Time to re-consider the meaning of BRAF V600E mutation in papillary thyroid carcinoma.

TIME TO RE-CONSIDER THE MEANING OF BRAF V600E MUTATION IN PAPILLARY THYROID CARCINOMA Greta Gandolfi1, Valentina Sancisi1, Simonetta Piana2 and Alessia Ciarrocchi1*

1) Laboratory of Translational Research, Research and Statistic Infrastructure, Azienda Ospedaliera Arcispedale S. Maria Nuova-IRCCS, Reggio Emilia, Italy 2) Pathology Unit, Department of Oncology, Azienda Ospedaliera Arcispedale S. Maria Nuova-IRCCS, Reggio Emilia, Italy

*Correspondence should be addressed to: Alessia Ciarrocchi Laboratory of Translational Research, Research and Statistic Infrastructure Azienda Ospedaliera Arcispedale S. Maria Nuova-IRCCS, Viale Risorgimento 80, 42123 Reggio Emilia, Italy Tel +39 0522 295668 Fax +39 0522 295743 [email protected]

ABBREVIATED TITLE: BRAF V600E MUTATION IN PTCs

KEY WORDS: BRAF, Papillary Thyroid Carcinoma, molecular marker

ABBREVIATIONS: BRAF, v-Raf murine sarcoma viral oncogene homolog B1; PTC, Papillary Thyroid Carcinoma; FTC, Follicular Thyroid Carcinoma; MAPK mitogen-activated protein kinase; LNM, lymph node metastases; CLND, central lymph node dissection; TSH, Thyroid Stimulating Hormone; TSHR, Thyroid Stimulating Hormone Receptor; TGFb, Transforming Growth Factor beta

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ijc.28976

International Journal of Cancer

Page 2 of 26

ABSTRACT

The BRAF V600E mutation, resulting from the BRAFT1799A transversion, is the most common genetic mutation in Papillary Thyroid Carcinoma (PTC), with a mean frequency close to 50% among all cases. A large number of studies in the past decade have tried to dissect the relevance and the function of the V600E mutation in controlling oncogenesis and progression of thyroid cancer. However, several works published in the latest years have provided new evidence, in partial conflict with the previous knowledge, suggesting the need of reconsidering the meaning of the BRAF V600E mutation in PTC. In this work, we attempt to discuss some of the most recent molecular, preclinical and clinical evidence in order to construct a more exhaustive model of function for the BRAF V600E in development, progression and therapeutic approach of thyroid cancer.

John Wiley & Sons, Inc.

2

Page 3 of 26


INTRODUCTION In the past 10 years, a great interest has developed around the biological function of the BRAF V600E mutation in driving cancers. This mutation and its association with different clinical features has been largely investigated in different tumor settings and in particular in melanoma, colon cancer and thyroid carcinoma (Figure 1) (1-10). In the field of thyroid oncology, the BRAF mutation is synonymous of malignancy, since benign lesions almost never harbor this mutation, and it is a specific mark of papillary thyroid carcinomas (PTCs), since its occurrence in the follicular histotype (FTCs) is basically missing (11-18). The frequency of this mutation in different types of thyroid tumors is represented in Figure 2. The high specificity of this mutation for the papillary malignant phenotype suggested its clinical application as marker for the detection of PTCs in patients with indeterminate fine needle aspiration (FNA) biopsies. Indeed, several studies have demonstrated that application of BRAF mutation analysis to cytological samples improves the accuracy of pre-surgical diagnosis (19-22). The high incidence of the BRAF V600E mutation and its association with the malignant phenotype of thyroid nodules has primed researchers to investigate a possible connection between this mutation and aggressive features of papillary cancers. In the past decade the occurrence of the BRAF V600E mutation has been associated with several morphological and clinical features of PTCs (including extrathyroidal extension, lymph node and distant metastases, higher TNM stage and recurrence), fostering the suggestion that this mutation could be considered a marker of aggressiveness and should be employed in a clinical setting to define high risk profile PTC patients. However, in spite of this large scientific effort, the BRAF V600E mutation still does not take off as clinical prognostic marker. This may be consequence of the fact that the available association studies are conflicting, contradictory and far from being exhaustive. Conflicts between different studies can stem from a variety of issues including different patients selection, different histological types included in the analysis, epidemiological factors, small number of cases, different detection methods applied (ranging from different strategies of allele-specific PCR to sequencing and pyrosequencing to the most recent immunohistochemistry detection) and different statistical approaches (23) . Several works published in the recent years have provided molecular and clinical data that suggest reconsidering the functional role of the BRAF V600E mutation in PTC development and progression. However, the impact of these observations is likely to be dimmed in the wide sea of information already available in the literature on this topic. In the present work, we attempt to discuss the most recent and controversial evidence on the BRAF V600E relevance in development and progression of thyroid tumors and to describe the latest results of the therapeutic use of the BRAF inhibitor in cancer treatment.

ONCOGENESIS It is well established that activating mutations in the MAPK cascade (including the BRAF V600E) lead to increased and un-controlled cell proliferation necessary for tumor formation (24). The oncogenic potential of this mutation in the thyroid is highlighted by the fact that transgenic mice overexpressing the mutated form of BRAF develop rapidly and consistently PTCs that in the majority of cases display a poorly differentiated


3


Page 4 of 26

phenotype (25-27). This observation and the strong association of BRAF V600E with malignancy indicate that the acquisition of this mutation is a driving event in thyroid tumorigenesis. According to this model, an original BRAF V600E mutation should induce transformation of one or few cells in an epithelial sheet within the thyroid, leading to development of a clonal PTC in which all tumor cells should harbor this mutation, at least in heterozygosis (Figure 3A). The rise of quantitative genotyping approaches has opened new opportunities to quantify the extent of clonality of the BRAF mutation in PTC samples. Quantitative sequencing analyses performed with pyrosequencing in two independent studies have shown that, unexpectedly, the BRAF mutation is occasionally a clonal event while in the majority of cases it may be detected only in a subset of tumor cells (on average 25% of total alleles) (28-31) . These observations would indicate that the BRAF mutation in human PTCs generally occurs as secondary genetic event, in the progression of already established tumors. As a consequence, the formed PTCs should be genetically heterogeneous and composed of a mixture of BRAF-mutated and wild type cells (Figure 3B). Genetic heterogeneity of the BRAF V600E mutation was previously described in melanoma and colon cancer suggesting that being a secondary genetic event is a common property of this mutation in human tumors (3234) . The subclonal distribution of the BRAF mutation within primary tumor hold profound implications both for the genetic asset of the metastasis, which can be different from the one of the originating primary lesion (Figure 3B and C), but also for the real feasibility of using pharmacological BRAF V600E specific inhibitor to treat PTC human patients. As such, extreme caution should be pay when addressing this issue. Indeed, the heterogeneity of the BRAF mutation in PTC is currently the object of an intense debate and it does not convince all the scientific community. One of the most relevant issues that have been raised concerns the possibility that the evaluation of mutated allele by quantitative sequencing approaches is biased by the presence of non-tumoral normal stromal cells that may alter the correct quantification of the mutation. In a recent study, Nikiforova and colleagues using a next generation sequencing approach observed that the percentage of BRAF mutated allele in a set of 19 PTC samples ranged from 18-44% of total allele and that the majority of samples display >25% of mutated allele. However, since tumor samples may have a certain amount of contamination by non-tumoral cells, the authors concluded that these numbers strongly support the idea that the BRAF mutation is a clonal driver alteration in PTCs (35). This hypothesis is fostered by the observation that immunostaining of PTCs using a BRAF V600E specific antibody shows heterogeneous staining only in a limited number of tumors, contrasting with the genetic heterogeneity detected by the quantitative sequencing analysis and with the hypothesis of a non-clonal distribution of the BRAF V600E mutation (30, 36, 37). It is likely that the presence of wild type stromal cells leads to a partial underestimation of mutated allele percentage in some of the tumors. However, it seems extremely unlikely that this account entirely for the large heterogeneity observed. Guerra and colleagues tried to address this issue by establishing single clones from primary BRAF V600E mutated thyroid tumors. Sequencing analysis showed that the BRAF V600E mutation was not uniformly distributed among all clones but could be detected only in subsets of the cultured cells supporting the hypothesis of a subclonal distribution of this mutation in the primary tumor (28). The issue of non-tumoral cells contamination was taken into


4

Page 5 of 26


consideration in a recent work by De Biase et al. that used two alternative quantitative techniques (the Allele Specific Locked Nucleic Acid PCR and the next generation sequencing) to evaluate the percentage of BRAF mutated allele in a set of 155 consecutive PTCs samples and normalized the data on the real amount of neoplastic cells within each tumor sample analyzed (31). Using this approach the authors showed that almost 60% of total cases had BRAF V600E allele percentage less than 40% confirming that this mutation may be heterogeneously distributed in PTCs. Intriguingly, all the quantitative sequencing studies published up to now (28-31, 35) are in accordance with the observation that only in rare PTC cases the BRAF V600E mutation is detected in less than 10% of tumor alleles, suggesting that this mutation tend to be acquired early during tumor progression. This hypothesis seems to be confirmed by the fact that also microPTCs and PTC at early stage harbor the V600E mutation (38-40) , and by the observation that the de novo insurgence of V600E mutation in metastases originating from wild type PTC occurs only rarely (30, 41, 42). The use of murine models offers functional insights on the role of BRAF V600E mutation in tumorigenesis. The phenotypic analysis of BRAF mutated transgenic mice showed that, while in the thyroid the overexpression of BRAF V600E leads to the rapid development of thyroid malignancies with a high penetrance (25), in melanocytes and lung epithelial cells this mutation is not sufficient to induce cell transformation. After an initial burst of proliferation, the transgenic overexpression of BRAF V600E in melanocytes and lung epithelial cells leads to the development of benign lesions, in which cells get stack in senescence (43, 44). In a recent work Franco et al. (26) have shown that ectopic expression of BRAF V600E mutated protein within the thyroid gland induces severe hypothyroidism preceding tumor development. This effect was associated with increased levels of Thyroid Stimulating Hormone (TSH). The authors crossed mice with the thyroid-specific knock-in of the BRAF V600E isoform with mice knock out for the Thyroid Stimulating Hormone Receptor (TSHR). In this model, they showed that the effect of the surrounding microenvironment and in particular the presence of TSH, which is a mitogen signal for thyroid, is crucial to allow BRAF V600E expressing thyrocytes to develop malignant lesions. Thus, as it has been reported for melanocytes and lung epithelial cells, the overexpression of BRAF V600E alone in a TSHR knock out background fails to transform the cells and drives formation of non-malignant lesions. Only in a later phase the BRAF V600E expressing thyrocytes escape the dependence from the TSH signal and develop low grade PTCs, likely through acquisition of new genetic alteration (26). Recently, this observation was extended and confirmed by a work by Shimamura and colleagues (45). These authors developed two distinct conditional approaches to induce the expression of the BRAF mutated protein in a restricted portion of the thyroid of adult mice thus avoiding thyroid disfunction and increased TSH levels. In this condition, the expression of BRAF V600E mutation in thyrocytes is not sufficient to drive formation of PTC in mice. Intriguingly, in line with this model, Cameselle-Teijero and colleagues, have described the occurrence of the BRAF V600E mutation in solid nest hyperplasia adjacent to a V600E mutated microPTC in a patient (46) . Even if not conclusive, all these observations suggest reconsidering the hypothesis of the BRAF V600E genetic alteration as the unique trigger of tumoral transformation in the thyroid. When gathered all together, these


5


Page 6 of 26

evidence seems to fit in a unique model in which the occurrence of a mutation within the BRAF gene is a favorable and early event in thyroid tumor development, even though it is not necessarily the initiating one. Questions remain open on the nature of additional molecular events taking place during genesis of PTC.

TUMOR AGGRESSIVENESS AND PROGRESSION In the past 10 years, the importance of BRAF V600E in driving aggressiveness in PTCs and its usefulness as prognostic marker for PTC patient stratification and management has been extensively investigated. A remarkable number of works have analyzed the association of this mutation with different clinical and morphological characteristics, with conflicting and contrasting results. Numerous studies reported a positive association between the BRAF V600E mutation and features of aggressiveness in PTCs (47-57). In 2005, a multicenter study described a significant association between BRAF mutation and extrathyroidal invasion, lymph node metastasis (LNMs), and advanced tumor stage III/IV at presentation in 219 patients (50). However, this association was lost when the tumor subtype was included in the model. These authors also showed that the BRAF mutation was an independent predictor of recurrence even in low-risk PTC patients. In 2007, Lupi et al. analyzed the frequency of the BRAF V600E mutation in 500 consecutive cases of PTC in a homogenous Italian cohort from a single institution (55). They reported a statistically significant association of the mutation with extrathyroidal invasion, multicentricity, presence of nodal metastases and absence of tumor capsule in a univariate analysis. However, when other variables were analyzed in a multivariate analysis the absence of tumor capsule was the only feature to remain associated with BRAF V600E mutation. Several studies also reported a positive association of the BRAF V600E mutation with advanced age of patients (14, 16, 19, 48, 54, 56, 58), which in turns has a significant negative impact on prognosis (17, 59). In parallel, several works did not report any association between this mutation and aggressive features of PTCs (14, 58, 60-65). In 2004, Fugazzola and colleagues analyzed a series of 260 sporadic PTCs and reported a correlation between the BRAF V600E mutation and older age of patients at the time of diagnosis, but did not find a statistically significant correlation between the mutation and other parameters like gender, TNM stage , multicentricity of the tumor, stage at diagnosis and outcome (60) . The same results were obtained by Puxeddu et al. in a separate study (61). In 2005 Trovisco et al. reported that mutated tumors did not displayed signs of higher aggressiveness (size, vascular invasion, extra-thyroid extension and nodal metastasis) as compared to the non-mutated ones, in a series of 315 tumors (14). It is quite difficult to reconcile such discrepant results. It is likely that case selection and statistical approaches used to analyze the data have an important impact on the outcome of the studies. The most powerful methodological instrument to analyze controversial data is the meta-analysis, which gather together results from multiple studies. Recently, four meta-analyses addressed the prognostic value of the BRAF V600E in PTCs (23, 66-68) . Three of these meta-analyses included respectively 12 articles and 1168 patients, 14 articles and 2,470 patients, 27 studies and 5,655 patients (66-68) . The largest and most recent meta-analysis analyzed results of 32 articles, published from 2003 to 2011, for a total of 6,372 patients (23).


6

Page 7 of 26


These meta-analyses established a positive association between the BRAF V600E mutation and most of the clinical features of aggressiveness that were analyzed, including advanced TNM stage, extrathyrodal extension, and LNMs. However, some of these authors raised concerns on the design of the studies analyzed and questioned the value of this mutation as independent prognostic factor. In particular, Li et al underlined that these results may be biased by the fact that the vast majority of the studies included in these metaanalyses were performed without taking into account the impact of possible confounding factors (23). For instance, several

studies included different PTC subtypes, but did not consider the heterogeneity of

histological types in their statistical analysis. Because the histotype is an independent risk factor associated with tumor outcome, and the association between BRAF genotype and different PTC histotype is well documented, this should be taken into account in the evaluation of BRAF as prognostic marker in PTC (14, 23, 69). Most of the studies were retrospective and did not analyze consecutive patients, with a possible bias toward larger tumor or better-documented diseases. Few studies evaluated only patients who have undergone routine central lymph node dissection (CLND), and thus had an unbiased evaluation regarding the presence of LNMs. Finally, because PTCs is frequently an indolent lesion, the association between BRAF mutation and higher mortality was only rarely documented (70). These important issues have been addressed in some recent studies. Two studies from Korean Institutions reported the predictive role of BRAF status for LNMs in two large series of patients who underwent CLND (71, 72). Contrasting findings were reported by two other studies, performed in U.S.A. institutions, which showed that the BRAF mutation was not an independent predictor of central LNMs in classic PTC and questioned the utility of BRAF mutation for patient stratification according to prognostic risk (73, 74). The low occurrence of deadly cases among the overall PTC population, and hence the difficulties in collecting large cohorts of clinically aggressive PTCs, has been so far an important limitation for studies that attempted to correlate genetic alteration to the outcome of PTC patients. In 2008, Elisei and colleagues investigated the prognostic significance of the BRAF V600E mutation in a series of 102 PTCs with an average follow-up of 15 years showing a significant correlation between the presence of the mutation and reduced survival (70) (. Recently, Xing and colleagues have published a retrospective multicenter study in which the BRAF V600E mutation was analyzed in a large cohort of 1,849 PTC patients from 13 medical centers and correlated with the PTC-related mortality (75). These authors, in a univariate analysis, observed a statistically significant association between the presence of the BRAF V600E mutation and the PTC-related mortality. However, when other well known aggressive parameters were included in the analysis, it emerged that the mutation per se was no longer predictive of tumor related mortality in PTC patients even if the hazard ratio for BRAF mutated versus non mutated patients remained elevated (OR=1.21). The major clinical feature known to be associated with a negative outcome of PTC patients is the presence of distant metastases at the time of diagnosis (17). However, little is known about the association of the BRAF V600E mutation with the tendency of PTCs to develop metastases to distant sites. In our study, we reported that the BRAF mutation is not associated with the development of distant metastases (76).


7


Page 8 of 26

We showed that the BRAF V600E frequency was less than 30% in a large cohort of 47 PTCs with distant metastases, a percentage lower than the one detected in the control group of PTCs without distant metastases (44%) and than the average occurrence reported in the literature for this mutation (~ 45%). Our study reported the experience of a single institution and it was focused on a subset of highly selected cohort of well-differentiated PTCs with distant metastasis. However, these data are not distant from the data published by other institutions. Indeed, other studies, while analyzing large cohorts of PTCs (58, 63, 65, 70, 77-79)) have reported the BRAF status in PTCs cases with distant metastases, and gathering together the data, it emerges that of a total of 74 distantly metastatic PTCs, 32 were BRAF-positive (43%) while 42 were non mutated in this gene (57%). Recently, Xing et al. (75) have reported a BRAF V600E frequency of 66% in a total of 110 patients with distant metastases, higher than the frequency of 44% observed in patients without distant metastases in the same study. Gathering all data together, it emerges that the BRAF V600E mutation is present in about 51 % of distantly metastatic PTCs, a frequency that does not differ from the overall BRAF V600E occurrence described in PTCs. In the light of this evidence, the use of BRAF V600E mutation for PTC patients risk stratification and management is being debated by part of the scientific community (80, 81). It seems established that the BRAF V600E mutation is a marker of aggressiveness but it seems also that this mutation does not add predictive value for PTC-related mortality and risk of recurrence beyond the information collected for tumor staging, including histopathology and clinical evaluation. The lack of correlation between the BRAF V600E mutation and distant metastases in PTC patients is well reflected by the phenotype displayed by genetically modified BRAF V600E mouse models. While the transgenic expression of BRAF V600E in the thyroid of mice leads to a rapid development of PTCs with an histologically aggressive phenotype, these BRAF mutated tumors do not develop either distant or LNMs (2527). Intriguingly, the tendency of PTCs to progress independently from the BRAF V600E is supported by the observation that about 6-20% of LNMs (depending on series) that originate from BRAF mutated PTCs do not show the mutation, suggesting that they likely originated from non-mutated cells of the primary tumor (30, 41, 42). Considering the possible heterogeneous distribution of the V600E mutation within the tumors, the metastatic potential of BRAF mutated PTC could be dependent on the amount of cells that indeed hold the mutation. However, when we attempted to correlate the percentage of BRAF V600E mutated alleles with the tendency of PTCs to develop distant metastasis, we did not observe any significant difference comparing distantly metastatic PTCs with PTCs without distant metastases, or with PTCs, which develop metastases to local lymph nodes (30). As well, three independent studies (28-31) reported no correlation between the percentage of mutated allele and the presence of LNMs. Conversely, Guerra et al. observed that PTCs with a BRAF V600E mutated allele percentage > 30% have a significantly lower disease free survival than tumors with a mutated allele percentage < 30% (29) and De Biase and colleagues observed a positive trend between the percentage of mutated cells and the size of the tumor even if not statistically significant (31). The contrasting


8

Page 9 of 26


results of these preliminary analyses indicate the need of further studies in order to establish the potential impact of BRAF V600E mutated allele percentage on thyroid tumor progression. When considered all together, these observations suggest that the BRAF mutation does not confer an indisputable advance to thyroid tumor cells in the metastatic process. But what do we know on the molecular functions of the BRAF mutation in this process? One would argue that if BRAF mutation conferred a metastatic advance to tumor cells then BRAF mutated tumor cells should display a more aggressive phenotype. The vast majority of molecular data on the BRAF V600E function were obtained in vitro (and extensively described in previous reviews [16, 17]). By contrast the availability of BRAF V600E associated gene expression profiling data are still limited. Frattini and colleagues have performed a gene expression analysis to identify a distinctive gene signature associated with specific genetic mutations. They failed to find a gene expression profile specifically associated with the BRAF V600E, suggesting that PTCs share a common expression profile, independently from their genetic alterations (82). Using a different approach, Cheng and colleagues get to the same conclusion. On a series of 410 PTCs samples, assembled with calculated risk estimates for several aggressive features, including vascular invasion and LNMs, they showed that the BRAF mutational status does not perform as an independent prognostic factor of aggressive features and does not correlate with the expression of selected proteomic biomarkers (83). These data are in conflict with the ones of Giordano et al, which described the existence of a BRAF V600E specific gene expression signature (84). Among the BRAF V600E associated genes, these authors found TM7SF4, encoding a transmembrane protein expressed in dendritic cells, suggesting a role of BRAF mutation in controlling immune response in PTC. The ability of BRAF V600E mutation to condition microenvironement favoring tumor progression, emerged also from other studies (53, 85, 86). Several additional molecular factors (i.e. TGF-b/Smad pathway, miRNA expression such as miR-146b, expression of RAC1b and uPA) have been proposed as associated with PTC aggressiveness and outcome. In none of these studies, the presence of BRAF V600E influenced the association between these factors and PTC aggressive features, nor the BRAF V600E status alone was associated with worse tumor phenotype (87-90). More convincing is the association of the BRAF V600E mutation with the acquisition of radio-iodine refractoriness, which may hold profound implication for patients treatment. This issue is addressed in detail in the following section. Recently, mutations in the proximal promoter of the Transcriptase component of the Telomerase complex (TERT) have been identified as a highly frequent event in aggressive cancer including thyroid (91, 92). A positive association between TERT promoter and BRAF mutation has been described in thyroid cancer suggesting the possibility of a cooperative effect between these two mutations (92-94). Even if larger prospective studies are needed in order to validate this hypothesis, it is possible that the presence of TERT and BRAF mutations may identify a subgroup of PTCs with a more aggressive behavior. In conclusion, considering the plethora of studies addressing the BRAF V600E prognostic value in PTC, the overall scenario still appears rather confusing. As a matter of fact, the BRAF V600E mutation occurs in as much as 45 % of all PTC tumors, which result in poor outcome in only 2-3 % of cases. This glaring


9


Page 10 of 26

discrepancy would not occur if the BRAF mutation had a pivotal role in driving cancer aggressiveness and suggests caution in the possible prognostic application of this mutation.

THERAPEUTICAL APPROACHES Beside its relevance as biological marker, the BRAF V600E mutation has gathered the attention of the scientific world as a possible target for drug development. In 2010, Flaherty et al. have published the encouraging results of a clinical trial in which a BRAF V600E specific inhibitor (PLX4032) was used on a cohort of metastatic melanoma patients and induced complete or partial tumor regression in about 81% of treated patients (4). Unfortunately, the high grade secondary effects of the drug, the development of resistance, and the existence of compensatory mechanisms have deluded the intrinsic promises that the encouraging results in melanoma hold for other type of BRAF mutated cancers. In the thyroid, the use of BRAF specific inhibitors has shown promising results in biological and preclinical studies but the efficacy of this drug is scarcely documented in patients (27, 95, 96). Targeting BRAF in mutated thyroid tumors is likely to be a correct strategy. Ideally, a proposed trial should take into account all that we have learned from the previous experiences. 1) The BRAF mutation is often an oligoclonal event in PTCs and metastases do not always maintain the mutation (28-31, 41). Therefore the use of solely BRAF inhibitors is likely to lead to a selection of non-mutated tumor cells that would finally confer resistance to the treatment (Figure 4A). Furthermore, evidence exists showing that, while inhibiting the mutated V600E isoform, PLX4032 exerts a stimulatory function upon the wild type BRAF isoform, increasing its signaling function and the activation of the downstream pathways (so called the BRAF inhibitor paradox) (97, 98). The development of MEK inhibitors and the combinatory use of these compounds with the BRAF inhibitors is already under clinical studies and holds promises of better efficacy and minor toxicity (99). 2) As learned from colon carcinomas, the constitutive inhibition of BRAF pushes the activation of compensatory pathways that confer resistance to the treatment. In particular, in colon cancer it has been shown that administration of BRAF inhibitors leads to an ectopic activation of the Epidermal Growth Factor Receptors and of its mitogen downstream signaling (100, 101). A similar mechanism has been described also in tumor thyrocytes. Blockade of BRAF and MEK by specific targeting drugs, results in a relief of the HER3/EGFR transcriptional repression with a consequent activation of the receptor activity and attenuation of the antitumoral effects of the two drugs (102). Based on these observations, the combination of BRAF inhibitor with anti-EGFR molecules could be suggested as an attractive therapeutic possibility for patients with mutated BRAF cancer. 3) Resistance to BRAF inhibitors can arise from any event that activates components of the BRAF downstream pathways, including loss of function mutation in the PI3K inhibitor PTEN (103). Preclinical data in colon cancer and thyroid tumors would suggest that blocking the PI3K pathway (for example through the mTOR inhibitor rapamacyn) would improve the efficacy of BRAF inhibition, shortcutting a possible compensatory mechanism (104, 105). This hypothesis is extremely intriguing since it has been shown that BRAF V600E mutation in thyroid tumors does not lead to a significant increase on the ERK phophorylation status but it is more often accompanied by increased activation of the AKT-mTOR pathway (106, 107). 4) Radio-iodine administration is the most


10

Page 11 of 26


effective treatment for thyroid cancer. However, advanced tumors tend to lose the ability of uptake iodine, primarily by the inactivation of Sodium/Iodine Symporter (NIS), becoming resistant to this treatment (53). The BRAF mutation has been shown to be frequently associated with loss of iodine uptake of thyroid tumors (53, 108, 109). In a recent work, Chakravarty et al. showed that the use of BRAF or MEK specific inhibitors restored radioiodine incorporation in the thyroid, in a murine BRAF V600E thyroid cancer model (27). More recently, this observation was translated into practice in a clinical trial, which showed that inhibition of MEK activity through Selumetinib administration could restore radioiodine uptake in advanced thyroid tumor patients (110). This confirms that in advanced thyroid tumors that have lost the ability to respond to radioiodine therapies, the administration of MAPK pathway may restore this therapeutic possibility. Unexpectedly, the MEK inhibition was more effective in NRAS than in BRAF mutated thyroid cancer. The molecular rationale of this difference remains to be elucidated but the authors suggest that MEK inhibition may not be sufficient to fully block the MAPK cascade in BRAF mutated tumors. In this case the use of a direct BRAF inhibitor in BRAF mutated tumors should lead to better results. In alternative, we may speculate that other BRAF-dependent signaling pathways (like the PI3K pathways) (53, 107) may play a more relevant role in inducing radioiodine refractoriness in BRAF mutated thyroid tumors.

CONCLUSION Thyroid carcinomas behave rarely as aggressive lesions (111). However, the small part of tumors that will turn dangerous is not distinguishable morphologically or clinically from the large majority of non-aggressive lesions. The great effort of researchers in this field is to identify molecular markers that may reliably select in advance the few tumors that will behave aggressively. This would ease the therapeutic approach for the majority of non-aggressive PTC patients with a significant amelioration of their life style and a great saving for health systems. In this context the BRAF V600E has been seeing for many years as the molecular marker that would fill the gap and make possible the early diagnosis of aggressiveness. At a decade from the beginning of this story, the scientific world seems to be divided between those that consider the BRAF V600E a reliable predictor of aggressiveness in PTCs and those that are skeptical on the prognostic value of this mutation. It is likely that the acquisition of a gain of function mutation in the BRAF gene represents a favorable event in the development and progression of thyroid cancer. However, the influence of this mutation in a disease like PTCs, characterized by a relative low aggressiveness and few deaths, is small and hard to measure when adjusted for other important factors. Time has come to move forward and look for new molecular determinants that alone or in association with BRAF V600E may be more reliable predictors of aggressive behavior in thyroid carcinomas.

ACKNOWLEDGEMENTS We wish to thank the Thyroid Research Audit for Innovation (T.R.A.I.N.) group of the Arcispedale S. Maria Nuova-IRCCS.


11


Page 12 of 26

REFERENCES 1)

Cantewell-Dorris ER, O’Leary JJ, Sheils OM. BRAFV600E: Implications for carcinogenesis and

molecular therapy. Mol Cancer Ther 2011;10:385-94. 2)

Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ,

Bottomley W, Davis N, Dicks E et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:94954. 3)

Fecher LA, Cummings SD, Keefe MJ, Alani RM. Toward a molecular classification of melanoma. JCO

2007;25:1606-20. 4)

Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O'Dwyer PJ, Lee RJ, Grippo JF,

Nolop K, Chapman PB. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010;363:809-19. 5)

Blokx WAM, Marcory van Dijk MCRF, Ruiter DJ. Molecular cytogenetics of cutaneous melanocytic

lesions –diagnostic, prognostic and therapeutic aspects. Hystopathology 2010;56:121-32. 6)

Yokota T. Are KRAS/BRAF mutations potent prognostic and/or predictive biomarkers in colorectal

cancers?Anti-Cancer Agents Med Chem 2012;12:163-71. 7)

Fu B, Yachida S, Morgan R, Zhong Y, Montgomery EA, Iacobuzio-Donahue CA. Clinicopathologic and

genetic characterization of traditional serrated adenomas of the colon. Am J Clin Pathol 2012;138:356-366. 8)

Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis:

RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934. 9)

Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol 2011;12:175-80.

10) Grisham RN, Iyer G, Garg K, DeLair D, Hyman DM, Zhou Q, Iasonos A, Berger MF, Dao F, Spriggs DR, Levine DA, Aghajanian C, Solit DB. BRAF mutation is associated with early stage disease and improved outcome in patients with low-grade serous ovarian cancer. Cancer 2013;119:548-54. 11) Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, Beller U, Westra WH, Ladenson PW, Sidransky D. BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst. 2003;95:625-7. 12) Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 2003;63:1454-7. 13) Soares P, Trovisco V, Rocha AS, Lima J, Castro P, Preto A, Máximo V, Botelho T, Seruca R, SobrinhoSimões M. BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC. Oncogene 2003;22:4578-80. 14) Trovisco V, Soares P, Preto A, de Castro IV, Lima J, Castro P, Máximo V, Botelho T, Moreira S, Meireles AM, Magalhães J, Abrosimov A, Cameselle-Teijeiro J, Sobrinho-Simões M. Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients' age but not with tumour aggressiveness. Virchows Arch 2005;446:589-95. 15) Xing M. BRAF mutation in thyroid cancer. Endocr Relat Cancer 2005;12:245-62.


12

Page 13 of 26


16) Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev 2007;28:742-62. 17) Handkiewicz-Junak D, Czarniecka A, Jarzab B. Molecular prognostic markers in papillary and follicular thyroid cancer: Current status and future directions. Mol Cell Endocrinol 2010;322:8-28. 18) Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol 2011;7:569-80. 19) Xing M, Tufano RP, Tufaro AP, Basaria S, Ewertz M, Rosenbaum E, Byrne PJ, Wang J, Sidransky D, Ladenson PW. Detection of BRAF mutation on fine needle aspiration biopsy specimens: a new diagnostic tool for papillary thyroid cancer. J Clin Endocrinol Metab 2004;89:2867-72. 20) Marchetti I, Lessi F, Mazzanti CM, Bertacca G, Elisei R, Coscio GD, Pinchera A, Bevilacqua G. A morpho-molecular diagnosis of papillary thyroid carcinoma: BRAF V600E detection as an important tool in preoperative evaluation of fine-needle aspirates. Thyroid 2009;19:837-42. 21) Kim SW, Lee JI, Kim JW, Ki CS, Oh YL, Choi YL, Shin JH, Kim HK, Jang HW, Chung JH. BRAFV600E mutation analysis in fine-needle aspiration cytology specimens for evaluation of thyroid nodule: a large series in a BRAFV600E-prevalent population. J Clin Endocrinol Metab 2010;95:3693-700. 22) Nikiforov YE, Ohori NP, Hodak SP, Carty SE, LeBeau SO, Ferris RL, Yip L, Seethala RR, Tublin ME, Stang MT, Coyne C, Johnson JT, Stewart AF, Nikiforova MN. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab 2011;96:3390-7. 23) Li C, Lee KC, Schneider EB, Zeiger MA. BRAF V600E mutation and its association with clinicopathological features of papillary thyroid cancer: a meta-analysis. J Clin Endocrinol Metab 2012;97:4559-70. 24) Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene 2007;26:3279-90. 25) Knauf JA, Ma X, Smith EP, Zhang L, Mitsutake N, Liao XH. Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res 2005;65:4238-45. 26) Franco AT, Malaguarnera R, Refetoff S, Liao XH, Lundsmith E, Kimura S, Pritchard C, Marais R, Davies TF, Weinstein LS, Chen M, Rosen N, Ghossein R, Knauf JA, Fagin JA. Thyrotrophin receptor signaling dependence of Braf-induced thyroid tumor initiation in mice. Proc Natl Acad Sci USA 2011;108:1615-20. 27) Chakravarty D, Santos E, Ryder M, Knauf JA, Liao XH, West BL. Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation. J Clin Invest 2011;121:4700-11. 28) Guerra A, Sapio MR, Marotta V, Campanile E, Rossi S, Forno I, Fugazzola L, Budillon A, Moccia T, Fenzi G, Vitale M. The primary occurrence of BRAF(V600E) is a rare clonal event in papillary thyroid carcinoma. J Clin Endocrinol Metab 2012;97:517-24.


13


Page 14 of 26

29) Guerra A, Fugazzola L, Marotta V, Cirillo M, Rossi S, Cirello V, Forno I, Moccia T, Budillon A, Vitale M. A high percentage of BRAFV600E alleles in papillary thyroid carcinoma predicts a poorer outcome. J Clin Endocrinol Metab 2012;97:2333-40. 30) Gandolfi G, Sancisi V, Torricelli F, Ragazzi M, Frasoldati A, Piana S, Ciarrocchi A. Allele percentage of the BRAF V600E mutation in papillary thyroid carcinomas and corresponding lymph node metastases: no evidence for a role in tumor progression. J Clin Endocrinol Metab 2013;98:934-42. 31) De Biase D, Cesari V, Visani M, Casadei GP, Cremonini N, Gandolfi G, Sancisi V, Ragazzi M, Pession A, Ciarrocchi A, Tallini G. High sensitivity BRAF mutation analysis: BRAF V600E is acquired early during tumor development but is heterogeneously distributed in a subset of papillary thyroid carcinomas. J Clin Endocrinol Metab. 2014; doi:29:jc20134389. 32) Baldus SE, Schaefer KL, Engers R, Hartleb D, Stoecklein NH, Gabbert HE. Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases. Clin Cancer Res 2010;16:790-9. 33) Wilmott JS, Tembe V, Howle JR, Sharma R, Thompson JF, Rizos H, Lo RS, Kefford RF, Scolyer RA, Long GV. Intratumoral molecular heterogeneity in a BRAF-mutant, BRAF inhibitor-resistant melanoma: a case illustrating the challenges for personalized medicine. Mol Cancer Ther 2012;11:2704-08. 34) Yancovitz M, Litterman A, Yoon J, Ng E, Shapiro RL, Berman RS, Pavlick AC, Darvishian F, Christos P, Mazumdar M, Osman I, Polsky D. Intra- and inter-tumor heterogeneity of BRAF(V600E) mutations in primary and metastatic melanoma. PLoS One 2012;7:e29336. 35) Nikiforova MN, Wald AI, Roy S, Durso MB, Nikiforov YE. Targeted next-generation sequencing panel (ThyroSeq) for detection of mutations in thyroid cancer. J Clin Endocrinol Metab 2013;98:E1852-60. 36) Capper D, Preusser M, Habel A, Sahm F, Ackermann U, Schindler G, Pusch S, Mechtersheimer G, Zentgraf H, von Deimling A. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol 2011;122:11-19. 37) Ghossein RA, Katabi N, Fagin JA. Immunohistochemical detection of mutated BRAF V600E supports the clonal origin of BRAF-induced thyroid cancers along the spectrum of disease progression. J Clin Endocrinol Metab 2013;98:E1414-21. 38) Ugolini C, Giannini R, Lupi C, Salvatore G, Miccoli P, Proietti A, Elisei R, Santoro M, Basolo F. Presence of BRAF V600E in very early stages of papillary thyroid carcinoma. Thyroid 2007;17:381-8. 39) Virk RK, Van Dyke AL, Finkelstein A, Prasad A, Gibson J, Hui P, Theoharis CG, Carling T, Roman SA, Sosa JA, Udelsman R, Prasad ML. BRAFV600E mutation in papillary thyroid microcarcinoma: a genotypephenotype correlation. Mod Pathol 2013;26:62-70. 40) Zheng X, Wei S, Han Y, Li Y, Yu Y, Yun X, Ren X, Gao M. Papillary microcarcinoma of the thyroid: clinical characteristics and BRAF(V600E) mutational status of 977 cases. Ann Surg Oncol 2013;20:2266-73. 41) Trovisco V, Couto JP, Cameselle-Teijeiro J, de Castro IV, Fonseca E, Soares P, Sobrinho-Simões M. Acquisition of BRAF gene mutations is not a requirement for nodal metastasis of papillary thyroid carcinoma. Clin Endocrinol 2008;69:683-5.


14

Page 15 of 26


42) Vasko V, Hu S, Wu G, Xing JC, Larin A, Savchenko V, Trink B, Xing M. High prevalence and possible de novo formation of BRAF mutation in metastasized papillary thyroid cancer in lymph nodes. J Clin Endocrinol Metab 2005;90:5265-9. 43) Dankort D, Filenova E, Collado M, Serrano M, Jones K, McMahon M. A new mouse model to explore the initiation, progression, and therapy of BRAFV600E-induced lung tumors. Genes Dev 2007;21:379-84. 44) Dankort D, Curley DP, Cartlidge RA, Nelson B, Karnezis AN, Damsky WE Jr, You MJ, DePinho RA, McMahon M, Bosenberg M. Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nat Genet 2009;41:544-52. 45) Shimamura M, Nakahara M, Orim F, Kurashige T, Mitsutake N, Nakashima M, Kondo S, Yamada M, Taguchi R, Kimura S, Nagayama Y. Postnatal Expression of BRAFV600E Does Not Induce Thyroid Cancer in Mouse Models of Thyroid Papillary Carcinoma. Endocrinology 2013;154:4423-30. 46) Cameselle-Teijeiro J, Abdulkader I, Pérez-Becerra R, Vázquez-Boquete A, Alberte-Lista L, Ruiz-Ponte C, Forteza J, Sobrinho-Simões M. BRAF mutation in solid cell nest hyperplasia associated with papillary thyroid carcinoma. A precursor lesion? Hum Pathol 2009;40:1029-35. 47) Namba H, Nakashima M, Hayashi T, Hayashida N, Maeda S, Rogounovitch TI, Ohtsuru A, Saenko VA, Kanematsu T, Yamashita S. Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endocrinol Metab 2003;88:4393-7. 48) Nikiforova MN, Kimura ET, Gandhi M, Biddinger PW, Knauf JA, Basolo F, Zhu Z, Giannini R, Salvatore G, Fusco A, Santoro M, Fagin JA, Nikiforov YE. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 2003;88:5399-404. 49) Kim KH, Kang DW, Kim SH, Seong IO, Kang DY. Mutations of the BRAF gene in papillary thyroid carcinoma in a Korean population. Yonsei Med J 2004;45:818-21. 50) Xing M, Westra WH, Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, Carson KA, Vasko V, Larin A, Tallini G, Tolaney S, Holt EH, Hui P, Umbricht CB, Basaria S, Ewertz M, Tufaro AP, Califano JA, Ringel MD, Zeiger MA, Sidransky D, Ladenson PW. BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 2005;90:6373-9. 51) Kim J, Giuliano AE, Turner RR, Gaffney RE, Umetani N, Kitago M, Elashoff D, Hoon DS. Lymphatic mapping establishes the role of BRAF gene mutation in papillary thyroid carcinoma. Ann Surg 2006;244:799-804. 52) Kim TY, Kim WB, Rhee YS, Song JY, Kim JM, Gong G, Lee S, Kim SY, Kim SC, Hong SJ, Shong YK. The BRAF mutation is useful for prediction of clinical recurrence in low-risk patients with conventional papillary thyroid carcinoma. Clin Endocrinol (Oxf) 2006;65:364-8. 53) Riesco-Eizaguirre G, Gutierrez-Martinez P, Garcia’-Cabezas M, Nistal M & Santisteban P. The oncogene BRAFV600E is associated with a high risk of recurrence and less differentiated papillary thyroid carcinoma due to the impairment of Na+/I−targeting to the membrane. Endocrine-Related Cancer 2006;13:257-69.


15


Page 16 of 26

54) Rodolico V, Cabibi D, Pizzolanti G, Richiusa P, Gebbia N, Martorana A, Russo A, Amato MC, Galluzzo A, Giordano C. BRAF V600E mutation and p27 kip1 expression in papillary carcinomas of the thyroid

Association between the BRAF V600E mutation and ultrasound features of the thyroid in thyroid papillary carcinoma.

Clinical significance of the BRAF V600E mutation in multifocal papillary thyroid carcinoma in Korea.

Prognostic value of the BRAF V600E mutation in papillary thyroid carcinoma.

Immunohistochemistry is highly sensitive and specific for detecting the BRAF V600E mutation in papillary thyroid carcinoma.

Significance of allelic percentage of BRAF c.1799T > A (V600E) mutation in papillary thyroid carcinoma.

Association between BRAF V600E mutation and regional lymph node metastasis in papillary thyroid carcinoma.

Clinicopathologic correlations of the BRAFV600E mutation, BRAF V600E immunohistochemistry, and BRAF RNA in situ hybridization in papillary thyroid carcinoma.

Increased CYP24A1 expression is associated with BRAF(V600E) mutation and advanced stages in papillary thyroid carcinoma.

Follicular variant of papillary thyroid carcinoma (FVPTC): histological features, BRAF V600E mutation, and lymph node status.

The prognostic significance of the BRAF V600E mutation in papillary thyroid carcinoma detected by mutation-specific immunohistochemistry.

BRAF Immunohistochemistry Using Clone VE1 is Strongly Concordant with BRAF(V600E) Mutation Test in Papillary Thyroid Carcinoma.

Correlation between the BRAF(v600E) gene mutation and factors influencing the prognosis of papillary thyroid microcarcinoma.

The role of BRAF(V600E) mutation as poor prognostic factor for the outcome of patients with intrathyroid papillary thyroid carcinoma.

Extended Antitumor Response of a BRAF V600E Papillary Thyroid Carcinoma to Vemurafenib.

Detection of plasma BRAF(V600E) mutation is associated with lung metastasis in papillary thyroid carcinomas.

The BRAF V600E mutation predicts poor survival outcome in patients with papillary thyroid carcinoma: a meta analysis.

Impact of clinical risk scores and BRAF V600E mutation status on outcome in papillary thyroid cancer.

Clinical significance of preoperative detection of serum p53 antibodies and BRAF(V600E) mutation in patients with papillary thyroid carcinoma.

MKP2 overexpression is associated with BRAF(V600E) mutation and aggressive behavior of papillary thyroid cancer.

Association between BRAF V600E mutation and recurrence of papillary thyroid cancer.

Absence of BRAF V600E in non-infiltrative, non-invasive follicular variant of papillary thyroid carcinoma.

Association between BRAF V600E Mutation and Ultrasound Features in Papillary Thyroid Carcinoma Patients with and without Hashimoto's Thyroiditis.

Detection of Circulating BRAF(V600E) in Patients with Papillary Thyroid Carcinoma.

BRAF V600E and TERT Promoter Mutations in Papillary Thyroid Carcinoma in Chinese Patients.