A next-generation sequencing (NGS) assay has shown promise as a new tool to aid in the diagnosis and treatment of indeterminate thyroid nodules, according to research presented at the 2014 American Thyroid Association (ATA) Annual Meeting.

“We developed a novel NGS-based assay designed to provide targeted mutation detection to aid in the diagnosis of indeterminate thyroid nodules from fine needle aspirate [FNA] and formalin-fixed paraffin-embedded [FFPE] tissue samples,” said lead author Shih-Min Cheng, PhD, of Quest Diagnostics in San Juan Capistrano, California.

Although FNA cytology provides a definitive diagnosis of either malignant or benign for the majority of thyroid nodules, about 25% of nodules have indeterminate cytomorphology. Based on draft 2014 ATA Guidelines, two categories of FNA cytology results may be clinically managed by conducting repeat FNA or molecular testing. Samples are categorized as atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS) in approximately 7% of cases and carry a risk of malignancy of 5% to 10%. Another 5% to 10% of cytology results are classified as follicular or Hürthle cell neoplasm (FN) or suspicious for FN (SFN) with a risk of malignancy of 20% to 30%. It is these two categories of indeterminate FNA cytology results for thyroid nodules that are particularly challenging for clinicians to decide how to appropriately manage.

The NGS panel for indeterminate thyroid nodules was based on a seven-gene polymerase chain reaction (PCR)—based assay that was then expanded to include additional molecular markers based on new gene mutations and gene translocations identified in the Cancer Genome Atlas (TCGA) or recent literature publications.

The types of specimen used in this study included indeterminate thyroid nodules obtained through FNA or FFPE or from lobectomy/thyroidectomy specimens (FFPE). DNA was extracted from thyroid FNA and FFPE samples and was quantified fluorometrically. The assay performed well with limited biopsy/tissue materials, as the mutational panel minimum DNA input was 1 to 10 ng, and it carried a 5% analytic assay sensitivity.

A two-step multiplex PCR was used to enrich the targeted exonic regions of 13 specific genes, including 

, plus the TERT promoter region. “The targeted amplification protocol covers all known hot-spot mutations,” said Cheng. Mutation frequency cutoff for clinical sample reporting was set at 5% to 10%.

After purification, the amplicons were sequenced using the Illumina MiSeq desktop sequencing platform. A TruSeq amplicon workflow was used to analyze coverage and detect mutations, and a positive control with known mutation frequencies was included in each run to ensure optimal assay performance. Data were analyzed using MiSeq analysis software, Quest Sequencing Analysis Pipeline (QSAP), and Integrative Genomics Viewer. A computer simulation of the assay calculated a clinical assay sensitivity of 89% or higher.

Using as little as 1 ng of DNA in the assay, the system was able to detect single-nucleotide variants and insertions and deletions to a 5% sensitivity level. Mutations in a cell line control mixture were also detected by the panel at the expected frequencies. Indeterminate FNA and FFPE tissue samples that had previously been tested for 

 mutations were also sequenced, and all previously identified mutations were identified with no false-negative results. However, this panel assay also detected mutations in the TERT promoter and others that had not been identified initially.

 mutation—positive results from the current assay and increased the mutation detection rate in FNA specimens. Cheng cautioned that these results are preliminary and that clinical validation is ongoing, however he is pleased with the outcomes thus far.

“Importantly, the assay [we developed] meets the challenges of limited DNA amounts and variable quality often associated with FNA and FFPE clinical specimens. The approach offers the opportunity to streamline the characterization of genomic alterations in thyroid cancers to aid in thyroid cancer diagnosis,” concluded Cheng.

	Cheng S, Lew D, Barlan C, et al. Development of a clinical targeted next-generation sequencing test for indeterminate thyroid nodules. Presented at: Annual Meeting of the American Thyroid Association; October 29 — November 2, 2014; Coronado, CA. Abstract 125.

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A next-generation sequencing assay has shown promise as a new tool to aid in the diagnosis and treatment of indeterminate thyroid nodules.

Novel NGS Test May Enhance Diagnosis and Management of Indeterminate Thyroid Nodules

The ThyroSeq v2 next-generation sequencing (NGS) assay was highly accurate at diagnosing cancer in thyroid nodules with follicular neoplasm or those suspected of follicular neoplasm cytology, according to Yuri Nikiforov, MD, presenting at the 2014 Annual Meeting of the American Thyroid Association.

The most common approach used to evaluate thyroid nodules is fine needle aspiration (FNA) cytology, but about 20% to 30% of FNA results have indeterminate cytology. A common indeterminate diagnosis, follicular (or oncocytic) neoplasm/ suspicious for a follicular (or oncocytic) neoplasm (FN/SFN), carries a risk of cancer of 15% to 30% and is typically managed with surgical lobectomy.

"In this study, we tested whether the most complete next-generation sequencing panel of genetic markers can significantly improve cancer diagnosis in these nodules," said Nikiforov, vice-chair for Molecular Pathology and director, Division of Molecular and Genomic Pathology at the University of Pittsburgh Medical Center.

NGS uses massively parallel sequencing of clonally amplified DNA templates to sequence up to 10

 bases with a sensitivity of 3% to 5% at a cost of about $0.05 to $1.00 per 10

 bases, and is able to investigate multiple regions of the genome up to the whole genome.

The objectives of this study were to create the most complete panel of genetic markers for thyroid FNAs based on NGS and to explore the panel performance for cancer diagnosis in nodules with FN/SFN (Bethesda System Diagnostic Category IV) cytology.

To do this, a total of 143 consecutive FNA samples with the cytologic diagnosis of FN/SFN and known surgical outcome were evaluated. Of these, 91 samples were studied retrospectively, and 52 samples were studied prospectively. The performance characteristics of the two cohorts were compared by testing for similarity in true positive fraction (TPF), false positive fraction (FPF), positive predictive value (PPV), and negative predictive value (NPV), and were found to be similar.

Molecular analysis was performed using targeted NGS on Ion Torrent PGM (318 chip) using the ThyroSeq v2 panel that simultaneously tests for point mutations in 13 genes, including BRAF, NRAS, HRAS, KRAS, PTEN TP53, TSHR, GNAS, CTNNB1, RET, PIK3-CA, AKT1, TERT, and 42 gene fusions involving the RET, BRAF, NTRK1, NTRK3, ALK, PPARG, and THADA genes. The cell type composition of FNA samples and the expression of oncogenes resulting from gene fusions were assessed by the expression of 15 genes.

In evaluating the rate of informative testing, 99.3% of samples worked for mutations, and 98% of samples worked for gene fusion and gene expression analysis. The overall analytical sensitivity was 3% to 5% of mutant alleles (6% to 10% of cells) with mutation and 1% of cells with gene fusion.

Using histology, 39 malignant and 104 benign nodules were detected, for an overall cancer rate of 27.3%. In these samples, the NRAS (n =16), KRAS (n = 6), TERT (n = 4), and TSHR (n = 3) genes were those that were most commonly affected by point mutations. These molecular alterations in FNA samples were associated with an 81%, 83%, 100%, and 33% risk of cancer, respectively. Two nodules displayed concurrent NRAS and TERT mutations. Three gene fusions were identified, including THADA (n = 5), PPARG (n = 4), and NTRK3 (n = 2). All three gene fusions were associated with a 100% risk of cancer.

In the overall cohort, the 60-gene ThryoSeq v2 panel performed well with a high sensitivity (90%; 95% CI, 80%—99%) and specificity (93% CI, 88%–98%), resulting in a PPV of 83% (95% CI, 72%–95%) and a NPV of 96% (92%–100%) in nodules with FN/SFN cytology. The overall accuracy of the panel was 92% (88%–97%).

"High NPV and PPV is expected in populations with variable disease prevalence (14% to 34%)," Nikiforov explained. This comprehensive genotyping panel may also have additional benefits in terms of both tumor prognostication (BRAF, TERT, and TP53) and in identifying targets for therapy (BRAF/RAS, ALK, NTRK3).

"The results of this study indicate that comprehensive genotyping of thyroid nodules using a broad NGS panel provides a highly accurate diagnosis for nodules with FN/SFN cytology and should facilitate optimal management of these patients," the researchers concluded.

The ThyroSeq v2 next-generation sequencing assay was highly accurate at diagnosing cancer in thyroid nodules with follicular neoplasm or those suspected of follicular neoplasm cytology.

Next-Generation Sequencing Effectively Detects Cancer in Thyroid Nodules

Preoperative vitamin D deficiency (VDD) is associated with postoperative symptomatic hypocalcemia in patients with thyroid cancer undergoing total thyroidectomy (TT) plus central compartment neck dissection (CCND), according to an oral presentation by Kee-Hyun Nam, MD, PhD, at the 2014 American Thyroid Association (ATA) Annual Meeting.

"Although some studies have reported that preoperative vitamin D deficiency is a risk factor for hypocalcemia after total thyroidectomy in patients with nontoxic multinodular goiter or Graves' disease, the association between VDD and postoperative hypocalcemia in thyroid cancer patients undergoing TT plus central compartment neck dissection remains unclear,” explained Nam, of the department of surgery at Severance Hospital, Yonsei University Health System in Seoul, South Korea. “We evaluated whether preoperative vitamin D deficiency was associated with postoperative symptomatic hypocalcemia."

According to World Health Organization (WHO) criteria, vitamin D levels below 10 ng/mL are defined as deficient, and vitamin D levels below 20 ng/mL are defined as insufficient. The prevalence of vitamin D insufficiency in Korea is high, affecting 47.3% of males and 64.5% of females. The results of a retrospective study conducted by Nam's institution found that, using these definitions, 25.7% of patients (104/404) had vitamin D deficiency and 52.0% of patients (210/404) had vitamin D insufficiency.

To determine whether or not there was, in fact, an association between preoperative vitamin D deficiency and the development of postoperative hypocalcemia, this study prospectively collected data from 267 consecutive thyroid cancer patients who underwent TT with CCND between September 2012 and May 2014. Patients were categorized by preoperative vitamin D levels as either deficient (<10 ng/mL) or not deficient (≥10 ng/mL). Symptomatic hypocalcemia was defined as the presence or signs or symptoms such as muscle cramps or spasms and a serum calcium level less than 8.2 mg/dL.

Overall, patients with thyroid cancer exhibited a high prevalence of vitamin D deficiency or insufficiency (25% and 52%, respectively). Preoperative vitamin D deficiency was associated with a significant increase in the rate of postoperative symptomatic hypocalcemia in patients with thyroid cancer undergoing total thyroidectomy with CCND (43.8% vs 30.4%, 

Postoperative serum parathyroid hormone (PTH) level is still the main predictor of postoperative hypocalcemia, based on a positive correlation between postoperative PTH and calcium level. Compared with patients without vitamin D deficiency, those with vitamin D deficiency were significantly more likely to develop symptomatic hypocalcemia in cases of postoperative intact parathyroid hormone (iPTH) levels less than 15 pg/mL (77.5% vs 53.2, 

 = .008). In an effort to identify an appropriate preoperative vitamin D threshold level, the investigators found that preoperative vitamin D levels greater than 20 ng/mL reduced the risk of symptomatic hypocalcemia by 72% (

"In conclusion, vitamin D deficiency is significantly associated with postoperative, symptomatic hypocalcemia in thyroid cancer patients undergoing TT plus CCND. In fact, vitamin D deficiency could predict symptomatic hypocalcemia in cases of postoperative iPTH levels <15 pg/mL,” said Nam. “We suggest that preoperative supplementation of oral vitamin D be considered as a way to minimize postoperative symptomatic hypocalcemia in thyroid cancer patients with preoperative vitamin D deficiency."

Postoperative hypocalcemia is the most common complication after TT, with transient hypocalcemia occurring in about 30% to 50% of patients and permanent hypocalcemia, lasting 6 months or longer, occurring in 0.5% to 2% of patients. In addition to uncomfortable symptoms, postoperative hypocalcemia also results in longer hospital stays and ongoing laboratory tests.

The parathyroid glands control calcium levels. Vitamin D

 is a principal regulator of both PTH secretion and the proliferation of parathyroid cells. The parathyroid glands have both vitamin D and calcium sensing receptors that together work as a part of a negative-feedback system. Patients with vitamin D deficiency absorb less calcium from the gastrointestinal tract, making them more reliant on PTH-mediated bone and nephron resorption of calcium. Temporary reduction of PTH secretion after TT predisposes patients with vitamin D deficiency to develop more marked hypocalcemia than those with normal vitamin D levels.

Preoperative vitamin D deficiency is associated with postoperative symptomatic hypocalcemia in patients with thyroid cancer undergoing total thyroidectomy plus central compartment neck dissection.

Vitamin D Levels Predict Hypocalcemia After Thyroid Cancer Treatment

In vivo models of anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC) show that downregulation of miR-30a-5p leads to overexpression of lysyl oxidase (LOX), a target which appears amenable to treatment with β-aminopropionitrile fumarate (BAPN). These findings suggest that a new therapeutic option could be on the horizon for patients with these aggressive forms of thyroid cancer.

"ATC, the most aggressive and lethal form of thyroid cancer, is resistant to current therapies. ATC commonly metastasizes to the lungs, bone, and brain, and patients with ATC typically have a median survival time of 1 year or less. Novel therapeutic approaches are needed to treat ATC and improve patient outcomes," said Myriem Boufraqech, PhD, of the Endocrine Oncology Branch of the National Cancer institute/National Institutes of Health in Bethesda, Maryland, at the 2014 ATA Annual Meeting.

This work sought to further elucidate the role of microRNAs (miRNA) and LOX expression in thyroid cancer progression. LOX initiates the process of collagen and elastin crosslinking, functions in remodeling and stiffening the fibrous deposits of proteins in the extracellular matrix of tumors, and, as such, is thought to play a key role in promoting tumor motility, migration, and rigidity, particularly in a hypoxic environment of a solid tumor.

To identify miRNA(s) and pathways involved in ATC, human thyroid cancer tissue samples were assessed using microRNA microarray and real-time polymerase chain reaction (RT-PCR). MiR-30a-5p was overexpressed in ATC cell lines, and luciferase assays were used to validate LOX as a direct target of miR-30a-5p. An in vivo metastatic ATC mouse model was then used to evaluate the role of miR-30a-5p and LOX in ATC progression.

Profiling of miRNA expression revealed that miR-30a-5p is significantly downregulated in both ATC and PDTC. Furthermore, 454 primary thyroid cancer samples from the Cancer Genome Atlas dataset showed that upregulation of LOX and downregulation of miR-30a were associated with more aggressive thyroid cancer and a higher mortality rate, suggesting that LOX and miR-30a may be useful as prognostic markers for aggressive thyroid cancer.

In the in vitro setting, ectopic expression of miR-30a-5p reduced the invasion and migration of ATC cells and downregulated the expression of epithelial mesenchymal transition (EMT) markers and LOX, which is a predicted target of miR-30a-5p.

Using an in vivo mouse model of ATC metastases, miR-30a-5p significantly reduced lung metastases with decreased vimentin, CD44, and LOX expression in lung metastases and tumor tissue. Additionally, both LOX and its downstream target TWIST-1 are highly expressed in ATC. In both ATC and PDTC samples, LOX overexpression was significantly inversely correlated with miR-30a-5p expression.

To demonstrate that miR-30a-5p directly targets LOX, the 3'-UTR region of LOX was cloned into a luciferase reporter plasmid and transfected along with miR-30a-5p into ATC cell lines. Compared with negative control miRNA, co-transfection of 3'UTR-LOX with miR-30a led to a decrease in luciferase activity, which demonstrates that LOX is, in fact, a direct target of miR-30a-5p.

Furthermore, direct knockdown of LOX not only inhibited cell proliferation, but also increased cellular apoptosis and decreased the invasion and migration of ATC cells. In the in vitro setting, treatment of four ATC cell lines with the irreversible LOX inhibitor BAPN did not affect cell proliferation but significantly inhibited cell invasion and migration and reduced EMT markers and CD44 expression. In vivo, mice with metastatic human ATC tumors that were treated with BAPN exhibited significantly fewer tumors, and lung metastases were smaller in size.

In general, miR-30a-5p suppresses migration, invasion, and in vivo metastases in ATC. Its role in thyroid cancer correlates with other work that suggests that LOX may play a role in other solid tumors, such as those in cervical and breast cancers, by promoting metastasis.

"Together, our data support an important role of the miR-30a-5p-LOX axis in thyroid cancer progression. The current development of agents targeting LOX could be useful for ATC and PDTC cancer therapy," Boufraqech concluded.

	Boufraqech M, nilubol N, Zhang L, et al. Tumor suppressor miR-30a-5p inhibits LOX expression and cancer progression in thyroid cancer. Presented at: the Annual Meeting of the American Thyroid Association; October 29 — November 2, 2014; Coronado, CA. Abstract 121.

In vivo models for two aggressive thyroid cancers have shown that downregulation of miR-30a-5p leads to overexpression of LOX, a target which appears amenable to treatment with Î²-aminopropionitrile fumarate.


Novel Therapeutic Target Identified in Aggressive Thyroid Cancers

The detection of BRAFV600E in patients with papillary thyroid cancer (PTC) using a blood-based assay was shown to be feasible in a cohort of patients undergoing thyroidectomy, according to a presentation by Carrie C. Lubitz, MD, MPH, at the 2014 Annual Meeting of the American Thyroid Association.

The objective of this research was to assess the feasibility and utility of this novel blood-based assay for identifying PTC patients with a high-risk tumor mutation. The researchers hypothesized that a blood BRAFV600E assay would be able to detect circulating BRAFV600E in patients with PTC and that the blood assay would correlate with tumor burden.

"BRAFV600E is an important driver of mutation in thyroid cancer and is found in half of patients with papillary thyroid carcinoma. Detection of this mutation in tumor DNA obtained cytologically or pathologically is useful in the work-up of nodules or for determining therapeutic options for using novel targeted therapies," explained Lubitz, an assistant professor of surgery at Harvard Medical School and a surgeon at Massachusetts General Hospital.

BRAFV600E is correlated with worse outcomes, including an increased risk of recurrence, and independently correlates with persistent PTC in patients with stage I/II disease and with mortality on univariate analysis. Therefore, knowledge of BRAF status is clinically actionable for surgical and adjuvant therapy. However, conventional BRAF testing requires a tissue sample.

"We have previously developed a novel, highly sensitive, and specific blood-based BRAFV600E assay for patients with melanoma," Lubitz said. This assay allowed for the detection of mutant BRAF in RNA from mixed populations with as few as 0.1% BRAFV600E-containing cancer cells. It was found that BRAFV600E levels decreased following BRAF inhibitor initiation, and blood BRAF was detectable prior to clinical or radiologic recurrence in stage IV patients.

For the study, blood samples were collected from 100 serial patients undergoing thyroidectomy for thyroid disease prior to undergoing surgery at Massachusetts General Hospital from September 2013 to July 2014, and for up to 4 weeks postoperatively. No patient had a history of colon cancer or melanoma. Investigators were blinded to both clinical and tissue-BRAF status. Circulating BRAFV600E levels were compared to BRAF mutation status from surgical pathologic assessment with conventional assays.

Circulating BRAFV600E levels were quantified for each blood sample, using a unique restriction enzyme site and a series of polymerase chain reaction (PCR) amplifications and wild-type BRAF digestions. Extracted RNA was then quantified using real-time PCR before beginning the assay to normalize all of the samples. Results were then analyzed using a receiver-operated curve and calculating the area under the curve. In determining a positivity criterion, the researchers sought to maximize the likelihood ratio (sensitivity/1-specificity) and considered the prevalence of "disease" (ie, BRAF positivity) and the consequences of false negative and false positive results.

Preoperative and postoperative blood samples were analyzed from 36 patients undergoing thyroidectomy for PTC (stages I-III) and 10 non-PTC patients (nodular hyperplasia [n = 6], Hurthie cell adenoma [n = 3], or medullary thyroid cancer [n = 1]). Mean tumor size was 1.9 ± 1.1 cm.

Tissue BRAFV600E results were positive for 24 (67%) of PTC patients and 2 (20%) of non-PTC patients. Using these data, a threshold value of 1.0 picogram was established as "positive" and separated the cohort into two distinct groups. Furthermore, tumor volume correlated with circulating tumor cell level.

One limitation of this study is that BRAF tissue status is considered to be the gold standard. Also, there is a potential for pathological missed or misdiagnosis, for example, in cases of coincident melanoma or colorectal cancer. Cytological BRAF testing was not done. Finally, postoperative and longitudinal follow-up would be needed for all patients, the researchers noted.

"Our findings confirm the feasibility of a novel blood assay in detecting circulating mutant BRAF in the blood of patients with papillary thyroid carcinoma. This rapid assay has potential as a more cost-effective method for diagnosis, surveillance in thyroglobulin-antibody positive patients postoperatively, and in assessing evidence of disease over time in the adjuvant setting, for example, during treatment with BRAF inhibitors," Lubitz concluded.

The detection of BRAFV600E in patients with papillary thyroid cancer using a blood-based assay was shown to be feasible in a cohort of patients undergoing thyroidectomy.

New Assay Effectively Detects Circulating BRAFV600E in Patients With Papillary Thyroid Cancer

Long-term moderate thyroid hormone suppression therapy (THST) is associated with improved outcomes in patients with differentiated thyroid cancer (DTC), according to data from 1987 to 2012 presented by Aubrey A. Carhill, MD, on behalf of the National Thyroid Cancer Treatment Cooperative Study Group (NTCTCSG) registry analysis.

"In the absence of prospective studies detailing the ultimate longitudinal effects of initial and long-term therapy for patients with differentiated thyroid cancer, clinicians have had to rely on retrospective studies and expert opinion,” said Carhill, as Assistant Professor in the Endocrine Neoplasia and Hormonal Disorders group at the University of Texas, MD Anderson Cancer Center in Houston. “Consequently, debate remains as to the appropriate extent of surgery, post-operative radioiodine, and thyroid-stimulating hormone-suppressive therapy."

The NTCTCSG registry prospectively collects data related to clinical outcomes from multiple institutions following treatment of patients with DTC. The predictive value of the NTCTCSG Registry clinicopathologic staging system for thyroid cancer, which uses patient age at diagnosis, tumor size and histology, and differentiation, metastases, intrathyroidal multifocality, and extraglandular invasion, has been consistently shown to be among the highest for disease-free survival (DFS) and overall survival (OS).

The registry data showed that stage III patients who received RAI (relative risk [RR] 0.66, 

 = .04) and stage IV patients who received both total/near-total thyroidectomy (T/NTT) and RAIs (RR 0.66 and 0.70, combined 

 = .049) exhibited improved OS. Patients of all stages exhibited significantly improved OS (stage I-IV: RR=0.13, 0.09, 0.13, 0.33, respectively) and DFS (stage I-III: RR=0.52, 0.40, 0.18, respectively) with moderate THST but not aggressive THST treatment. Furthermore, when distant metastatic disease was diagnosed in long-term follow-up, moderate THST was associated with significantly improved OS, and moderate THST and lower initial disease stage were independently predictive of improved OS. In follow-up of patients who were rendered disease free after initial therapy, moderate THST was shown to be independently predictive of DFS for at least the first 3 years of follow-up.

In multivariate analysis of primary treatments for DTC, in all stages, only THST was associated with both improved OS and DFS. Like others, this study has its limitations, including institutional bias, assignment of disease status at entry, treatment selection, and others. However, when examining the degree of THST, aggressive THST confers no additional survival advantages as compared with moderate THST, even when limiting the analysis to patients with distant metastatic disease.

"This registry update increases by more than 2,000 the number of patients followed to 4,941 in the overall cohort and 3,238 patients in the THST cohort, with a mean follow-up time of 6 years," Carhill said. Since the last registry update in 2001 with data from 14 institutions, the registry now includes a total of 34,631 person-years of documented follow-up time with some patients followed for as long as 25 years. For all registry patients, the effects of initial therapies, including thyroidectomy and radioactive iodine (RAI), as well as long-term THST were documented, the NTCTCSG staging system was applied, and OS and DFS were assessed. Moderate THST was defined as a TSH level in the subnormal to normal range, while aggressive THST was defined as a TSH level maintained in the undetectable to subnormal range.

"We confirm previous findings that T/NTT followed by RAI is associated with survival benefit in high-risk but not low-risk patients. In contrast with our earlier reports, only moderate THST is associated with better outcomes in all stages, including low-risk stage I patients, and aggressive THST may not be warranted even in patients diagnosed with distant metastatic disease during follow up," she said. "The challenge for clinicians is to balance the benefits and risks of treatment. Moderate THST continued at least 3 years after diagnosis may be indicated in higher risk patients," Carhill concluded.

	Carhill A, Litofsky DR, Ain K, Brierley J, Cooper DS, Fein, H, et al. Long-term moderate thyroid hormone suppression therapy is associated with improved outcomes in differentiated thyroid carcinoma: national thyroid cancer treatment cooperative study group registry analysis 1987—2012. Presented at: the 84th Annual Meeting of the American Thyroid Association; October 29-November 2, 2014; Coronado, CA.Clinical Oral Abstract #11.

Long-term moderate thyroid hormone suppression therapy is associated with improved outcomes in patients with differentiated thyroid cancer.

Moderate THST Improves Outcomes in Differentiated Thyroid Cancer

Numerous disease-related variables could serve as prognostic and predictive factors for treatment outcomes with sorafenib (Nexavar) in patients with radioactive iodine (RAI)-refractory differentiated thyroid cancer (RAI-rDTC), according to a new multivariate Cox model analysis of the DECISION trial data presentation by Martin Schlumberger, MD, at the 2014 ATA Annual Meeting.

"Patients with RAI-rDTC have a poor prognosis, and there is a lack of effective treatments. Median survival for patients with metastatic RAI-rDTC is estimated to be 2.5 to 3.5 years," explained Schlumberger, of the Service de Médecine Nucléaire et Cancérologie Endocrinienne, Gustave Roussy, in Villejuif, France. “Overall, subgroup analyses indicated that patients whose maximum individual target tumor size was <1.5 cm had a longer progression-free survival and appeared to have less benefit from sorafenib treatment than patients with lesions ≥1.5 cm. Additionally, lesions >1.5 cm, as well as lung-only metastases with no bone metastases, were predictive for better treatment effect with sorafenib.”

The FDA approved sorafenib in November 2013 for the treatment of RAI-rDTC based on the results of the phase III, randomized, placebo-controlled, double-blind DECISION trial. In the trial treatment with sorafenib extended the median progression-free survival (PFS) time by 5 months, from 5.8 months with placebo to 10.8 months with sorafenib (HR = 0.59; 95% CI, 0.45-0.76, 

<.0001). In a predefined subgroup analysis, sorafenib showed benefit across all patient subgroups. Specifically, 

 mutations were not independently prognostic of outcome, nor did these mutations predict PFS benefit from sorafenib.

In the new analysis, a number of baseline or disease-related variables were prognostic for longer PFS for all patients enrolled in the study. These included papillary histology, individual target lesion size <1.5 cm, a lower tumor burden, baseline thyroglobulin levels below the median (486 ng/mL), site of metastases (lung-only), and geographic region (Asia versus Europe or North America).

Tumor burden and histology were both prognostic and predictive factors for PFS in patients treated with sorafenib. The relative benefit of sorafenib increased in patients with a higher tumor burden. Patients with maximum target lesions <1.5 cm appeared to derive less benefit in terms of relative risk in PFS from sorafenib treatment.

Papillary histology was a positive prognostic factor for all patients and a predictive factor for benefit from sorafenib. Thyroglobulin levels at baseline were prognostic for all patients, but were not predictive for improved relative risk in PFS; both low and high thyroglobulin groups benefited from sorafenib. An absence of bone metastases and presence of lung-only metastases was also a positive predictive factor for relative risk in PFS.

Additional post-hoc exploratory analyses of PFS outcomes by presence or absence of thyroid cancer-related symptoms at study entry were conducted. From a total of 417 patients who received either sorafenib (n=207) or placebo (n=210), 48 sorafenib- and 45 placebo-treated patients were identified as having symptoms that were consistent with thyroid cancer, which included dyspnea, exertional dyspnea, pleural effusion, dysphagia, hemoptysis, chest, bone, or tumor pain, spinal cord compression, cough, obstructive airway disorder, or pulmonary embolism. The rest of the patients were asymptomatic.

"Here, both symptomatic and asymptomatic patients at entry had improved PFS following treatment with sorafenib," said Schlumberger. "Patients appeared to benefit from sorafenib treatment, irrespective of disease-related symptoms at baseline. Thus, based on these post-hoc exploratory analyses, patients with progressive RAI-rDTC and maximum tumor size <1.5 cm appear to have a good prognosis and may be candidates for "watch and wait" before initiating sorafenib.”

Numerous disease-related variables could serve as prognostic and predictive factors for treatment outcomes with sorafenib in patients with radioactive iodine-refractory differentiated thyroid cancer.

Disease-Related Variables Predictive of Outcomes With Sorafenib in DTC

Treatment with programmed cell death 1 (PD-1) immunotherapy across various oncology clinical trials has been associated with the induction of painless thyroiditis, characterized by transient thyrotoxicosis and hypothyroidism, in patients with metastatic malignancies, according to a presentation by Paul G. Walfish, CM, OOnt, of the Department of Endocrinology at Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases at Mount Sinai Hospital in Toronto, Canada, during the 84th Annual Meeting of the American Thyroid Association. With the evolving use of PD-1, it is important for clinicians to monitor patients for the clinical and biochemical signs of thyroid dysfunction and treat potential adverse consequences.

Walfish and colleagues identified a subgroup of 10 patients with documented metastatic malignancies who received immunotherapy with PD-1 receptor antibodies as part of a clinical trial. In the subgroup, 7 patients had malignant melanoma and 3 patients had non-small cell lung cancer. These clinical trial participants had abnormal thyroid function tests and were referred to a tertiary thyroidology clinic for diagnosis and management.

Overall, 60% of patients were female, and the average patient age was 55 years. Sixty percent of patients displayed transient thyrotoxicosis and negative thyrotropin binding inhibitory immunoglobulins and required treatment with beta blockers. Anti-thyroid antibodies, including both anti-thyroglobulin and anti-thyroid peroxidase, were identified in 4 patients (67%). After approximately 4 weeks, thyroidotoxicosis spontaneously resolved, but patients required thyroid hormone replacement therapy with levothyroxine to treat their hypothyroidism. The remaining 4 patients presented with hypothyroidism that was not preceded by a thyrotoxic phase. These patients displayed serological evidence of anti-thyroid antibodies an average of 6 to 8 weeks following initial PD-1 drug exposure. No relationship was consistently seen between the occurrence of painless thyroiditis syndrome and a patient's oncological response to immunotherapy.

Novel immunotherapies that have recently been investigated for the treatment of several malignancies have now been recognized to have selective immunotoxic effects on the thyroid gland and several other endocrine organs. Since these effects do not occur in all patients exposed to such therapy, it is likely that affected individuals have an immunogenetic susceptibility to such adverse consequences.

Previously, Walfish's group and other researchers have demonstrated that polymorphic variations of the cytotoxic T lymphocyte antigen-4 (CTLA-4) receptor gene can predispose individuals to a variety of autoimmune endocrine disorder such as Grave's disease, Addison's disease, Hashimoto's thyroiditis, and type 1 diabetes mellitus. In this case, a similar mechanism (polymorphic variants of the PD-1 receptor gene) may account for the occurrence of painless thyroiditis in a subgroup of patients treated with anti-PD-1 monoclonal antibodies (mAbs). Perhaps similar alterations in immune checkpoints may account for cases of sporadic or postpartum thyroiditis.

Like CTLA-4, PD-1 is a negative immune regulator that prevents T-cell proliferation, activation, and cytokine release in response to foreign antigens. Negative regulation is mediated by the PD-1 receptor and its PD-L1 or PD-L2 ligands. Both CTLA-4 and PD-1 receptors are important checkpoint inhibitors, and mAb blockade of these targets is a promising novel immunotherapy for the treatment of several metastatic malignancies.

Upregulation of PD-1 ligands occurs in some tumors and can contribute to the inhibition of active T-cell immune surveillance. Binding of PD-1 ligands to PD-1 receptors found on T cells inhibits T-cell proliferation and cytokine production to prevent tumor attack. Anti-PD-1 receptor mAbs release the blockade and permit an anti-tumor immune response to metastases. Although adverse endocrinopathies, including thyroid dysfunction, have recently been described secondary to treatment with anti-CTLA-4 mAb therapy, the potential for similar effects on thyroid dysfunction following anti-PDâ€‘1 mAb immunotherapy have not been well characterized.

Some cases of painless thyroiditis have occurred sporadically or in the postpartum period, while other cases have been reported in response to other endogenous stimuli such as Grave's disease, Addison's disease before treatment, and Cushing's disease after remission. Additionally, a number of medications, including treatment with interferon-alfa and ribavirin, kinase inhibitors, interleukin-2, amiodarone, lithium, and iodine, have been associated with the development of painless thyroiditis. Walfish also noted that some patients develop painless thyroiditis after radiation treatment or following a surgical procedure in which the thyroid has been too vigorously palpated.

Treatment with PD-1 immunotherapy across various oncology clinical trials has been associated with the induction of painless thyroiditis, characterized by transient thyrotoxicosis and hypothyroidism, in patients with metastatic malignancies

Katherine Hasal, MS