Intratumor BRAF V600E Heterogeneity and Kinase Inhibitors in the Treatment of Thyroid Cancer: A Call for Participation

Dear Editor:

In general, differentiated thyroid cancer (DTC) patients have an excellent prognosis, and even in the presence of metastatic and radioiodine-resistant disease, the life expectancy of most DTC patients is not reduced. Nevertheless, in ∼3% of DTC patients, the disease can become (rapidly) progressive, refractory to radioiodine treatment and fatal (1).

The therapeutic management of this small group of patients remains a challenge for the clinician whose tools are limited to cytotoxic agents and kinase inhibitors (KIs). Although unusual complete responses to cytotoxic chemotherapy have been described, this treatment is associated with significant toxicity and varying efficacy (2 –4). Clinical trials on KIs have shown promise for patients with metastatic radioiodine-resistant (MRR)–DTC and now represent a new treatment strategy for these refractory patients. Small molecules with tyrosine kinase inhibitory capacity have been developed with two main goals: the inhibition of neoangiogenesis and the inhibition of the mitogen-activated protein kinase pathway, which is abnormally activated by mutated kinases. Angiogenesis plays a critical role in tumor-cell growth and metastasis, supplying nutrients and oxygen, and facilitating distant metastasis. Therefore, its inhibition is a strategy for the treatment of cancer (5). Vascular endothelial growth factor (VEGF) is overexpressed in thyroid tumors, and the inhibition of its receptor (VEGF-R) signaling has been shown to inhibit the growth of aggressive thyroid cancer in xenograft models, thereby providing a strong rationale for targeting VEGF-Rs in this disease (6,7). More recently, the regulation of the VEGF-R2 protein expression by ubiquitination and its effect on angiogenesis and cell migration observed in papillary thyroid cancer (PTC)–derived cells also suggested a direct effect of VEGF-R inhibitors on PTC cells (8).

Advanced cancer results from the progressive increase in favorable genetic alterations. Once established, the survival of cancer cells is tied to the continued expression of the activated genes (oncogenes) or the inactivation of tumor suppressor genes (anti-oncogenes). Accordingly, targeting even a single genetic mutation may affect tumor-cell viability. Activating mutations of BRAF and, less frequently, RAS, as well as RET/PTC and PTEN rearrangements, are the most frequent genetic alterations occurring in MRR-DTC. The serine/threonin kinase BRAF^V600E, the most frequently altered kinase in DTC, appears as the most promising target for KI-based therapy (9). Preclinical studies suggest that tumors with the BRAF ^V600E mutation are dependent on BRAF^V600E for proliferation and survival this indicates the protein (10 –12). To date, the inhibition of VEGF-Rs and BRAF^V600E is providing at least a partial rationale for the efficacy of KIs in the treatment of MRR-DTC.

Vandetanib, sunitinib, and pazopanib are KIs that affect VEGF-Rs and other kinases, while sorafenib also inhibits BRAF^V600E. Clinical trials with these KIs have shown encouraging results in a relevant fraction of patients, although the effect is of a short duration and shows frequent relapses. The encouraging results obtained with sorafenib in many BRAF ^V600E-positive tumors, including melanomas and thyroid cancers, are consistent with the hypothesis that BRAF inhibition is a promising strategy in MRR-DTC that has become tied to BRAF mutations. The recent progress in the successful application of KIs represents an exciting new option for the treatment of MRR-DTC, but it is important not to lose sight of the dangers that could stem from the underestimation of the limits of this treatment.

In recent years, the role of other known oncogenes (i.e., RET/PTC and mutated RAS) in thyroid carcinogenesis has been reconsidered, leading to the conclusion that more crucial genetic events in PTC are still to be discovered (13 –16). More recently, it has been demonstrated, by means of a quantitative assay, that conventional PTCs often consist of a mixture of tumor cells expressing wild-type and mutant BRAF (17,18). The subclonal or even oligoclonal occurrence of BRAF ^V600E supports the concept that the BRAF mutation may not be the initiating event in thyroid tumorigenesis and this may have important therapeutic implications. Before this direct demonstration, a possible mixed composition of PTCs with respect to the BRAF mutation was suggested by the observation that in some cases, patients with BRAF ^V600E primary tumors also had or developed BRAF ^wild-type metastases, and BRAF ^V600E metastases co-existed or developed in patients with BRAF ^wild-type primary tumors (19,20). More recently, the study of Sancisi et al. documented the genetic discordance between primary tumors and metastases in two of five metastatic PTCs analyzed, suggesting that this is not a sporadic occurrence (21). Jovanovic et al. performed a microsatellite allelic imbalance and BRAF ^V600E mutation analysis of synchronous tumor foci from multifocal PTC patients (22). They found that BRAF ^V600E was not always present in all tumor foci with concordant allelic imbalance changes, indicating that BRAF ^V600E can occur as a subclonal evolution in PTC. Regardless of whether the heterogeneity of BRAF ^V600E is the consequence of a secondary event or the effect of the DNA repair mechanisms as hypothesized (23), the subclonality of BRAF ^V600E in MRR-DTC has important therapeutic implications. A therapeutic strategy based upon BRAF inhibitors in tumors bearing subclonal BRAF ^V600E could be initially successful in targeting tumor cells expressing the oncogene. After the initial tumor growth arrest and/or shrinkage, oncogene-negative cells that are insensitive or less sensitive to the treatment could restart the growth of the tumor, causing the progression of the disease and escape from the clinical response during treatment.

Another consequence of the subclonal presence of BRAF ^V600E is the occurrence of response at some metastatic sites and a progression in others as a result of different populations of tumor clones with individual drug sensitivity. Therefore, a therapeutic strategy based upon BRAF inhibitors might be inadequate for tumors that contain mutated BRAF in only a subpopulation, and clinical trials should consider this aspect for patient stratification. The BRAF mutation status of the tumor is a parameter that is considered in the majority of the clinical trials of new oral KIs in advanced DTC, particularly for sorafenib and other more selective KIs, to elucidate better the relationship between BRAF activity and outcome with KI treatment. In these trials, the BRAF mutation status was assessed by Sanger sequencing or real-time polymerase chain reaction. The possibility of a mixed-tumor cell population was not taken into consideration, and its therapeutic significance remains unclear. Moreover, concurrent BRAF and RAS mutations, in the same tumor but not necessarily in the same cell, was found in a few cases (17). In light of the heterogeneous composition of PTC, the association between BRAF mutation status and the response to KI treatment must be re-evaluated, taking into account the clonal/subclonal status of BRAF ^V600E. A stratification of patients based on the percentage of BRAF ^V600E-bearing cells in the tumor could reveal a significant association with outcomes, enabling the selection of patients who are most likely to respond and reducing the rate of nonresponding patients. With this aim, a new clinical trial has been registered at clinicaltrials.gov (NCT01700699). The study is designed to assess the percentage of BRAF ^V600E alleles by a quantitative assay in primary tumor tissues, synchronous metastases, and metachronous metastases in MRR-DTC patients treated with a TKI. The study could reveal an association between tumor response to TKIs and a defined BRAF ^wild-type/BRAF ^V600E ratio in the primary tumor and metastasis. The study is currently recruiting participants.