0.000 – ns – 0.000
Costa et al. (2008) 49 Univariate 27 (55%) – – ns ns – ns ns ns
Elisei et al. (2008) 102 Univariate 38 (37%) ns ns 0.02 ns – ns ns 0.03

Wang et al. (2008a,b) 108 Univariate 54 (50%) ns – 0.02 0.02 – ns ns 0.04

Multivariate – – – 0.005 – – – –

Frasca et al. (2008) 323 Univariate 125 (39%) 0.005 – – 0.0001 – 0.0001 – 0.05
Multivariate – – – 0.0003 – 0.007 – ns

Lee et al. (2009) 64 Univariate 24 (38%) ns ns ns 0.001 – 0.003 – 0.001 (only T status)

Ito et al. (2009) 631 Univariate 242 (38%) – ns 0.05 ns – 0.005 ns ns

Multivariate ns (for tumors >1 cm) 0.033 (for ≥ 55 years) – – 0.0001 (for N1b) – –

Guan et al. (2009) 1032 Univariate 639 (62%) – ns ns 0.003 – 0.005 – 0.001
ns, not significant.
 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
Table 4
Association of BRAF with disease-free and overall survival.
Author Number of patients
Kim et al. (2006a,b) 203
Fugazzola et al. (2004) 260
Kebebew et al. (2007) 274
Abubaker et al. (2008) 536
Elisei et al. (2008) 102
Ito et al. (2009) 631
Costa et al. (2008) 202
with overall survival. A higher rate of persistent disease, mortality,
or both in was reported in BRAF-positive PTC patients (P = 0.005).
Multivariate logistic regression, that also included well-accepted
clinicopathological factors, found significant correlation with nega-
tive outcomes only for BRAF status. Log-rank analysis confirmed the
negative prognostic impact of BRAF mutation on overall survival,
with deaths occurring practically exclusively in patients harbor-
ing BRAF-positive tumors. It must be stressed that patients in the
Elisei study were followed substantially longer than were patients
in studies negative for prognostic significance of BRAF mutation
(Fugazzola et al., 2004; Ito et al., 2009; Kim et al., 2006b). However,
genetic background differences also may explain the discrepant
results of the studies.
A recent study on the molecular biology of recurrent PTC
(Henderson et al., 2009) is far less convincing than the Elisei et
al. study (Elisei et al., 2008). The authors report that the BRAF
mutation was found in 77.8% of recurrences, which far exceeds
the frequency seen historically in primary PTCs. However, in the
majority of patients, these investigators did not compare the BRAF
status in the recurrent tumors versus in the primary tumors.
Additionally, they analyzed only lymph node recurrences, and
the lower limit of their range of times-to-recurrence started at
4 months, an interval short enough to suggest that the disease
might be attributable to primary treatment failure rather than true
recurrence.
3.2. Is it the time to apply BRAF as clinically relevant
prognostic/predictive factor in PTC?
The question, whether we are ready to implement the BRAF-
oncogene as a molecular prognostic factor in PTC is answered
positively by the majority of recent publications. However, the
authors of this review would like to adopt a more cautious position
and indicate what we believe is still necessary before BRAF might
be used as a clinically relevant factor of poor prognosis in DTC. Let
us list our major doubts:
1. As stated earlier, the frequency of BRAF mutation in PTC is
high—45% on average, with values over 70–80% in some popula-
tions. This high prevalence will mean that implementing BRAF
mutation as a factor of poor prognosis will shift many DTC
patients, considered up to now as low risk patients, in the group
of patients who require more extensive therapy (Table 5). Our
evaluation indicates that as many as 31% of all PTC patients
and 39% of those diagnosed with stage I–II disease, will face
the risk of overtreatment if the decision will be based on the
BRAF-positivity of their tumors, among them at least of (1/4) of
patients with micro-PTC, who constitute up to 44% of patients
with tumor ≤2 cm (Bonnet et al., 2009) and who were consid-
ered until now as having an excellent prognosis. We think that,
as of now, the evidence-based support for such consequences
is very weak and the risk of overtreatment is significant. As an
example, a 32-year-old pregnant woman (10th week of preg-
nancy) with an incidentally detected hypoechoic nodule 8 mm
15
Disease-free survival (decrease) Overall survival (decrease)
ns –
ns –
0.03 by multivariate logistic regression analysis –
0.01 by Kaplan-Meier analysis –
0.03 by multivariate logistic regression analysis 0.015 by log-rank analysis
ns –
ns –
in diameter was referred to our department because of cyto-
logic diagnosis of PTC. No enlarged lymph nodes were palpated
or seen by sonography. The recommendation based on current
guidelines (Cooper et al., 2009) would be to monitor carefully
and, if no signs of distinct progression are observed, to perform
thyroidectomy after delivery. However, for better evaluation,
BRAF mutation was sought and confirmed to be present in the
cytologic material obtained by fine needle aspiration biopsy. Due
to the presence of this molecular factor, the consulting surgeon
decided to perform surgery in the second trimester. In our opin-
ion, the risk of overtreatment based on molecular diagnosis in
this case was significant, since at least 20% of women of this age
presenting with PTC would bear BRAF-positive tumors, while the
clinical data clearly show that the prognosis is excellent in these
cases.
On the other hand, the risk of undertreatment in the young
BRAF-negative patients should be considered.
2. The relevance of BRAF status to the indications for radioio-
dine treatment seems still not well explained. On the one side,
there are plenty of data showing that BRAF mutation corelates
with a poor response to radioiodine due to a lack of proper
membrane sodium–iodine symporter expression (see further
discussion below). On the other side, there are proposals to
apply more radioiodine in BRAF-positive patients due to the
poorer prognosis. What we lack is evidence that more radioio-
dine will be helpful in these patients. Approaches to increase
tumor radiosensitivity or to apply complementary pharmaco-
logical therapy in these patients would appear to be more
reasonable.
For the evaluation of the prognostic significance of BRAF in thy-
roid cancer, a comparison with one of the most aggressive cancers,
melanoma, may be of value. As already mentioned, BRAF mutations
are frequent in both cancers and are found at early disease stage—in
thyroid in micro-PTC and in skin, in the majority of benign nevi, the
precursors of melanomas. In benign nevi, the clonal activation of
MAPK by BRAF is followed by induction of senescence which pro-
vides a barrier against tumor progression (Wajapeyee et al., 2008).
Similarly, the expression of mutated BRAF in lung epithelium results
in development of benign tumors that express senescence markers
and only rarely progress to malignant tumors unless functional p53
or p16 are absent (Dankort et al., 2007). BRAF-induced senescence
has not been analyzed in thyroid tissues and can difficult due to lack
of benign counterpart to PTC. However, this hypothesis may explain
why BRAF is surprisingly frequent in indolent micro-PTCs—only the
next molecular event(s) is/are necessary to accelerate its growth to
clinically evident disease. In this case, we should focus on identi-
fying events, secondary to BRAF mutation, which may be of better
prognostic/predictive value then BRAF itself. According to Knauf
and Fagin (2009), in PTC, mechanisms for overcoming senescence
probably do not require loss of functional p53, which occurs late in
progression to poorly differentiated thyroid carcinoma, nor rise in
p16.
16 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28

Although molecular changes observed in other cancer cannot

General evaluation
be directly translated to PTC, we should also take into account
that in other types of cancer (e.g., colorectal, ovarian, lung) BRAF

Considerable

Acceptable

Acceptable

Acceptable
mutation is not related to poorer prognosis, on the contrary, it is
of the risk observed in cases with better outcome. In fact, the generally more
favorable outcome of BRAF-induced cancers may be the cause of
the generally good prognosis in PTC. This stresses once more the
need to search for molecular markers which shall stratify BRAF-
=A × (1 − B) × (1 − C)

positive PTC cases and help to specify patients with truly poor
In all PTC patients

prognosis.

3.3. Other molecular initiating events specific for PTC—do they

3%

3%
1%

3%

2%

9%
have prognostic potential?
Risk of undertreatment

3.3.1. RET/PTC rearrangements

=(1 − B) × (1 − C)

26%

6%
9%

5%

13%
In the subgroup

RET is a transmembrane tyrosine kinase receptor, the consitu-

tive activation of which by point mutation in parafollicular C cells
leads to medullary thyroid cancer. RET/PTC rearrangements, where
the 3 or tyrosine kinase domain of the RET gene is fused with the
5 domain of one of several constitutively expressed genes, cause
the constitutive activation of the RET gene, which otherwise is nor-
General evaluation

mally silent in follicular cells (Santoro et al., 2004). The fusion leaves
Theoretical consideration of the risk of over- and undertreatment if treatment of PTC is to be tailored according to the BRAF mutation status of the tumor.

intact the tyrosine domain (TK) of the RET receptor and enables
Not evaluable
Considerable

Considerable
d
Acceptable

Acceptable

the RET/PTC oncoprotein to activate the MAPK cascade (Knauf et

of the risk

al., 2003b). Several studies suggest that the oncogenic effects of

RET/PTC rearrangements require signaling along the MAPK path-
way in the presence of functional BRAF kinase (Knauf et al., 2003a;
Mitsutake et al., 2006).
In all PTC patients

RET/PTC rearrangements may arise from fusion with a wide

range of house-keeping genes (until now, 12 affected genes and
Therapeutic decisions based on

=A × C × B

circa 15 rearrangements have been described), and constitute an

event specific both for organ (thyroid) and histotype (PTC) (Fagin
1%

20%
4%

21%

10%

32%
the tumor BRAF status

and Mitsiades, 2008; Santoro et al., 2004). However, some data indi-
Risk of overtreatment

cate the possibility of RET/PTC rearrangements also in lymphocytic

In the subgroup

thyroiditis (Rhoden et al., 2006).

Transfection of the RET/PTC1 gene in normal rat thyroid cells
resulted in loss of differentiation and of TSH growth dependency.
=C × B

However, the cells were totally transformed only after transfection

9%

39%
37%

39%

39%

with RET/PTC and mutated RAS genes, suggesting that simultaneous

activation of several genes was necessary for tumor progression.
5 Years DFSa

This observation, together with the frequent occurrence of RET/PTC

CSS, cancer-specific survival; DFS, disease-free survival; PTC, papillary thyroid cancer.

rearrangements in papillary microcarcinoma, suggests that such

70%

90%
85%

70%

rearrangements are an early event in thyroid carcinogenesis (Jhiang

∼90%

–
(C)

et al., 1996; Viglietto et al., 1995).

A wide range of RET/PTC prevalence in PTC, ranging from 3% to
5 Years CSSa

85% has been reported (Kondo et al., 2006; Nikiforov, 2002; Tallini
and Asa, 2001). In general, RET/PTC prevalence is higher in young
100%

100%
98%

99%

95%
70%

patients and in radiation-induced PTC, and decreases with patients’

Data extracted from the meta-analysis of Lee et al. (2007).

age (Collins et al., 2002; Fenton et al., 2000). However, apart from
study population variations, the wide range of RET/PTC prevalence
Younger than 20 years, usually in >15 years patients.
13%

43%

56%

also reflects the different procedures used to identify RET/PTC rear-

% of patients with
BRAF mutationsb

rangements (Zhu et al., 2006). RET rearrangement is frequently

Because of poorer prognosis in this subgroup.

found in micro-PTC, an observation that is usually interpreted as

Data estimated from Jonklaas et al. (2006).

the evidence in favor of an initiating role in PTC. However, doubts

about this interpretation have been raised by the recent reports on
(B)

RET/PTC multiclonality (Unger et al., 2004), which may suggest that

Young patients (≤20 years of age)

at least in some PTCs, the recombinations may have arisen later in

% of patients

tumor progression.
with PTCa

The majority of RET/PTC studies did not find any correlation

between the rearrangements and other clinicopathological factors
10%

40%
20%

53%

26%
4%
(A)

related to PTC course or outcome. In few of the studies, though,

Adult patients

an increased frequency of lymph node metastases was noticed in

All stages

patients with RET/PTC rearrangements (Adeniran et al., 2006). Ini-

tially, this observation was interpreted as evidence for correlation
I + IIc

I + II
Table 5

Stage

IV
III

with poorer prognosis. Later on, when the more frequent occur-
II
I

rence of RET/PTC in younger of PTC patients has been known, the

 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
higher occurrence of lymph node metastases was correlated with
younger age rather than with RET/PTC rearrangements (Collins et
al., 2002).
3.3.2. NTRK rearrangements
NTRK1 is another membrane tyrosine kinase receptor which reg-
ulates growth, differentiation and apoptosis in the peripheral and
central nervous system, with nerve growth factor acting as its lig-
and. Similarly to RET, NTRK-3 is activated by rearrangements with
genes constitutively expressed in thyroid follicular cells. Activation
of the oncoproteins TRK1-3 is ensured by autophosphorylation of
the tyrosine kinase domain (Pierotti et al., 1996). NTRK rearrange-
ments are rare and are found in <10% of PTCs (Brzezianska et al.,
2007; Roque et al., 2001). Their rarity precludes the studies on their
prognostic roles.
3.3.3. Gene rearrangements are frequent in thyroid cancers
Until recently, gene rearrangements were regarded as charac-
teristic of hematological malignancies. Among solid tumors, they
were found only in sarcomas and thyroid carcinomas. This picture
is now being changed by the detection of very small chromoso-
mal deletions/amplifications in other solid tumors, e.g., prostate
cancer (Carver et al., 2009; Tomlins et al., 2009). Nevertheless,
predisposition of thyroid cells to undergo gene rearrangement is
not completely understood (Teixeira, 2006; Wang et al., 2008b).
The juxtaposing of the loci participating in RET/PTC and NTRK1
rearrangements in interphase nuclei of thyroid follicular cells has
been reported (Gandhi et al., 2006; Nikiforova et al., 2000; Roccato
et al., 2005). Interestingly, these rearrangements are more fre-
quently found in young patients (Collins et al., 2002; Fenton et
al., 2000; Wang et al., 2008a). Of note, though, the decisive fac-
tor(s) explaining the good prognosis of PTC in young patients,
remains unclear: is it the biology of the tumor (which is caused
by a gene rearrangement, not BRAF mutation) or of the host
(different immunity in young patients) or of both (Jarzab et al.,
2005a).
3.4. Is there some multiclonality in papillary thyroid cancer?
Many data indicate that, despite the general rule of non-overlap
between the main PTC-inducing mutations, which are regarded as
mutually exclusive (Fagin, 2004), there is a substantial degree of
multiclonality in this type of thyroid cancer. Both BRAF mutations
and RET/PTC rearrangements can be found as non-clonal changes
and different types of RET/PTC rearrangement may be found in dif-
ferent tumor foci from the same patient (Aherne et al., 2008; Oler et
al., 2005; Sugg et al., 1998; Unger et al., 2004; Vasko et al., 2005; Zhu
et al., 2006). Concomitant presence of BRAF mutation and RET/PTC
rearrangement in one tumor is possible and this is also true for
other mutations (Nikiforova et al., 2004; Smyth et al., 2005; Wang
et al., 2008b). This fact is of a great potential impact on the power
of these molecular changes as prognostic or predictive factors.
3.5. Gene mutations important for development of both papillary
and follicular thyroid cancer
3.5.1. RAS mutations
The RAS–RAF–MEK–ERK1/2 pathway is hyperactivated in 30%
of human cancers (Balmanno and Cook, 2009). RAS is a membrane-
associated, small protein with guanosine triphosphate (GTP)
binding ability, involved in proliferation, differentiation and cell
survival. Translocation of RAS to the cytoplasmatic membrane is
an important step in the protein’s activation. Farnesylation of RAS
is the first obligatory step in a series of post-translational modifica-
tions leading to membrane association, which, in turn, determines
the switch from an inactive to an active form. RAS is activated by
17
a variety of membrane receptors, mainly belonging to the tyrosine
kinase receptor family, rarely G proteins. Non-membrane coupled
tyrosine kinases can also to activate RAS, with the down-stream
activation of different effector pathways.
The RAS protein is encoded by three genes, H-, N- and K-RAS
and each of them may underlie activating point mutations, occur-
ring most frequently at codon 12 or 61. Such mutations lead to the
change in conformation of the protein so that it remains bound
to GTP, i.e., constitutively active (while in normal conditions GTP-
ase activity of RAS leads to RAS deactivation). In some cancers, like
colon cancer, RAS mutation is regarded as the first oncogenic event
necessary but not sufficient for the cell to cross over the benign ade-
noma/cancer barrier (Takayama et al., 2006) as RAS mutations are
found with the same frequency in colonic adenomas as in colonic
cancers. In some other types of cancer RAS mutations occur at later
stages of carcinogenesis.
In thyroid gland, RAS mutations are found already in follicu-
lar thyroid adenomas (FAs) and the transition from the follicular
adenoma to follicular cancer requires the next molecular step
(although, unlike in colon cancer, adenomas are not regarded as
precancerous stage for FTC). RAS mutations were described in some
19% of FAs and in up to about 50% of FTC (Esapa et al., 1999; Shi et
al., 1991; Suarez et al., 1990), however, in more recent studies RAS
mutations are evaluated with an overall frequency of about 20%
(Garcia-Rostan et al., 2003; Vasko et al., 2003).
Importantly, RAS mutations are not thyroid cancer histotype-
specific and are also found in PTC, with the prevalence ranging from
0% to 15% (Abrosimov et al., 2007b; Adeniran et al., 2006; Hou et al.,
2007; Vasko et al., 2003). These mutations are rarest in the classical
variant of PTC, and more frequent in the follicular variant, where
their prevalence may reach even 20–50% (Goutas et al., 2008; Zhu
et al., 2003). Some studies indicate that N2-RAS mutations are par-
ticularly frequent (Abubaker et al., 2008; Hou et al., 2007; Wang
et al., 2007), other focus on K-RAS (Goutas et al., 2008) which are
seen in FTC with lower frequency (Fagin and Mitsiades, 2008). Their
frequency in anaplastic thyroid cancer is estimated as to be about
20–25% (Smallridge et al., 2009).
RAS mutation can promote thyroid tumorigenesis through
the RAF–MEK–ERK1/2 pathway or through its interaction with
PI3K/AKT pathway (Xing, 2005). Garcia-Rostan et al. (2003)
reported the association of RAS mutations with aggressive thyroid
cancer phenotypes and poor prognosis: 55% (11/20) of patients
with RAS-positive well-differentiated thyroid cancers died in com-
parison to 15% (9/58) RAS-negative ones (P = 0.016). There are no
other reports on this issue, however, the observation that RAS muta-
tions are rather frequent in poorly differentiated thyroid cancers
(40–55%) supports this notion (Fagin and Mitsiades, 2008).
3.6. Follicular thyroid cancer-specific gene mutations
3.6.1. PAX8/PPAR rearrangements
This type of rearrangement results from the translocation
between the PAX8 (paired box gene 8) gene, which encodes a paired
domain transcriptional factor, and the peroxisome proliferator-
activated receptor (PPAR) gene. The resulting fusion protein (PPFP;
PAX8/PPAR␥ fusion protein) encodes a nearly full-length PPAR␥,
the expression of which is under the transcriptional regulation of
the PAX8 promoter (Kroll et al., 2000). The functional consequences
of this PAX8/PPAR rearrangement are yet not fully understood.
Some studies have shown negative effect of PAX8/PPAR rearrange-
ment on the function of wild-type PPAR (Gregory et al., 2004),
however other have questioned this negative effect and showed
the up-regulation of many genes which are transcriptional tar-
gets of PPAR (Giordano et al., 2005). Another possible oncogenic
mechanism of PAX8/PPAR rearrangement is deregulation of PAX8
function, which is critical for thyroid cell differentiation (Reddi
18 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
et al., 2007). In FTC, PAX8/PPAR rearrangement is expressed in
some 35–47% of cases (Cheung et al., 2003; Fagin and Mitsiades,
2008; Sahin et al., 2005). PAX8/PPAR rearrangement has also been
shown in follicular adenoma with a varying frequency (Cheung et
al., 2003; Marques et al., 2004), summed up to 13% (Sahin et al.,
2005), thus may be regarded as an early event in follicular tumori-
genesis, requiring additional mutations to induce FTC (Cheung et
al., 2003; Gregory et al., 2004). Again, RAS and PAX8/PPAR are
regarded mutually exclusive (Nikiforova et al., 2003b). There are
no very convincing data on PAX8/PPAR rearrangement prognos-
tic significance (Sahin et al., 2005). The rearrrangement does not
appear to be expressed in classical PTC, while in follicular variant
of PTC some authors describe it frequent occurrence (Castro et al.,
2006), while others (Sahin et al., 2005) negate this possibility.
3.6.2. RAS homolog 1 (ARH1) silencing
As already mentioned, the mechanisms of transition from a
benign to a malignant tumor, from FA to FTC, are not well known.
High frequency of loss of heterozygosity in imprinted genomic
regions has been suggested to contribute to this transition (Sarquis
et al., 2006). One of the proposed mechanisms is the reduction of the
number of ARH1 copies. Weber et al. (2005a) showed that relatively
few FAs but the majority of FTCs, including minimally invasive FTCs,
show marked ARH1 mRNA underexpression, due to deletion of the
non-imprinted second allele in conjunction with hypermethylation
of the genomically imprinted allele.
3.7. Mutations of other genes and their importance for thyroid
cancer and its outcome
3.7.1. PIK3CA mutations and amplification
PIK3CA mutation is not a common mechanism in the activation
of PI3K/AKT in thyroid carcinoma while amplification of this gene
is more frequent. In PTC, rare mutations and much more frequent
amplifications, comprise alltogether 15–53% of tumors (Abubaker
et al., 2008; Hou et al., 2007). PIK3CA amplication is observed also in
FTC and even in FAs (Hou et al., 2007; Wu et al., 2005); however, it
is the most frequent in anaplastic thyroid cancer. Its down-stream
target, AKT, is phosphorylated in at least half of PTC, but indepen-
dently of PIK3CA mutation or amplification (Abubaker et al., 2008).
3.7.2. PTEN mutations
PTEN (phosphatase and tensin homolog) is a dual-specific
phosphatase, a negative regulator of the AKT/PI3K pathway
by dephosphorylation of phosphatytylinositol-3,4,5-triphosphate
(PIP3), which can also influence the MAPK pathway. PTEN is a tumor
suppressor gene and its germline loss of function mutations pre-
dispose to multiple tumors including FTC (Yeh et al., 1999). Single
cases of loss of function somatic mutation of PTEN were described
in FTC (7%), somewhat more frequently in ATC (12–50%) and rarely
in PTC (2%) (Frisk et al., 2002; Garcia-Rostan et al., 2005; Gimm et
al., 2000; Hou et al., 2007; Smallridge et al., 2009). A novel rear-
rangement involving PTEN and the H4 gene, has been described as
the non-clonal event in 14/18 PTCs investigated (Puxeddu et al.,
2005) and as a feature present in 4.8% of a larger series of PTCs in
Chinese patients (Wang et al., 2008b). All these rearrangements are
paracentric inversions of chromosome 10q, the same chromosome
which is responsible for RET rearrangements.
3.7.3. ˇ-Catenin mutations
The ␤-catenin protein, when bound to E-cadherin, mediates
cell skeleton/adhesion interactions, and when non-sequestered
and non-degraded, plays a role in gene transcription regulation
of growth-promoting genes. It constitutes an element of Wnt sig-
naling pathway and is in fact a Wnt nuclear effector (Fagin and
Mitsiades, 2008). The Wingless (Wnt) family of secreted glyco-
proteins controls early developmental processes including cellular
migration, proliferation and differentiation. Non-canonical Wnt
signaling is associated with tumorigenesis and Wnt5a has been
reported to be increased in the majority of PTCs but in only some
FTCs (Kremenevskaja et al., 2005). Mutations of ␤-catenin gene
(CTNNB1) are most frequent in poorly differentiated and anaplastic
cancer (Garcia-Rostan et al., 1999; Garcia-Rostan et al., 2001).
3.7.4. TP53 mutations
TP53 encodes a multifunctional nuclear protein which is impor-
tant for cell cycle arrest at DNA damage, senescence or apoptosis
and is one of the best known tumor suppressor genes. Contrary
to what is seen in many other cancers, TP53 loss of function
mutations occur late in thyroid tumorigenesis and are practically
absent in DTC (0–9%), while the prevalence of these mutations
rises as the tumor grade worsens, reaching 17–38% in poorly dif-
ferentiated and 55–88% (Fagin and Mitsiades, 2008; Smallridge
et al., 2009) in anaplastic cancers. At this moment, the features
of poor differentiation are easily detected histologically, thus, the
immunohistochemical or molecular investigation of TP53, docu-
mented widely in other tumors, has not gained clinical significance
in thyroid cancer.
4. Prognostic aspects of other gene expression changes in
papillary and follicular thyroid cancer
Over the last two decades many gene expression changes have
been described in papillary and follicular thyroid cancers and
related to the stage of the disease or its outcome. The clear distinc-
tion between the changes characteristic for papillary and follicular
cancers is however not possible for many reasons: First, due to
the much higher frequency of PTC, most of the genes described
below were either not examined in FTC or investigated in small
groups of FTC cases, not allowing for specific distinction. Second,
many of those genes in which such comparison was performed,
showed only minor differences between histotypes because they
were late consequences of the neoplastic transformation of the fol-
licular thyroid cell. Thus, it is rationale to look for their prognostic
significance independently from the histotype or variant of thyroid
cancer. In the given below brief list of genes, expression changes of
which has putative or proved prognostic relevance, we inform on
histotype-specific changes only then, when they were investigated
with sufficient power.
4.1. Single genes expression changes
4.1.1. MET proto-oncogene
The MET proto-oncogene encodes a membrane tyrosine kinase
receptor for hepatocyte growth factor (HGF). HGF is a potent mito-
gen for epithelial cells and promotes cell motility and invasion.
About 50% of cases of PTC are characterized by MET overexpression
(Di Renzo et al., 1992), which is believed to be a sign of more aggres-
sive disease (Mineo et al., 2004; Ramirez et al., 2000). BRAF-positive
tumors were associated wit MET overexpression in aneuploid PTCs
(Rodrigues et al., 2007).
4.1.2. Epithelial growth factor receptor (EGFR)
Among receptor tyrosine kinases EGFR belongs to the best
known. EGFR (HER1) protein levels are increased in PTCs in com-
parison to normal thyroid tissue and the high expression of EGFR
has been suggested to be associated with worse outcome of PTC
after thyroidectomy (Akslen and Varhaug, 1995), an observation
not repeated in other studies (Ruan et al., 2008). Another early
study indicated on HER2 as the predictor of metastasis in PTC
(Kremser et al., 2003). The immunohistochemical evaluation of
 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
EGFRvIII showed its expression in the majority of PTCs (Omidfar
et al., 2009). In the study of Wiseman et al. (2008a), which investi-
gated expression of HER1–4 genes, overexpression of at least one
of them was observed in 76% of DTC and only HER3 was positively
correlated with DTC stage, while HER4 was inversely correlated
with T stage. In this context it is important that HER3 (ERBB3) is
characteristic for BRAF-induced PTCs (Giordano et al., 2005).
Until recently, it was assumed that activating mutations of EGFR
do not play any role in the molecular biology of DTC (Omidfar et
al., 2009). However, the recent finding of Masago et al. (Masago et
al., 2009) indicated that EGFR mutations commonly seen in pul-
monary carcinoma may be observed in a subset of papillary or
poorly differentiated thyroid cancers which may make tumor cells
oncogene-addicted.
4.1.3. Mitogen-inducible gene-6 (MIG-6)
MIG-6 is an immediate early response gene, expression of which
is induced by cellular stress, hormones or growth factors in many
cells. It exerts a negative effect on EGFR signaling that is mediated
by the MAPK cascade (Ferby et al., 2006). Because MIG-6 expres-
sion is induced by MEK–ERK activation, such expression may be
regarded as a negative feedback loop signal in normal cells, lost
in cancer. In PTC, MIG-6 directly correlated with EGFR expression
and its higher mRNA level was associated with better overall sur-
vival in a population of 106 PTC patients followed for six years.
Interestingly, MIG-6 expression was independently predictive of
disease-free survival in BRAF-positive patients (Ferby et al., 2006).
4.1.4. Vascular epithelial growth factor (VEGF) and VEGF receptor
VEGF overexpression is a characteristic feature of malignant
tumors, seen in thyroid cancer as in other cancers (Wiseman et al.,
2008b) and may be interpreted as marker of tumor hypoxia. How-
ever, not in all cancers it is correlated with HIF-1alpha expression.
Thyroid cancer belongs to those tumors in which hypoxia-inducible
factor-1␣ is up-regulated at the same time that VEGF is overex-
pressed (Jubb et al., 2004). The correlation of VEGF expression with
the expression of other angiogenic factors is strong (Tanaka et al.,
2002). Quantitative evaluation of VEGF expression in PTC showed
the association of this phenomenon with metastatic PTC (Klein et
al., 2001). VEGF expression was closely correlated with tumor size,
extrathyroidal invasion and BRAF presence (Jo et al., 2006), while
Tian et al. (2008) observed the correlation with lymph node metas-
tases both for VEGF and metalloproteinase MMP2. In the study of Jo
et al (Jo et al., 2006), VEGF was the only molecular marker associated
with tumor size, extrathyroidal invasion and cancer stage by uni-
variate analysis and the association with extrathyroidal invasion
was confirmed in multivariate analysis, while VEGFR expression
was only weakly associated with PTC recurrence in young patients
(Fenton et al., 2000).
4.1.5. E-cadherin and catenins
E-cadherin (CDH1) is a calcium-dependent transmembrane gly-
coprotein that functions as a cell-cell adhesion molecule. It has an
intracellular domain that complexes with catenin proteins for regu-
lation of further Wnt-mediated signal transduction to the nucleus.
CDH1 expression is reduced in thyroid carcinomas and has been
associated with poor prognosis, not only in PTC (von Rhein et al.,
1997) but also in FTC (Brecelj et al., 2005) as well as with PTC-to-
ATC transition (Wiseman et al., 2007). Dislocalization of catenins in
thyroid cancer may be secondary to loss of E-cadherin (Bohm et al.,
2000; Rocha et al., 2003). However, the study of Kapran et al. (2002)
did not show any association of E-cadherin or catenins expression
with PTC recurrence.
19
4.1.6. Cyclin D1 and other cell cycle regulators
Association of overexpression of cyclin D1, one of the target
genes of Wnt signaling pathway, with more advanced PTC, has
been adressed for a long time (Ferenc et al., 2005; Ito et al., 2005).
Although cyclin D1 and other cell cycle regulators, such as cyclin E,
p16, p21 were overexpressed in thyroid cancer, only p16 expres-
sion correlated significantly with extrathyroidal tumor extension
and the presence of lymph node metastases in the study of Melck
et al. (2007). Also, in the study of Pesutic-Pisac et al. (2008) loss
of p27 was more essential than cyclin D1 overexpression, while
Khoo et al. (2002) suggested the contribution of both markers. The
study of Hoos et al. (2002) was negative for correlation of cyclin
D1 expression with indices of prognostic relevance in oncocytic
thyroid cancers.
4.1.7. Mucin 1
Mucin 1 (MUC1) is a glycoprotein important for cell adhe-
sion, overexpressed in about 25% of PTC (Wreesmann et al., 2004).
Preclinical studies support the role of MUC1 in promoting an
aggressive phenotype of PTC (Patel et al., 2005). MUC1 overexpres-
sion is especially frequent in the tall-cell variant of PTC, known
for its poor prognosis (Ghossein and LiVolsi, 2008). Identified as
an independent prognostic marker by genome-wide gene expres-
sion profiling of PTC, MUC1 overexpression was described to be
significantly associated with the treatment outcome (Wreesmann
et al., 2004), independent of extrathyroidal extension or of the PTC
variant. However, other reports question the correlation of MUC1
overexpression with PTC prognosis (Abrosimov et al., 2007a; Min
et al., 2008). The significance of MUC1 overexpression for cancer
outcome may be more essential if we consider MUC1 contribution
to the survival network, causing resistance to genotoxic agents in
thyroid cancer (Siragusa et al., 2007).
4.1.8. S100A4 calcium-binding protein
This small calcium-binding protein also called calvasculin has
been reported to promote metastasis and was indicated as a molec-
ular factor of poor prognosis in PTC (Zou et al., 2004, 2005). In
papillary microcarcinomas, the expression of S100A4 correlated
more strongly with lymph node metastases than did any other
molecular factor investigated and was more significant for asso-
ciation with stage than BRAF status (Min et al., 2008).
4.1.9. Osteopontin and CD44v6
Osteopontin (SPP1) is a cytokine regulating cell trafficking
within the immune system, binding a splice variant of CD44,
that is overexpressed in PTC and contributes to the mitogenesis
(Figge et al., 1994), survival and motility of PTC thyrocytes and their
invasion potential (Guarino et al., 2005). In PTC, the gene expres-
sion level of SPP1 was associated with metastasis but not with the
BRAF status of the tumor (Oler et al., 2008).
4.1.10. Immunity-related genes
Activation of the MAPK pathway induces up-regulation of
chemokines and their receptors on tumor cells, relevant for their
sustained proliferation and cell motility. Tumors use molecules of
the innate immune system for their growth, survival and metasta-
sis (Melillo et al., 2005). Expression of toll-like receptor 3 (TLR3) is
characteristic for PTC but not FTC (McCall et al., 2007). The basally
expressed receptors are functional and able to activate the NF␬B
pathway and to increase the expression of interferone IFN␤ and
CXCL10 chemokine. Interestingly, TLR5 overexpression is coordi-
nated with WNT5a expression.
Chemokine receptors CXCR2–CXCR4 and CCR7 are expressed on
PTC cells (Melillo et al., 2005; Sancho et al., 2006; Wagner et al.,
2008) and CCR7 was reported to be associated with pathologic fac-
tors of PTC aggressiveness such as extrathyroidal tumor extension,
20 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
angiolymphatic invasion and presence of lymph node metastases
(Wagner et al., 2008). Chemokine CXCL14 was described to be asso-
ciated with metastatic PTC (Oler et al., 2008).
Interestingly, expression of HLA-DR and DQ molecules is
inversely associated with PTC recurrence risk (Jo et al., 2008).
4.2. Prognosis-related gene signatures in thyroid cancer
4.2.1. Gene expression profiling and prognosis of thyroid cancer
Several DNA microarray studies show distinct differences in the
molecular profiles of not only papillary versus follicular cancer, but
also between subtypes of papillary cancer (Aldred et al., 2004; Baris
et al., 2005; Chevillard et al., 2004; Finley et al., 2004a,b; Giordano
et al., 2005; Oler et al., 2008). It is to be stressed that some gene
ontology groups are characteristic of PTC, mainly cell adhesion-
related genes involved in cancer invasion and metastasis, or the
gene signature related to the immune response to this DTC his-
totype (Huang et al., 2001; Jarzab et al., 2005b). While the gene
expression profile of PTC differs from that of normal thyroid tissue
by a huge number of overexpressed genes, in FTC, down-regulated
genes far outweigh up-regulated genes (Aldred et al., 2003). In the
recent study by Oler et al. (2008) based on SAGE (serial analysis of
gene expression) and followed by QPCR (quantitative polymerase
chain reaction), there were 548 differentialy expressed genes when
PTC was compared with FTC and FA. Of the 548, 131 genes had a
more than 10-fold increase in expression in PTC, and 61 genes, an
over 15-fold increase.
Despite the very characteristic morphologic phenotype of
PTC, surprisingly few single genes were described as unique for
this histotype. CITED1 (Cbp/p300-interacting transactivator, with
Glu/Asp-rich carboxy-terminal domain, 1), encoding a transcrip-
tional transactivator nuclear protein, is one of the few identified
until now (Prasad et al., 2004). Other examples are nicotinamide-
N-methyl-transferase (NNMT), encoding a liver enzyme involved
in the biotransformation of many drugs and xenobiotics (Xu et al.,
2003), or chemokine (C–X–C motif) ligand 14 (CXCL14), cystatin
E/M (CST6) and DHRS3 (dehydrogenase/reductase (SDR family)
member 3), a protein involved in retinol storage, all three found
to be characteristic of PTC by Oler et al. (2008). It seems that rather
than single genes, gene groups (gene clusters or gene signatures)
are characteristic of PTC, among them, the already-mentioned gene
classes related to adhesion/motility or invasion (Fig. 2). On the other
hand, many genes have similarly altered expression in both PTC
and FTC, which helps to define these changes as common conse-
quences of different neoplastic transformation pathways (Polanski
et al., 2007).
In FTC, according to Aldred et al. (2003), three of the most down-
regulated genes localized to previously reported regions of loss of
heterozygosity (LOH) were caveolin-1 and -2 and bone morpho-
genetic protein 3b (GDF10). Takano et al. (2004) identified the trefoil
factor 3 gene as down-regulated in FTC, but this gene should not
be regarded as FTC-specific, as it is down-regulated in PTC as well
(Hamada et al., 2005).
Despite the publication of numerous thyroid cancer gene
expression profile analyses (see the meta-analysis of Griffith et
al., 2006), only a few studies addressed prognosis-related gene
signatures. Finley et al. (2004b) were the first to assess whether
gene expression profiling was capable of differentiating aggressive
thyroid carcinomas from non-aggressive ones. They characterized
their group of 29 DTC tissue samples according to the patients’
AMES criteria and obtained a list of 339 differentially expressed
genes. By this criterion, all of an additional 6 samples for which
AMES status was unknown were correctly clustered, while there
were 6/29 misclassified samples in the test group, yielding a sen-
sitivity of 85.7% and a specificity of 80.9% for high-risk tumors.
The same procedure done for extrathyroidal extension was even
more sensitive. Unfortunately, this gene signature has not yet been
verified by another group.
4.2.2. Metastasis-associated genes in thyroid cancer
Although the survival rate in well-differentiated thyroid cancer
is excellent, that is not the case in the 5–10% of patients with dis-
tant metastases. Thus, identification of a metastasis-related gene
signature is desirable. One of the first approaches was carried out
by Zou et al. (2004), who established a thyroid cancer cell line with
high metastatic capacity and specified genes characteristic of this
line, among them MET, ezrin, integrin, motility-related protein-
1, cadherin P, NEDD5 and S100A4. In their quantitative real-time
PCR (QPCR) validation study, mentioned already above, they con-
firmed S100A4 to be associated with poor thyroid cancer prognosis.
Two other genes were listed by Oler et al. (2008) as metastasis-
associated; CXCL14 and cystatin M. However, the definition of
metastatic PTC was very poor in this study. Some comparisons
were also performed by Rodrigues et al. (2007) who reported low
expression of many genes in M1 aneuploid PTCs.
4.2.3. Epithelial-to-mesenchymal transition in PTC
In the search for key molecular prognostic factors in thyroid
cancer, the mechanisms of tumor invasion must be considered.
The process of epithelial-to-mesenchymal transition (EMT) has
been implicated in the conversion of early-stage tumors to inva-
sive malignancy and has been extensively studied in invasive PTC
(Vasko et al., 2007). After loss of cell-to-cell contacts and remodel-
ing of the cytoskeleton, tumor cells are more prone to migration and
this process is hallmarked by loss of cadherin-E expression. Inte-
grin pathway, Notch, MET, TGF-beta, NF␬B, PI3K and p21-activated
kinase belong to EMT-regulating proteins, also fibronectin 1 is
considered as EMT related. In the study of Vasko et al. (2007),
microarray analysis of the central part and of the invasive front
of selected PTC tumors has been performed, showing in the lat-
ter the overexpression of TGF-beta, NF␬B and integrin pathway as
well as of small G protein regulators and CDC42. Overexpression of
vimentin, the hallmark of EMT, was associated with tumor invasion
and nodal metastasis and was correlated with RUNX2 expres-
sion, as judged by immunohistochemistry. Interestingly, there was
no correlation of expression of these invasion-related proteins
with tumor genotype, e.g., BRAF status. However, in the study of
Watanabe et al. (2009) silencing of BRAF reduced the increased
expression of vimentin. For PTC cell invasion, enhanced AKT activ-
ity is especially important, but there was no correlation of AKT with
BRAF presence in PTC (Vasko et al., 2007). According to Wang et al.
(2007), BRAF mutation and AKT pathway activation are mutually
exclusive in PTC.
4.3. Genetic germline background and the prognosis of papillary
and follicular thyroid cancer
Some 5–10% of all cancers developing in follicular cells occur on
the background of familial predisposition, referred as familial non-
medullary thyroid carcinoma (FNMTC), with autosomal dominant
mode of inheritance and penetrance increasing with age (Cavaco
et al., 2008; Handkiewcz-Junak et al., 2006; Malchoff and Malchoff,
2006). This clinical entity develops most often (in over 85% of
cases) as familial PTC and differs from sporadic cancer by earlier
age of onset, more frequent multifocal disease, higher degree of
aggressiveness and higher recurrence rate. Not all involved family
members suffer from thyroid cancer, some develop multinodular
goiter or follicular adenomas. The diseases are genetically heteroge-
nous, with some genes identified in single families but with only
a few general susceptibility genes described (Canzian et al., 1998;
Gudmundsson et al., 2009; Jazdzewski et al., 2008; McKay et al.,
2001; Ngan et al., 2009). Somatic BRAF or RAS mutations are found in
 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28 21

Fig. 2. A large cluster of cell adhesion-related genes are homogenously up-regulated in PTCs. By their expression nearly all PTCs may be separated from follicular tumors and
benign tissues. Hierarchical cluster analysis based on 100 PTC and 99 FTC, FA and non-neoplastic thyroid samples from own material (49 PTC tumors) and those published
by Giordano et al. (2005) and Weber et al. (2005b). The intensity of the color refers to the intensity of gene expression; red refers to the up-regulation, and green refers to
the downregulation. Color coding of tissues: blue, PTC; red, FTC; green, benign tumors.

familial PTCs with similar frequency as in sporadic tumors (Cavaco
et al., 2008). In this aspect it is important to add that there are no
data suggesting that any particular genetic background facilitates
the BRAF mutation what could be suspected considering the pres-
ence of a unique BRAF mutation present in a large proportion of PTC
cases. However, environmental influences are also conceivable, as
suggested by recent Chinese data showing that BRAF-positive PTCs
are significantly more frequent in areas of extremely high iodine
intake (Guan et al., 2009) and by comparison of different Sicilian
areas (Frasca et al., 2008).
5. Final considerations on prognostic impact of genetic
factors in papillary and follicular thyroid cancer
5.1. Relation between BRAF status and gene expression profile
5.1.1. Is BRAF-positive DTC a distinct molecular subtype?
The mutational status of PTC correlates well with its gene
expression profile, allowing bioinformatical analysis of genome-
wide data to clearly differentiate BRAF, RAS or RET/PTC mutants,
even if an unsupervised approach, Principal Component Analysis,
is applied. The most important paper on this issue, published by
Giordano et al. (2005), showed distinctly that PTC mutational sta-
tus was even more strongly correlated with gene expression profile
than with morphology as evaluated by subdivision into PTC vari-
ants. When comparing RET/PTC mutants versus BRAF mutants, 3891
probesets with P < 0.01 were specified, with a similar distance of
both of these mutant types from RAS mutants, the number of dif-
ferent genes being on average approximately 20 times as many as
obtained for permuted data sets. By further selection of genes for
those that yielded P < 0.01 as well as a fold-change <0.5 or >2 for any
group compared to any other, the BRAF mutants were characterized
by 82 probesets (31 up-, 51 down-expressed). These probesets had
a very strong significance, verified, as required in genomic studies,
by the False Discovery Ratio (FDR), which was 0.5 (it is well accepted
in genomic studies that significant genes should show FDR under
5–10%). Many genes were down-regulated in RAS mutants, while
many increases were common between BRAF and RET/PTC mutant
tumors. The most differentially expressed gene in the BRAF mutants
was TM7SF4. Its product is a dendritic cell protein, likely related to
the immune response in these tumors.
Although the other studies published were less powerful
(Frattini et al., 2004; Musholt et al., 2006), they did not contra-
dict the basic message of the Giordano study: the gene expression
profile of BRAF-positive PTC is very distinct and easily recognizable.
Oler et al. (2008) described that the expression of a newly discov-
ered chemokine, CXCL14, and of cystatin M, a secreted inhibitor
of lysosomal cysteine proteases, was not only markedly elevated
in PTC, but also significantly higher in BRAF-positive versus BRAF-
negative tumors. Both genes were simultaneously associated with
metastatic PTC. Also, expression of such PTC-characteristic genes
as fibronectin 1, CITED1 and vimentin were positively correlated
22 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
with the expression of BRAFV600E (Watanabe et al., 2009). The
conditional expression of BRAFV600E and RET/PTC showed that the
more invasive potential of BRAF-mutated tumors may be at least
partially explained by more distinct overexpression of metallopro-
teinases MMP3, MMP9 and MMP13 (Mesa et al., 2006).
All these data, especially regarding genome-wide differences,
substantiate the hypothesis that BRAF-induced PTC is in fact a dis-
tinct molecular subtype of PTC, with a different, more aggressive
disease course and a poorer outcome, which demands a subtype-
specific treatment. However, prospective data still are necessary to
prove this theory.
5.1.2. BRAF-mutated PTCs exhibit fewer thyroid-specific gene
expression features
Many authors have confirmed the lower expression of thyroid-
specific genes in BRAF-positive PTCs: the gene expression profiling
study by Giordano et al. (2005) noted a down-expression of TPO
and the study of Durante et al. (2007) confirmed the earlier obser-
vations of the more intensive loss of gene expression of NIS (−82%),
AIT-B (−86%), TPO (−90%), and TG (−46%) in BRAF-mutated PTCs.
When BRAF-positive and BRAF-negative PTCs are compared, the
level of NIS gene expression is at least 5 times lower in the former
(Durante et al., 2007). Not only is there a BRAF-dependent down-
regulation of NIS expression on the transcriptional level, but also,
the trafficking of the NIS protein to the cell membrane is impaired in
BRAF-positive PTCs (Riesco-Eizaguirre et al., 2006). Moreover, sup-
pression of the BRAF/MEK/MAPK pathway restores expression of
iodide-metabolizing genes (Liu et al., 2007a). Unlike the case with
NIS or TPO genes, the expression of TSH-R and PAX8 seems not to
be different in BRAF-positive tumors in comparison to in other PTCs
(Durante et al., 2007).
To interpret these data, some more general remarks seem neces-
sary: the down-expression of thyroid-specific genes is not specific
to BRAF-induced tumors, but is seen on the RNA level, the pro-
tein level or both in every case of thyroid cancer (Ambroziak et
al., 2005; Huang et al., 2001; Krause et al., 2007; Lazar et al., 1999;
Skubis-Zegadlo et al., 2005). Especially, the direct attribution of the
poorer outcome of BRAF-positive PTC to the down-regulation of
thyroid differentiation-related genes seems precocious. These two
facts are only associated, the causative relation is not proven. It
appears to be more aggressive tumor biology rather than poorer
response to radioiodine therapy which causes poorer outcome
in BRAF-positive tumors. The impact of the lower expression of
NIS protein in BRAF-mutated tumors is diminished by the fact
that both BRAF-positive and BRAF-negative tumors exhibit mainly
cytoplasmic localization of NIS, which precludes that protein’s
functionality. The down-expression of NIS in BRAF-positive PTCs
is seen also in microcarcinomas (Oler and Cerutti, 2009) and
even in these low-risk tumors, BRAF mutation has been related
to development of more advanced disease, while the prognosis is
excellent.
5.1.3. BRAF mutation is associated with the more aggressive
tumor phenotype in PTC
The exact molecular mechanisms of the invasive potential of
BRAF-induced tumors are still not known. As mentioned above,
the poorer outcome in patients bearing BRAF-positive tumors is
with certainty not merely the result of poorer efficacy of radioio-
dine treatment, due to impaired radioiodine uptake and retention.
The prominent activation of the MEK–ERK1/2 pathway in BRAF-
mutated thyrocytes is well known, but the cascade of subsequent
events is not well defined. Interestingly, MET overexpression, an
event related to poorer PTC prognosis, is seen mainly in BRAF-
positive tumors (Giordano et al., 2005). Also, the mean Ki-67 index,
an indicator of tumor cell proliferation, has been reported to be sig-
nificantly higher in BRAF-positive than in BRAF-negative patients
(1.01% versus 0.1%) (Nakayama et al., 2007); importantly, both
these Ki-67 values are rather low, which is a known feature of PTC
(Saltman et al., 2006). The increased GLUT-1 expression in BRAF-
positive tumors may be interpreted as the sign of such tumors’
higher aggressivity. A multivariate analysis concluded that BRAF-
positive tumors have higher VEGF expression (Jo et al., 2006).
Recent reports indicate that prohibitin, a multifunctional protein,
is overexpressed only in PTCs bearing a BRAF mutation. The most
plausible mechanism for prohibitin overexpression is the increased
gene promoter activity by the BRAF oncoprotein (Franzoni et al.,
2009).
Nevertheless, recent publications indicate that some molecu-
lar markers correlate more closely with clinicopathological factors
of poor prognosis than does BRAF-oncogene presence itself. In
contrast to BRAF-positivity, S100A4 and cyclin D1 overexpression
predicted lymph node metastasis in a series of nearly 200 PTC
microcarcinomas (Min et al., 2008). The S100A4 gene was also
investigated in a more detailed study and shown to be significantly
associated with metastases (Zou et al., 2005).
5.2. Major questions in the therapeutic strategy in DTC which can
be solved by molecular diagnosis
In fact, currently, the major clinically relevant problems we face
in DTC are:
1. How to differentiate between indolent and progressing micro-
PTC? BRAF estimation may help to find the progression-potent
tumors, but obviously at the present status of knowledge is not
powerful enough to predict the need for more aggressive treat-
ment than is presently recommended.
2. How to specify the more precise indications for lymphadenec-
tomy in DTC?
Until now, the decision regarding lymphadenectomy is based
on the following rules: routine central lymphadenectomy and
selective lateral lymphadenectomy meaning that the latter pro-
cedure is performed only when evidence or at least strong
suspicion exists for the presence of lymph node metastases
(Cooper et al., 2006; Pacini et al., 2006). Knowing the BRAF status
in the primary tumor may prompt omission of central lym-
phadenectomy in BRAF-negative cases, however, this may lead
to undertreatment of RET/PTC-positive young patients. On the
other side, detection of a BRAF mutation would prompt a deci-
sion to perform central lymphadenectomy in small tumors, a
strategy that is not obligatory today, and hence could lead to
overtreatment.
3. How to optimize the decision for adjuvant radioiodine after
surgery, especially in the “gray zone” of T1-T2N0-N1a patients?
Basing the decision on BRAF status would mean that about in
2/3 of the patients in this stage adjuvant treatment after surgery
would be necessary. However, the indications for a standard
adjuvant treatment with radioiodine are doubtful in this group
in view of decreased expression of genes responsible for iodine
metabolism in BRAF-positive cancers.
4. How to treat patients with advanced DTC?
We do not feel that the information on BRAF status changes
anyhow the present treatment rules—even the success of
sorafenib, a tyrosine kinase inhibitor which is able to target BRAF
as well, has not been shown dependent on BRAF mutation status
(Kloos et al., 2009). On the other hand, data from other cancers
suggest that tumors bearing a BRAF mutation may be particularly
prone to respond to MEK inhibitors (Ball et al., 2007), an obser-
vation that deserves confirmation in the context of advanced
thyroid cancer.
 D. Handkiewicz-Junak et al. / Molecular and Cellular Endocrinology 322 (2010) 8–28
5.3. Conclusions from the transcriptome-wide analyses of DTCs
1. Among the genetic factors analyzed for their prognostic signif-
icance, the presence of activating BRAF mutation in papillary
thyroid cancer seems the only one to be considered in clinical
practice. However, the putative significance of BRAF-positivity
is limited by the fact of its frequent occurrence. Further studies
are necessary to stratifiy BRAF-positive papillary thyroid cancers
and to indentifiy molecular factors related to the subset with
poor prognosis.
2. By gene signature, the difference between BRAF-positive and
BRAF-negative PTC is so distinct that BRAF-positive cancer should
be regarded as a molecular subtype of PTC. There is an urgent
need to investigate, which gene signature within the BRAF-
related gene expression profile is specifically correlated with
poor prognosis.
3. Differences between papillary and follicular cancers shown until
now by gene expression profiling are profound, but they are
still not sufficiently well characterized, thus, their relation to
the putative differences in prognosis between these histotypes
is precocious.
Acknowledgments
The authors thank Robert J. Marlowe for very engaged and com-
petent editorial assistance and Emilia Wilk and Monika Kowal for
excellent general assistance. Daria Handkiewicz-Junak was partly
supported by Polish Ministry of Science and Higher Education
grant no. N40110132/2138 and Barbara Jarzab was supported by
European FP6 project no. 036495, acronym: Genrisk-T and Polish
Ministry of Science and Higher Education grant no. PBZ-MNiI-
2/1/2005.
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