Progression free survival by the treatment with Cabozatinib in a case of BRAF mutated Radioiodine refractory recurrent metastatic Papillary Carcinoma Thyroid

The prognosis of the follicular variant of papillary thyroid carcinoma (FVPTC) falls between that of classical papillary thyroid carcinoma (cPTC) and follicular thyroid carcinoma (FTC) (1). FVPTC has lower mortality and less frequent distant metastases than FTC, but higher mortality and more frequent distant metastases than cPTC. FVPTC has fewer lymph node metastases and less frequent infiltrative disease and extrathyroidal extension than cPTC, but more than FTC. But is it a hybrid disease or a mixture of diseases? The pathological appearance of a follicular-patterned tumor with the nuclear features of cPTC suggests that FVPTC is a hybrid. However, the heterogeneous nature of the disease and the mutational profile of FVPTC strongly suggest that it is a mixture of diseases (2). It is the intersection of classical descriptive pathology and modern molecular biology that permits an understanding of this group of diseases.

Distant metastases uncommonly occur in differentiated thyroid carcinoma (DTC), but they are a frequent cause of thyroid cancer-related death. Genomic alterations in metastatic tumors, and the relationship with their corresponding primary tumors in DTC, are poorly understood. The objective of this study was to investigate whether genetic alterations in primary tumors are concordant with distant metastases in DTC patients. Surgical samples from primary and matched distant metastatic tumor pairs from 17 DTC patients, and three additional unpaired metastatic tumors from two patients, were analyzed using targeted next-generation sequencing (Ion Torrent Ampliseq cancer panel) with a focus on known recurrent somatic mutations in thyroid cancer. Additionally, TERT promoter mutations were assessed by direct sequencing. BRAF mutations were found in 8/10 patients with papillary thyroid carcinoma (PTC). A NRAS mutation was detected in one patient with follicular variant PTC. TERT promoter mutations were detected in 8/10 patients with PTC, and most were coexistent with a BRAF mutation (7/8 BRAF-positive PTC patients, and one BRAF-negative PTC patient). In follicular thyroid carcinoma, NRAS was the most frequently observed mutation (4/9 patients), followed by HRAS (two patients) and KRAS (one patient). TERT promoter mutations were found in 6/7 RAS-positive follicular thyroid carcinoma patients. Key somatic alterations such as BRAF and RAS mutations were highly concordant between primary and matched metastatic tumors without discrepancies. The BRAF or RAS mutant allelic frequency was higher in matched metastatic tumors than in the corresponding primary tumors (35% vs. 25% for BRAF mutation, p = 0.04; and 40% vs. 34% for RAS mutation, p = 0.002). TERT promoter mutations were also mostly concordant in matched tumors (concordance rate 93%). BRAF, RAS, and TERT mutations are highly prevalent in metastatic DTC, and are concordant between primary and metastatic DTC. This high concordance suggests that primary tumors may reflect the key somatic alterations in matched metastatic DTC. Frequent coexistent TERT promoter and BRAF or RAS mutations in metastatic DTC also suggests its important role in the progression of DTC.

81st Annual Meeting of the American Thyroid Association SHORT CALL ABSTRACTS

TheRomanphilosopherSenecareferstotheideathateveryso-called sin or outcome is the result of a series of complex interconnected factors. This rings true of much of biology, particularly cancer biology. Substantial developments have occurred in the last 5 to 10 years to help us better understand the molecular complexity of thyroid cancer. Among all thyroid cancers, the most common type is papillary thyroid carcinoma (PTC), which comprises approximately 75% of all thyroid cancers. Somatic BRAF mutation was first discovered in thyroid cancer in 2003. Since then, we have learned that BRAF mutation is uniquely seen in PTC and PTC-derived undifferentiated thyroid carcinomas. Prevalence of somatic BRAF mutation in adultonset PTC is approximately 45%, and in PTC-derived undifferentiated thyroid carcinoma, approximately 25%. In subtypes of PTC, the highest percentage of BRAF mutation (77%) is found in the tallcell variant of PTC, and the lowest (12%) in the follicular variant of PTC. In conventional PTC (cPTC), the BRAF mutation is found in approximately 60%. However, BRAF mutation has not been reported in follicular thyroid carcinoma, medullary thyroid carcinoma, thyroid adenoma, or nodular hyperplasia. Results of a multicenter study in PTC showed a strong association of BRAF mutation with lymphnodemetastasis,extrathyroidalextension,advanceddisease(stages III and IV), and disease recurrence. These findings have since been replicated in many independent studies. This knowledge has led to proposed BRAF-stratified surgical and medical management of PTC. In the article that accompanies this editorial, Xing et al extend our current understanding of PTC natural history by demonstrating that coexistence of BRAF p.V600E and TERT c.228C T ( 124C T; previously referred to as C228T) is associated with the worst clinicopathologic characteristics in cPTC as well as PTC subtypes. In previous publications, the authors as well as others had already demonstrated the association of TERT promoter mutations with clinically more aggressive differentiated thyroid cancers. In this study, the authors examined the presence of BRAF p.V600E and TERT 124C T mutations in a large cohort (N 507) of patients with PTC who were treated over a 12-year period at Johns Hopkins Hospital. The median follow-up was 24 months (interquartile range, 8 to 78 months). BRAF and TERT mutations were seen together in 7% of all subtypes of PTC and 7% of cPTC. Tumor recurrences per 1,000 person-years were three-fold greater in patients with either BRAF p.V600E or TERT 124C T mutations, but 8.5-fold if they had both mutations compared with patients with neither mutation. This suggests a synergistic interaction between BRAF and TERT. This is further reflected in the result thatthecoexistenceofbothmutationswasassociatedwithhigh-risk clinicopathologic characteristics, including significantly reduced tumor recurrence-free survival, even after adjusting for other risk factors. MutationsintheTERTpromoterwerefirstreportedinbothfamilial and sporadic melanomas and provided a new mechanism by which cells could acquire increased telomerase activity. There were two highly recurrent somatic mutations ( 124C T [C228T] and 146C T [C250T]) seen in sporadic melanomas, and these mutations have subsequently been associated with other cancers including thyroid, bladder, and glioblastoma. TERT promoter mutations result in twoto four-fold increased promoter activity with the additional unique featurethatthesemutationscreateadenovoCCGGAA/Tgeneralbinding motif for E-26 transcription factors/ternary complex factors (ETS/TCF), which differ from the natural state (ie, wild type) GGAA/T ETS binding site within the TERT promoter. This suggests that upregulation of ETS/ TCF transcription factors will likely enhance TERT activity. The effect of the promoter mutations on TERT expression is only possible in the presence of ETS/TCF transcription factors that bind to the newly created consensus binding site. Some of the ETS/TCF transcription factors are downstream targets of the mitogen-activated protein kinase pathway, where BRAF plays an important role. It is therefore biologically plausible that in both melanoma and thyroid cancer, activated BRAF, by enhancing the effects of TERT promoter mutation, results in poorer outcomes. The study by Xing et al is important because it provides evidence that the coexistence of both mutations is a poor prognostic marker in PTC. This suggests that any molecular-based stratification for treatment or care will need to take both mutations into account and not either BRAF p.V600E or TERT 124C T alone. Although mortality from differentiated thyroid cancer is low, patients deserve to receive the treatment that not only minimizes the likelihood of progression or recurrence, but also has the best risk-benefit and toxicity profiles. Molecular markers could enable clinicians to spare many individuals from invasive surgery and therapies that have many adverse effects. Enrollment of patients with JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 32 NUMBER 25 SEPTEMBER 1 2014

Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma

Epidemiology of Thyroid Cancer.

The pathogenesis of thyroiditis caused by immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-CTLA-4, is incompletely understood. To gain mechanistic insights, we developed a mouse model of ICI-related thyroiditis and assessed clinical, hormonal, and cytokine profiles. Forty-three NOD-H2 h4 mice (24 M, 19 F), 112 days old at the time of the first i.p. injection, were divided into four experimental groups: 20 mice received combined anti-PD-1 and anti-CLTA-4; 8 only anti-PD-1; 8 only anti-CTLA-4; and 7 an isotype control. Mice were sacrificed on day 172 (2 months after the initial injection) to collect thyroid gland (for histopathology and flow cytometry) and spleen (for flow cytometry). Mice were also studied before sacrifice to determine thyroid area and structure (by ultrasound using a MS700 transducer), thyroid function (by serum total T4 and TSH using bead-based Luminex technology), and the serum levels of numerous cytokines/chemokines (by Luminex).Thyroiditis was more severe in mice receiving anti-PD-1 than anti-CTLA-4 (p = 0.01), and associated significantly with the absolute number of CD45+ infiltrating cells (cumulative OR 1.25, 95% CI 1.1-1.4, p < 0.001). On the contrary, thyroiditis was more prevalent (100% vs 63%, p < 0.01) in the anti-CTLA-4 mice, which also showed a larger thyroid area (17 -8.2 vs 11 -4.2 mm 2 , p < 0.01) than those treated with anti-PD-1 and controls. Interestingly, mice treated with PD-1 that developed thyroiditis showed a striking increase in systemic IL-6 (40 pg/mL at baseline, 268 pg/mL on day 172), an increase not seen in the anti-CTLA-4 group (p = 0.01). IL-6 mirrored thyroiditis severity, with highest serum values found in greatest histopathology scores (cumulative OR 1.1, 95% CI 1.02-1.15, p = 0.009). GM-CSF and MIP1b increased more in the anti-CTLA-4 group (p < 0.001 for both), whereas the other cytokines/chemokines did not differ among the groups. The study reports the first mouse model of thyroiditis induced by PD-1 blockade and, comparing it to the anti-CTLA-4 model, uncovers distinctive histopathological, sonographic, hormonal, and immunological features. The study also offers biomarkers, such as serum IL-6, that could be used in the clinical setting.

Activating BRAF mutations have recently been reported in 28-83% of papillary thyroid carcinomas (PTCs). However, it is not known whether aberrant BRAF splicing occurs in thyroid carcinoma. To investigate aberrant BRAF splicing and its association with BRAF mutation in thyroid tumours, we studied aberrant BRAF splicing and BRAF mutation from 68 thyroid tumours. BRAF(V600E) mutation was detected in 20 of 43 PTCs and all three anaplastic thyroid carcinomas (ATCs). There is a higher frequency of BRAF mutation in PTC patients with stage III and IV tumours compared with stage I and II. Novel BRAF splicing variants were detected in 12 PTCs, three follicular variants of PTC (FVPTCs), and one ATC, as well as in two thyroid carcinoma cell lines, ARO and NPA. These variants did not have the N-terminal auto-inhibitory domain of wild-type B-Raf, resulting in an in-frame truncated protein that contained only the C-terminal kinase domain and caused constitutive activation of B-Raf. These variants were significantly associated with advanced disease stage and BRAF(V600E) mutation (p < 0.001, Fisher exact test). Furthermore, expression of these variants in NIH3T3 and CHO cells could activate the MAP kinase signalling pathway, transform them in vitro, and induce tumours in nude mice. These data suggest that BRAF splicing variants may function as an alternative mechanism for oncogenic B-Raf activation. Combination of the BRAF(V600E) mutation and its splicing variants may contribute towards disease progression to poorly differentiated thyroid carcinoma.

Iodine or Not (IoN) for Low-risk Differentiated Thyroid Cancer: The Next UK National Cancer Research Network Randomised Trial following HiLo

While the association of the BRAF(V600E) mutation with aggressive histopathological tumor features and clinical behavior has been extensively studied in papillary thyroid carcinoma (PTC), the BRAF(K601E) mutation has not been well characterized. This study reports what is currently the largest series of BRAF(K601E) mutated thyroid nodules. Histopathologic, cytologic, and molecular reports over a period of seven years (June 2007 to June 2014) were reviewed to identify thyroid cases with various types of BRAF mutations. All cases positive for the BRAF(K601E) mutation were reviewed to confirm histopathologic diagnosis and establish tumor variant, and clinical charts were reviewed to obtain clinical characteristics and follow-up information. The BRAF(K601E) mutation was identified in 39 patients and comprised 5.3% of all BRAF mutations noted in thyroidectomy specimens. Twenty-seven out of 29 nodules (93%) with BRAF(K601E) mutated tumors with surgical pathology results available for review were PTC, one (3.4%) was a follicular thyroid carcinoma, and one (3.4%) was a follicular adenoma. The majority of K601E-mutant PTCs (20 cases) were follicular variant PTC. Encapsulation was present in all but one case, and one case showed capsular invasion. Coexisting mutations overall were not identified in BRAF(K601E) mutated thyroid nodules except in a case that exhibited a complex K601E + T599I mutation and had a classic PTC phenotype. The majority of K601E mutant nodules were T1 lesions (69%) and T2 lesions (28%) by TNM staging. With a median follow-up of 19.6 months, no structural or biochemical recurrence or metastases were found in patients with an isolated BRAF(K601E) mutation. The BRAF(K601E) mutation is the second most common BRAF mutation found in thyroid nodules. Unlike BRAF(V600E), the most common mutation, K601E is strongly associated with follicular-patterned cancer, particularly with the encapsulated follicular variant of PTC, and may also be found in follicular thyroid carcinomas. Overall, BRAF(K601E) mutant tumors show better clinical outcomes than BRAF(V600E) positive tumors, and preoperative BRAF(K601E) analysis may provide important prognostic information for use in clinical management.

Whether mutation status should be used to guide therapy is an important issue in many cancers. We correlated mutation profile in radioiodine-refractory (RAIR) metastatic thyroid cancers (TCs) with patient outcome and response to tyrosine kinase inhibitors (TKIs), and discussed the results with other published data. Outcome in 82 consecutive patients with metastatic RAIR thyroid carcinoma prospectively tested for BRAF, RAS and PI3KCA mutations was retrospectively analyzed, including 55 patients treated with multikinase inhibitors. Papillary thyroid carcinomas (PTCs) were the most frequent histological subtype (54.9 %), followed by poorly differentiated thyroid carcinoma [PDTC] (30.5 %) and follicular thyroid carcinoma [FTC] (14.6 %). A genetic mutation was identified in 23 patients (28 %) and BRAF was the most frequently mutated gene (23 %). Median progression-free survival (PFS) on first-line TKI treatment was 14.6 months (95% CI 9.9-18.4). BRAF mutation positively influenced median PFS, both in the entire TKI-treated cohort (median PFS 34.7 months versus 11.6 months; hazard ratio [HR] 0.29; 95% CI 0.09-0.98; p = 0.03) and in the TKI-treated PTC cohort (n = 22) [log-rank p = 0.086; HR 2.95; 95 % CI 0.81-10.70). However, in TKI-treated patients, PDTC histologic subtype was the only independent prognostic factor for PFS identified in the multivariate analysis (HR 2.36; 95% CI 1.01-5.54; p = 0.048). Patients with BRAF-mutant PTC had a significantly longer PFS than BRAF wild-type when treated with TKIs. However, due to the small number of BRAF-mutant patients, further investigations are required, especially to understand the potential positive effect of BRAF mutations in RAIR TC patients while having a negative prognostic impact in RAI-sensitive PTC patients.

To explore the invasiveness of papillary thyroid microcarcinoma(PTMC)with BRAF mutation. Totally 99 patients with PTMC with BRAF mutation were enrolled in this study, meanwhile another 97 patients with papillary thyroid carcinoma (PTC) (tumor size>1 cm)with BRAF mutation were included as controls. The clinicopathologic factors including extrathyroidal invasion, multifocality, and distant metastasis were analyzed. The rates of extrathyroidal invasion and nodal metastasis in PTMC group were as high as 16.10% and 71.74%, respectively. In the PTMC group and PTC group,the extrathyroidal invasion rate was 16.10% and 39.18%, cervical lymph node metastasis rate was 71.74% and 91.75%, and distant metastasis rate was 1.01% and 9.28%, respectively. In the PTMC subgroups with tumor sizes ≤0.3 cm, 0.3-0.6 cm, and 0.6-1.0 cm, the cervical lymph node invasion rate was 60.00%, 72.50%, and 73.81%, the extrathyroidal invasion rate was 10.00%, 9.09%, and 24.44%, and the multifocality rate was 60.00%, 38.64%, and 57.78%, respectively. Univariate analysis showed that the tumor size was not significantly correlated with multifocality (Χ (2)=3.752, P=0.153), cervical lymph node metastasis (Χ (2) = 0.780,P = 0.677), extrathyroidal invasion (Χ (2) = 4.182, P = 0.124), and distant metastasis (Χ (2)=1.212, P = 0.545). While the BRAF group and PTC group were not significantly different in multifocality (Χ (2) = 1.742, P=0.187), they were significantly different in terms of extrathyroidal invasion (Χ (2) = 13.000, P = 0.000), nodal involvement (Χ (2) = 12.819, P = 0.000), and distant metastasis (Χ (2) = 5.316, P = 0.021). Multivariate analysis showed that nodal metastasis was independently associated with size>1 cm (P=0.001) and extrathyroidal invasion (P=0.003). BRAF mutant PTMC manifests relative high extrathyroidal involvement and nodal metastasis, and the similar multifocality as BRAF mutant PTC. Radioactive iodine should be considered in PTMC with the presence of BRAF mutation combined with extrathyroidal invasion or nodal metastasis.

BackgroundThe molecular etiology of thyroid carcinoma (TC) and other thyroid diseases which may present malignant precursor lesions is not fully explored yet. The purpose of this study was to estimate frequency, type and clinicopathological value of BRAF exon 15 mutations in different types of cancerous and non-cancerous thyroid lesions originating in an ethnically diverse population.MethodsBRAF exon 15 was sequenced in 381 cases of thyroid lesions including Hashimoto´s thyroiditis, nodular goiters, hyperplastic nodules, follicular adenomas (FA), papillary TC (PTC), follicular variant PTC (FVPTC), microcarcinomas of PTC (micro PTC; tumor size ≤ 1 cm), follicular TC (FTC), and non-well differentiated TC (non-WDTC).ResultsWe identified BRAF mutations in one of 69 FA, 72 of 115 (63%) PTC, seven of 42 (17%) FVPTC, 10 of 56 (18%) micro PTC, one of 17 (6%) FTC, and one of eight (13%) non-WDTC. Most of the cases showed the common V600E mutation. One case each of PTC, FVPTC, and FTC harbored a K601E mutation. A novel BRAF mutation was identified in a FA leading to deletion of threonine at codon 599 (p.T599del). A rare 3-base pair insertion was detected in a stage III PTC resulting in duplication of threonine at codon 599 (p.T599dup). Patients with PTC harboring no BRAF mutation (BRAFwt) were on average younger than those with a BRAF mutation (BRAFmut) in the PTC (36.6 years vs. 43.8 years). Older age (≥ 45 years) in patients with PTC was significantly associated with tumor size ≥ 4 cm (P = 0.018), vessel invasion (P = 0.004), and distant metastasis (P = 0.001). Lymph node (LN) involvement in PTC significantly correlated with tumor size (P = 0.044), and vessel invasion (P = 0.013). Of notice, taken the whole TC group, family history of thyroid disease positively correlated with capsular invasion (P = 0.025).ConclusionsOlder age is manifold associated with unfavorable tumor markers in our series. The K601E identified in a PTC, FVPTC, and FTC seems to be more distributed among different histological types of TC than previously thought. The T599del is a yet undescribed mutation and the rare T599dup has not been reported as a mutation in PTC so far.

Mutational analysis is reshaping the practice of fine-needle aspiration cytology for the diagnosis of thyroid nodules. The v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) valine (V) to glutamic acid (E) substitution at codon 600 (BRAF(V600E)) is the most effective diagnostic/prognostic marker and is used mainly for papillary thyroid carcinomas (PTCs). Although BRAF(V600E) represents 95% of all BRAF mutations, uncommon BRAF mutations have been identified in thyroid carcinomas. For the current study, the authors evaluated morphologic (plump pink cells and sickle-shaped nuclei) anti-BRAF(V600E) antibody (VE1) immunocytochemical and molecular findings of BRAF mutations in PTCs and in the follicular variant of PTC (FVPC). Between January 2013 and June 2014, there were 150 cytologic samples with surgical follow-up at the authors' institution. BRAF mutations, which were identified using liquid-based cytology, were classified into wild-type BRAF, BRAF(V600E), and uncommon BRAF mutations. All clinicopathologic correlations between BRAF and FVPCs were analyzed. Forty-four of 150 samples were identified as benign histologic lesions, and the authors focused on the 106 cytologic samples from patients who had malignant outcomes (60 PTCs and 46 FVPCs). The series included 16 follicular neoplasms, 36 samples diagnosed as suspicious of malignancy, and 54 samples diagnosed as positive for malignancy. The BRAF(V600E) mutation was detected in 17.4% of FVPCs and in 66.6% of PTCs, whereas uncommon BRAF mutations were detected only in FVPCs. Plump pink cells and VE1 expression were not identified in samples that had uncommon BRAF mutations. VE1 immunocytochemistry yielded positive results in all 36 samples that had the BRAF(V600E) mutation. Uncommon BRAF mutations were observed only in FVPCs and were linked to less aggressive behavior. Negative/weak VE1 expression was observed in both wild-type and uncommon BRAF mutations. The current investigation did not reveal any plump cells or morphologic BRAF findings in samples that had uncommon BRAF mutations. In the authors' experience, BRAF mutations detected by DNA methods were more accurate in identifying FVPCs.

Rearrangement of RET proto-oncogene is the major event in the etiopathogenesis of papillary thyroid carcinoma (PTC). We report a high prevalence of BRAF(V599E) mutation in sporadic PTC and in PTC-derived cell lines. The BRAF(V599E) mutation was detected in 23 of 50 PTC (46%) and in three of four PTC-derived cell lines. The prevalence of the BRAF(V599E) mutation in PTC is the highest reported to date in human carcinomas, being only exceeded by melanoma. PTC with RET/PTC rearrangement as well as the TPC-1 cell line (the only one harboring RET/PTC rearrangement) did not show the BRAF(V599E) mutation. BRAF(V599E) mutation was not detected in any of 23 nodular goiters, 51 follicular adenomas and 18 follicular carcinomas. A distinct mutation in BRAF (codon K600E) was detected in a follicular adenoma. Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC. BRAF(V599E) mutation did not coexist with alterations in any of the RAS genes in any of the tumors. These results suggest that BRAF(V599E) mutation is frequent in the etiopathogenesis of PTC. The BRAF(V599E) mutation appears to be an alternative event to RET/PTC rearrangement rather than to RAS mutations, which are rare in PTC. BRAF(V599E) may represent an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation.