 may be associated with an aggressive phenotype and higher recurrence rates for patients with locally advanced well-differentiated thyroid cancers, according to findings presented at the 2015 International Thyroid Congress.

"These advanced thyroid cancers seem to have a high percentage of multiple mutations," lead investigator Umamaheswar Duvvuri, MD, PhD, assistant professor of otolaryngology specializing in head and neck surgery at the University of Pittsburgh School of Medicine, told 

. "Most thyroid cancers have a mutation in the gene 

, which is very common; however, most thyroid cancers, small ones at least, tend to only have one mutation, whereas these advanced ones have multiple mutations, most commonly 

. We think these genes corroborate to allow bad thyroid cancers to form."

In recent years, the incidence of well-differentiated thyroid cancers has increased at an alarming rate, with a near doubling of incidence over the past 6 years, Duvvuri highlighted. As a result, researchers sought to uncover the distinct molecular profiles that could be used as prognostic indicators of future aggressiveness using next-generation sequencing (NGS). 

For the study, researchers at the University of Pittsburgh put together a series of mutational panels focusing on the major signal-transducing players controlling papillary thyroid carcinoma oncogenes. This started with a 7-gene panel in 2007, followed by a 15-gene panel by 2013, and then a 56-gene panel in 2014 that employs NGS. Through this process, over 95% of thyroid cancer alterations have been identified, according to Duvvuri.

For the study, researchers selected a group of T4 well-differentiated thyroid cancers that did not have any anaplastic or poorly differentiated areas. All patients were treated with curative intent. Molecular testing was conducted using the ThyroSeq v2.1 panel, which explores 14 point mutations, 42 gene fusions, and 7 cell composition genes.

Overall, 25 samples were procured, processed, and analyzed from patients at a mean age of 58 years (range, 29-86) with well-differentiated T4 thyroid carcinomas. Findings from these samples were compared with a control group of 102 patients with T1-3 well-differentiated thyroid tumors.

 alterations (56%). Only one patient out of 25 had no identifiable mutation, suggesting that most of the pertinent mutations were currently identifiable, since alterations were detected in 96% of patients. In the control group, a total of 45 patients had 

Nearly 48% of the patients with T4 tumors had coexisting mutations in 

. In contrast, in the control group, the rate of coexisting 

 <.001). In the advanced cohort, coexisting mutations in 

 were associated with an increased risk of recurrence when compared to tumors without both alterations. Overall, 58.3% of those with both alterations had a recurrence compared with just 18.3% with a separate 

 mutations are associated with more aggressive cancers, the most aggressive thyroid cancers tend to have multiple mutations. These findings may indicate that comutation in 

 are more distinct in advanced thyroid cancer, Duvvuri pointed out.

	Duvvuri U, Grimes M, Mady L, et al. Multi-gene next generation sequencing (thyroseq) assay on locally invasive T4 well differentiated thyroid cancer.  Presented at: International Thyroid Congress; October 18-23, 2015; Orlando, FL. Abstract 461.

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Coexisting mutations in BRAF and TERT may be associated with an aggressive phenotype and higher recurrence rates for patients with locally advanced well-differentiated thyroid cancers.

Coexisting BRAF/TERT Mutations Predict Aggressive Thyroid Cancer

A phase II Mayo Clinic consortium study confirmed the significant clinical activity seen with pazopanib (Votrient) for patients with radioactive iodine (RAI)-refractory metastatic differentiated thyroid cancer (DTC); however, the trial was unable to uncover an effective predictive biomarker for the multikinase inhibitor, according to findings presented by Keith Bible, MD, PhD, at the 2015 International Thyroid Congress.  

In the final analysis of the phase II study, pazopanib demonstrated a partial response rate of 37% in patients with pretreated RAI-refractory DTC (95% CI, 24.6-50.1), which Bible labeled as a high-level of response. The primary biomarker explored in the study was serum thyroglobulin (Tg), which did not serve as a predictor of outcome.

Pazopanib is an inhibitor of c-KIT, FGFR, PDGFR and VEGFR that is FDA-approved as a treatment for patients with soft tissue sarcoma or renal cell carcinoma. Investigators are attempting to identify markers that can help with early prediction of outcomes, “so as not to have to continue treatment for a longer period of time,” according to Bible, professor of oncology at the Mayo Clinic.

“Most tyrosine kinase inhibitors are promiscuous, having a footprint across the kinome that is very messy and seems to have activity for thyroid cancer, but I think we are still struggling to know how that footprint varies,” he said. “It is striking to me that we can often go from one kinase inhibitor to the next and get sequential responses, when in theory they are all at least primarily targeting VEGFR. So I think we have a lot to learn about off-target effects and what is really most important in these agents and what is most beneficial in these patients.”

At the meeting, Bible presented data that focused on the primary endpoint of changes in serum Tg post-initiation of pazopanib. Response was analyzed as a secondary endpoint. The study enrolled 60 patients with RAI-refractory DTC from across the United States, Asia, and Australia. A majority of those treated with pazopanib (91.7%) had received prior systemic therapy following RAI.

Following the first cycle of pazopanib, Tg changes did not differ between those who did and did not respond to therapy. Among those with a partial response, the overall Tg nadir level represented a median of decline of 86.8% (interquartile range [IQR], 70.9-90.7). In those with stable disease, the nadir was reduced by 69% from baseline (IQR, 27.7-78.1). A comparison of the two arms was significant (

Despite this difference, changes in Tg could not be confirmed as a robust predictor of response to pazopanib, particularly since an initial decline was not evident. In addition to Tg levels, mutations in 

 were identified in 11 of 16 patients examined (69%). However, none of these mutations correlated with treatment response.

Overall, adverse events (AEs) in the trial were consistent with expectation, and no deaths were attributed to pazopanib, Bible emphasized. Three of the 60 patients discontinued the study without tumor burden reevaluation.

Consistent with the class, Bible pointed out that hypertension was the most common AE associated with pazopanib for patients with RAI-refractory DTC. Other grade ≥3 AEs included fatigue (n = 8; 13.3%), decreased neutrophil count (n = 8; 13.3%), diarrhea (n = 7; 11.6%), hand-foot syndrome (n = 7; 11.6%), and increased AST/ALT liver enzymes (n = 6; 10%). 

Investigators will continue to work toward identifying a marker that can serve as a useful indicator response for VEGFR tyrosine kinase inhibitors. Additionally, studies are continuing to assess pazopanib in patients with thyroid cancer. In addition to DTC, a phase II study sponsored by the NCI is looking at intensity-modulated radiation therapy and paclitaxel with or without pazopanib for patients with anaplastic thyroid cancer. This study hopes to enroll 121 participants (NCT01236547).

	Bible KC, Suman VJ, Molina JR, et al. Pazopanib in patients with progressive, radioactive iodine-refractory, metastatic differentiated thyroid cancer lacking thyroglobulin antibodies: Mayo Phase 2 Consortium Study Results. Presented at: International Thyroid Congress; 18-23, 2015; Orlando, FL. Abstract 103.

Pazopanib demonstrated significant clinical activity for patients with RAI-refractory differentiated thyroid cancer; however, a predictive biomarker for the therapy could not be uncovered.

Pazopanib's Activity in Thyroid Cancer Not Predicted by Thyroglobulin

Researchers based at the Institute of Pathology, University Hospital Essen, Germany, identified molecular markers in medullary thyroid cancer (MTC) that were associated with response to vandetanib (Caprelsa) and the development of metastases.

The TKI vandetanib was approved in 2011 for the treatment of symptomatic or progressive MTC in patients with unresectable, locally advanced, or metastatic disease. The goal of the research, which was presented at the 2015 International Thyroid Congress, was to identify biomarkers that can be used to identify patients early in treatment who will derive a benefit from the drug.

An early determination of likely drug responsiveness may assist in decisions to avoid unnecessary serious adverse events (AEs) associated with TKIs and inform medical oncologists that another drug should be considered. Alternatively, with a favorable early-response molecular profile, the therapeutic course would be maintained.

Presenting at the Congress, University Hospital Essen physician Vera Tiedje, MD, described molecular profiling from 32 patients with MTC at different tumor stages (n = 10 pN0cM0; n = 9 pN1cM0; n=14 pN1p/cM1) with a median age of 56 years and a follow-up time of 42 months. Nine of the patients died over this observation period. Ten of the patients were started on vandetanib treatment due to progressive disease, based on RECIST criteria.

Molecular profiling comparisons were made between the patients’ own primary versus more progressive tumor tissues. Tiedje et al examined the mRNA expression level of 33 different tyrosine kinase genes using nCounter NanoString. Next-generation sequencing was used to determine the 

Twenty-five percent of MTCs are caused by germline mutations in the 

 gene. These are considered familial MTCs. While sporadic MTCs are not familial, they do most often possess acquired somatic 

 mutations that are detectable in some cells of sporadic MTC.

 mutations in 65% (21/32 samples) of patients. The 

mutation is the highest risk mutation, and it was also the most common mutation detected, at 57% (8/14) of patients in the pN1p/cM1 MTC arm.

  = .042) mRNA expression was significantly lower in MTC of the pN1cM0/pN1pcM1 arm compared with the pN0cM0 arm. By contrast, 

 = .026) mRNA expression was significantly higher in MTC of the pN1cM0/pN1pcM1 arm compared with the pN0cM0 arm.

Five of 10 patients treated with vandetanib showed a partial response. All of these responders had the 

  =.042) mRNA expression was distinctly higher in primary tumor tissues of patients responding to vandetanib therapy.

Together these are encouraging results, which suggest that molecular profiling may be useful to determine the likelihood of progressive metastatic MTC development. Most significantly, the early determination of whether or not the cancer is responding to vandetanib therapy. Data indicates that the early monitoring of 

may offer early hints about whether or not the patient should stay on vandetanib therapy.

Tiedje et al identified distinct characteristic changes in the levels of tyrosine kinases associated with progression from primary tumor to metastatic progression encoding mRNAs for 

Of even more immediate practical significance, changes in the level of 

 were seen in the patients that favorably responded to vandetanib therapy.

	Tiedje V, Ting S, Walter R, et al. Tiedje V. Prognostic markers and response to vandetanib therapy in sporadic medullary thyroid cancer patients. Presented at: 2015 International Thyroid Congress; October 18-23, 2015; Orlando, FL. Abstract 492.

Researchers have identified molecular markers in medullary thyroid cancer that were associated with response to vandetanib and the development of metastases.

Tumor Profiling Identifies Potential Biomarkers for Vandetanib in MTC

Dual targeting of BRAF V600E—positive papillary thyroid carcinoma (PTC) with the anti-

 agent vemurafenib (Zelboraf) and the angiogenesis inhibitor sorafenib (Nexavar) showed promising clinical activity, according to research presented at the 2015 International Thyroid Congress.

Sorafenib was approved in 2013 for the treatment of locally recurrent or metastatic, progressive differentiated thyroid carcinoma refractory to radioactive iodine treatment. Antitumor activity with vemurafenib in 

 V600E—positive PTC was previously demonstrated in a phase II study presented at the 2013 European Cancer Congress. In the study, the median progression-free survival in treatment-naïve patients who received vemurafenib was 15.6 months.

Carmelo Nucera, MD, PhD, assistant professor at Harvard Medical School, and colleagues used cell cultures of human metastatic/recurrent 

 -PTCs and tested the effects of vemurafenib versus sorafenib used as single agents or in combination. To date, they have used predominately three types of assays. These include biochemical assays of enzyme activity along with two dynamic cellular assays—measures of cell-cycle proliferation and in vitro microenvironmental analysis: migration and motility.

 PTCs responded to vemurafenib than sorafenib. In contrast, with wild-type 

 cells, the BRAF inhibitor does not work and sorafenib works very well. Ultimately the combination overlaps in activity, Nucera said.

The key, according to Nucera, is tumor heterogeneity, and therefore, the tumor microenvironment needs to be considered. Macrophages, for example, are an important component in the anaplastic pericyte microenvironment. Macrophages and endothelial cells exist in this microenvironment.

Nucera’s group observed that the pericytes act like stem cells, interacting with endothelial cells to regulate angiogenesis. Pericytes spread after exposure to various signaling molecules like PDGFR-β, according to Nucera. Pericytes have a strong capability to produce an extracellular matrix, which may ultimately confer advantage to PTC cells to promote their migration and invasion.

Higher PDGFR-β was observed by Nucera et al in BRAF V600E cells than in the wild-type equivalent, but addition of a BRAF inhibitor caused down-regulation of PDGFR-β activation. No response was seen after sorafenib treatment, while the combination belies the treatment of the single sorafenib agent.

Nucera’s study preliminarily suggests that the combination of an antiangiogenic compound and a BRAF inhibitor may be more effective than either agent alone, not only in blocking the proliferation of PTC cells, but also in preventing cell types like pericytes in the microenvironment from promoting otherwise reproductive advantages to PTC cells.

Nucera highlighted that these results indicate for the first time that PTC cells express VEGFR2, and this dual combination treatment with anti—

 and antiangiogenesis therapy is likely to improve therapeutic response in metastatic 

—positive PTC cells by directly reducing cell survival and migratory capacities via pERK1/2 and pPDGFR-β down-regulation.

Richard Kloos, MD, cochair for the session and professor of Medicine at Ohio State University, stated that the value of adding sorafenib must be that the BRAF activity is not strong enough. Kloos then asked whether or not the sorafenib effect is really a dose effect working through 

Nucera elaborated that the BRAF inhibitor is working on the BRAF system. Therefore, if you use a pan¬—tyrosine kinase inhibitor (TKI) like sorafenib, you can improve the activity to kill any tumor cell, said Nucera. The rationale for using an antiangiogenic agent is that you have a human system with many different cells with many different TK targets. In Nucera’s observations, combining the BRAF inhibition with a pan-TKI will fundamentally work on any tumor cell.

	Sisms J, Antonello Z, Nucera C, et al. Combinatorial targeting with anti-BRAFV600E and anti-angiogenesis therapy enhances cell death and suppresses cell migration in metastatic papillary thyroid carcinoma. Presented at: 2015 International Thyroid Congress; October 18-23, 2015; Orlando, FL. Abstract 106.

Dual targeting of BRAF V600Eâ€“positive papillary thyroid carcinoma with the anti-BRAF V600E agent vemurafenib and the angiogenesis inhibitor sorafenib showed promising clinical activity.

Vemurafenib/Sorafenib Combo Shows Promise in Papillary Thyroid Carcinoma

A large survey of endocrinologists found wide variation in management strategies for thyroid nodules that were frequently misaligned with current guidelines, particularly for biopsies by fine-needle aspiration (FNA), according to findings presented by Nicole O. Vietor, MD, at the 2015 International Thyroid Congress.

In the survey, which was the first to document current management strategies for thyroid nodules, respondents provided feedback on 6 cases, including 5 low-risk nodules and 1 high-risk case. For the high-risk example, which was the only scenario indicated for FNA in most guidelines, 93.8% of respondents said they would use FNA. For the low-risk groups, which were not indicated for FNA, 40.5% said they would conduct the biopsy.

In addition to variations in the use of biopsy, the survey revealed greater utilization of molecular screening of thyroid nodules, particularly in North America, where 73.9% of respondents said they would use routine molecular analysis. In Europe, Asia, and other international locations, molecular analysis utilization dropped to approximately 40%.

"Management of a thyroid nodule is highly variable and differs from societal guidelines in multiple areas," Vietor, of the Walter Reed National Military Medical Center, Bethesda, and colleagues wrote in their abstract. "Regional differences exist with regard to FNA, particularly the use of molecular analysis."

To perform the study, surveys were collected from 897 members of the American Thyroid Association (ATA), American Association of Clinical Endocrinologists (AACE), and The Endocrine Society (TES). Respondents were from North America (63%), Europe (12%), Asia (6.5%), the Middle East/Africa (5.6%), and Oceania (1.9%).

For the use of FNA, respondents were asked whether they would perform the procedure on the following nodules: 1.5-cm hypoechoic nodule (94% said, yes); 0.7-cm hypoechoic nodule with calcifications (67%); 1.4-cm complex-cystic nodule (55%); 1.2-cm isoechoic nodule (42%); 1.8-cm spongiform nodule (37%); or, a 2.5-cm purely cystic nodule (24%). 

Consistent with current guidelines, 99.4% of respondents indicated that they would test for thyroid stimulating hormone (TSH). However, vast regional differences were seen for the use of a serum calcitonin assay, which is used to detect C-cell hyperplasia and medullary thyroid cancer.

Several guidelines for the evaluation of thyroid nodules recommend routine serum calcitonin testing; however, the overall utilization in the survey was just 8.4%. Of those surveyed, roughly 32% of European respondents said they would use a serum calcitonin assay compared with just 2.3% of those from North America. For Asia, Latin America, and the Middle East, calcitonin testing rates were 14%, 9%, and 6% respectively.

Approximately half of those surveyed responded that they would perform a thyroid ultrasound, which is advocated for in most guidelines. Ultrasounds were recommended for 83% of FNAs. A 25-gauge needle was selected most commonly (44%) followed by 27-gauge (15%). Approximately a third of respondents did not use any pain reduction methods for the FNA procedure, while some physicians used a topical gel (9%), ice pack (8%), or ethyl chloride spray (8%) to decrease pain.

For follicular lesions of undetermined significance, the majority of respondents (38%) would use molecular analysis, followed by repeated cytology (20%) and lobectomy (20%). For a follicular neoplasm, about 47% would perform a lobectomy with 29% performing molecular analysis and 15% performing a total thyroidectomy. For a suspicious malignancy the results were “much more split,” according to Vietor, with 43% performing total thyroidectomy and another 43% performing lobectomy. Roughly 9.55% would perform molecular analysis.

Cytopathologists were primarily responsible for interpreting FNA results (73%). Assessment of FNA was also frequently performed by a pathologist (18%), a commercial laboratory (8%), or in rare cases, an endocrinologist (1%).                   

In addition to commenting on each case scenario, respondents were asked about pregnancy issues, for example, whether or not physicians would reconsider performing FNA in a 22-year-old woman at 8 weeks gestation with a 1.5-cm nodule. “Results were split, with 34% stating that they would postpone the procedure and 37% stating they would perform the procedure now,” Vietor explained.

Additionally, to account for age, respondents were asked if they would reconsider performing the FNA procedure on an 82-year-old person with otherwise similar criteria involving no previous history of thyroid disease but a 1.5-cm thyroid nodule. Approximately 64% said they would no longer perform the FNA given the advanced age.

	Vietor NO, Burman K, Cooper D, et al. A 2015 survey of endocrinologists on the management of thyroid nodules. Presented at: International Thyroid Congress; October 18-23, 2015; Orlando, FL. Abstract #10.

A large survey of endocrinologists found wide variation in management strategies for thyroid nodules that were frequently misaligned with current guidelines, particularly for biopsies by fine-needle aspiration.

W. Todd Penberthy, PhD

Articles

Coexisting BRAF/TERT Mutations Predict Aggressive Thyroid Cancer

Pazopanib's Activity in Thyroid Cancer Not Predicted by Thyroglobulin

Tumor Profiling Identifies Potential Biomarkers for Vandetanib in MTC

Vemurafenib/Sorafenib Combo Shows Promise in Papillary Thyroid Carcinoma

Survey Reveals Potential Overuse of Thyroid Nodule Biopsy