Tumor size and molecular risk group are associated with differentiated thyroid cancer recurrence.

BACKGROUND: The threshold at which active surveillance can be considered is variable, with some algorithms proposing nonoperative treatment for differentiated thyroid carcinomas ≤2 cm and lobectomy alone for lesions 2.1-4 cm. To inform both decision for and extent of initial surgery, we aim to evaluate whether molecular results can complement tumor size to identify differentiated thyroid carcinomas associated with disease recurrence. METHODS: Patients from 2007-2013 and 2017-2021 who had initial thyroidectomy (differentiated thyroid carcinoma size 1-4 cm, clinical N0M0) were included. When available, molecular testing results were categorized into 3 previously described molecular risk groups (low, intermediate, and high). Primary outcome was structural recurrence. RESULTS: Recurrence was diagnosed in 3.8% of 1,739 patients with differentiated thyroid carcinomas. Preoperative variables including size (1-2 cm vs 2.1-4 cm, P = .43), age >55 years (P = .92), and male sex (P = .31) were not associated with recurrence. Molecular testing results were available for 1,020, and after excluding molecular risk group high-risk differentiated thyroid carcinoma, structural recurrences were associated with molecular risk group intermediate risk (7.2% vs molecular risk group low, 0.7%, P < .001), and most likely in differentiated thyroid carcinoma, which were both 2.1-4 cm and molecular risk group intermediate risk (11.3% vs size 1-2 cm 5.8%, P = .04). CONCLUSION: Overall, structural recurrences for differentiated thyroid carcinomas ≤4 cm were low (<5%) and molecuar testing was the only preoperative variable associated with recurrence. However, when molecular risk group intermediate risk was present, larger tumor size (2.1-4 cm) had a 2-fold greater risk of recurrence compared with tumors 1-2 cm, and size may still be helpful to guide management. When considering de-escalated treatment for the proposed guidelines with a cutoff of 2 cm, initial decision-making may be further optimized with identification of preoperative molecular risk groups.

Reappraisal of BRAFK601E-positive thyroid tumors in the NIFTP era.

BRAFK601E is an uncommon mutation typically found in encapsulated follicular-patterned thyroid tumors. Previous studies on BRAFK601E-positive thyroid tumors were conducted before implementation of the non-invasive follicular neoplasm with papillary-like nuclear features (NIFTP) diagnosis. This study aimed to characterize BRAFK601E-positive tumors and evaluate changes in diagnosis and management of these patients after introduction of NIFTP. We evaluated 25 thyroid tumors that were positive for BRAFK601E and diagnosed considering the NIFTP criteria. Clinicopathologic characteristics and recurrence rates of these tumors were compared to 29 BRAFK601E-positive tumors diagnosed prior to the acceptance of NIFTP diagnosis. RNA-seq analysis was performed on 10 BRAFK601E-positive tumors. In the current study, 72% of BRAFK601E-positive tumors were diagnosed as non-invasive tumors on resection, with NIFTP (48% of all tumors) being the most common diagnosis. BRAFK601E-positive tumors exhibited a RAS-like gene expression profile with BRAF-RAS score (BRS) and thyroid differentiation score (TDS) distinct from BRAFV600E-positive tumors (P<0.001). Since 2016, patients with BRAFK601E-positive tumors less frequently underwent total thyroidectomy (41% vs 100%, P<0.001) and received radioiodine (7% vs 75%, P<0.001). None of the tumors positive for an isolated BRAFK601E mutation from the current or 2016 studies showed recurrences on follow-up. Our study demonstrates that most BRAFK601E-positive tumors are low risk, RAS-like tumors, which were diagnosed as NIFTP in half of all study cases. Since 2016, patients with BRAFK601E-positive nodules receive less aggressive treatment. The risk of recurrence of BRAFK601E-positive tumors without other, high-risk features appears to be low, and lobectomy without radioiodine is likely sufficient treatment for these patients.

Parsing the thyroid cytopathology suspicious for malignancy diagnosis through molecular-derived risk of malignancy and other related parameters: Insights into nodule characteristics and practice patterns.

INTRODUCTION: Thyroid cytopathology cases with suspicious for malignancy (SFM) diagnosis often result in resection. However, molecular testing offers details that may provide additional insights. In this study, the molecular profiles of SFM cases from two institutions that routinely used ThyroSeq v3 (TSV3) were examined. MATERIALS AND METHODS: Following institutional review board approval, SFM thyroid cytopathology cases with TSV3 results were retrieved from the databases of two institutions. Molecular information including molecular-derived risk of malignancy (MDROM), cytologic-histologic correlation data, and other related parameters were calculated. Statistical comparisons were made with a p <.05 considered significant. RESULTS: The core data set comprised 114 SFM cases that passed TSV3 quality assurance. All TSV3 results were reported as positive or negative for genomic alterations and all except five cases provided a probability of malignancy estimate. The overall combined baseline MDROM of 75.7% (95% CI, 70.0-81.4) was comparable to the risk of malignancy (74%) published in the Bethesda System. There was a statistically significant difference between the combined MDROMs of resected and unresected cohorts (79.0% vs 58.6%; p = .0153). Interestingly, the MDROMs of the resected cohorts from the two institutions were statistically different (75.0% vs 85.3%; p = .020). Cytologic-histologic correlation revealed malignant outcome in 88.5% of resected cases. CONCLUSIONS: Molecular analyses of SFM cases demonstrated higher risk genomic alterations that were associated with histologically overt neoplasms, resulting in increased malignancy outcome compared to baseline. MDROM analysis revealed differences in the cytopathologic practice patterns regarding follicular-patterned neoplasms at the two institutions.

Combination Targeted Therapy with Pembrolizumab and Lenvatinib in Progressive, Radioiodine-Refractory Differentiated Thyroid Cancers.

PURPOSE: Lenvatinib, a potent multikinase inhibitor, improves progression-free survival (PFS) in patients with radioiodine (RAI)-refractory differentiated thyroid cancer; however, most patients experience disease progression, warranting further therapy. We evaluated the efficacy and safety of lenvatinib plus pembrolizumab in these patients. PATIENTS AND METHODS: We enrolled patients with progressive, RAI-refractory differentiated thyroid cancer who were either naïve to multikinase inhibitors (cohort 1) or who had progressed on lenvatinib (cohort 2). Patients received oral lenvatinib daily (cohort 1, 20 mg; cohort 2, dose at progression) and intravenous pembrolizumab (200 mg) every 21 days. RESULTS: In cohorts 1 and 2, 30 and 27 patients were enrolled, respectively. Adverse events were consistent with those observed in other cancers. In cohort 1, the confirmed overall response rate was 65.5%. There were no complete responses (primary endpoint). The 12- and 18-month PFS were 72.0% and 58.0%, respectively, and the median PFS was 26.8 months. In cohort 2, the confirmed overall response rate was 16% (primary endpoint), and the median PFS was 10.0 months (95% confidence interval, 7.0-17.9 months). Tumor histology, driver mutations, and immune-related biomarkers, including PD-L1 expression, thyroid-specific antibody levels, and CD8+ T-cell tumor infiltrate, did not correlate with response to therapy. Increased baseline peripheral blood monocytes and neutrophil to lymphocyte ratio were associated with a worse PFS in cohort 1. CONCLUSIONS: Lenvatinib plus pembrolizumab may enhance the durability of lenvatinib monotherapy in lenvatinib-naïve patients. Furthermore, the addition of pembrolizumab may be a viable salvage therapy for patients who have progressed on lenvatinib.

Seven years of Non-invasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features (NIFTP): Rate of Acceptance and Variation of Diagnostic Approaches Across Different Continents.

CONTEXT: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was introduced as a new entity replacing the diagnosis of noninvasive encapsulated follicular variant of papillary thyroid carcinoma (PTC). Significant variability in the incidence of NIFTP diagnosed in different world regions has been reported. OBJECTIVE: To investigate the rate of adoption of NIFTP, change in practice patterns, and uniformity in applying diagnostic criteria among pathologists practicing in different regions. METHODS: Two surveys distributed to pathologists of the International Endocrine Pathology Discussion Group with multiple-choice questions on NIFTP adoption into pathology practice and whole slide images of 5 tumors to collect information on nuclear score and diagnosis. Forty-eight endocrine pathologists, including 24 from North America, 8 from Europe, and 16 from Asia/Oceania completed the first survey and 38 the second survey. RESULTS: A 94% adoption rate of NIFTP by the pathologists was found. Yet, the frequency of rendering NIFTP diagnosis was significantly higher in North America than in other regions (P = .009). While the highest concordance was found in diagnosing lesions with mildly or well-developed PTC-like nuclei, there was significant variability in nuclear scoring and diagnosing NIFTP for tumors with moderate nuclear changes (nuclear score 2) (case 2, P < .05). Pathologists practicing in North America and Europe showed a tendency for lower thresholds for PTC-like nuclei and NIFTP than those practicing in Asia/Oceania. CONCLUSION: Despite a high adoption rate of NIFTP across geographic regions, NIFTP is diagnosed more often by pathologists in North America. Significant differences remain in diagnosing intermediate PTC-like nuclei and respectively NIFTP, with more conservative nuclear scoring in Asia/Oceania, which may explain the geographic differences in NIFTP incidence.

The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

Performance of a multigene genomic classifier and clinical parameters in predicting malignancy in a Southeast Asian cohort of patients with cytologically indeterminate thyroid nodules.

BACKGROUND: Most thyroid nodules are benign. It is important to determine the likelihood of malignancy in such nodules to avoid unnecessary surgery. The primary objective of this study was to characterize the genetic landscape and the performance of a multigene genomic classifier in fine-needle aspiration (FNA) biopsies of cytologically indeterminate thyroid nodules in a Southeast Asian cohort. The secondary objective was to assess the predictive contribution of clinical characteristics to thyroid malignancy. METHODS: This prospective, multicenter, blinded study included 132 patients with 134 nodules. Molecular testing (MT) with ThyroSeq v3 was performed on clinical or ex-vivo FNA samples. Centralized pathology review also was performed. RESULTS: Of 134 nodules, consisting of 61% Bethesda category III, 20% category IV, and 19% category V cytology, and 56% were histologically malignant. ThyroSeq yielded negative results in 37.3% of all FNA samples and in 42% of Bethesda category III-IV cytology nodules. Most positive samples had RAS-like (41.7%), followed by BRAF-like (22.6%), and high-risk (17.9%) alterations. Compared with North American patients, the authors observed a higher proportion of RAS-like mutations, specifically NRAS, in Bethesda categories III and IV and more BRAF-like mutations in Bethesda category III. The test had sensitivity, specificity, negative predictive value, and positive predictive value of 89.6%, 73.7%, 84.0%, and 82.1%, respectively. The risk of malignancy was predicted by positive MT and high-suspicion ultrasound characteristics according to American Thyroid Association criteria. CONCLUSIONS: Even in the current Southeast Asian cohort with nodules that had a high pretest cancer probability, MT could lead to potential avoidance of diagnostic surgery in 42% of patients with Bethesda category III-IV nodules. MT positivity was a stronger predictor of malignancy than clinical parameters.

Molecular-derived risk of malignancy and the related positive call rate of indeterminate thyroid cytology diagnoses as quality metrics for individual cytopathologists.

BACKGROUND: Indeterminate thyroid cytopathology diagnoses represent differing degrees of risk that are corroborated by follow-up studies. However, traditional cytologic-histologic correlation may overestimate the risk of malignancy (ROM) because only a subset of cases undergo resection. Alternatively, some molecular tests provide probability of malignancy data to calculate the molecular-derived risk of malignancy (MDROM) and the positive call rate (PCR). The authors investigated MDROMs and PCRs of indeterminate diagnoses for individual cytopathologists as quality metrics. METHODS: This study was approved by the Department of Pathology Quality Improvement Program. Thyroid cytopathology diagnoses and ThyroSeq v3 results were retrieved for each cytopathologist for a 2-year period with at least 3 years of follow-up for the atypia of undetermined significance (AUS), follicular neoplasia (FN), and follicular neoplasia, oncocytic-type (ONC) cytopathologic diagnoses. MDROMs and PCRs were compared with reference ROMs and cytologic-histologic correlation outcomes. RESULTS: The overall MDROMs (and ranges for cytopathologists) for the AUS, FN, and ONC categories were 13.4% (range, 5.8%-20.8%), 28.1% (range, 22.1%-36.7%), and 27.0% (range, 19.5%-41.5%), respectively, and most individual cytopathologists' MDROMs were within reference ROM ranges. However, PCRs more effectively parsed the differences in cytopathologists' ROM performance. Although the overall PCRs were not significantly different across cytopathologists (p = .06), the AUS PCRs were quite different (p = .002). By cytologic-histologic correlation, six of 55 resected cases (10.9%) were falsely negative, and there were no false-positive cases. CONCLUSIONS: MDROMs and PCRs evaluate concordance with reference ROMs and with one another and provide individual feedback, which potentially facilitates quality improvement.

Progress in Thyroid Cancer Genomics: A 40-Year Journey.

Background: Very little was known about the molecular pathogenesis of thyroid cancer until the late 1980s. As part of the Centennial celebration of the American Thyroid Association, we review the historical discoveries that contributed to our current understanding of the genetic underpinnings of thyroid cancer. Summary: The pace of discovery was heavily dependent on scientific breakthroughs in nucleic acid sequencing technology, cancer biology, thyroid development, thyroid cell signaling, and growth regulation. Accordingly, we attempt to link the primary observations on thyroid cancer molecular genetics with the methodological and scientific advances that made them possible. Conclusions: The major genetic drivers of the common forms of thyroid cancer are now quite well established and contribute to a significant extent to how we diagnose and treat the disease. However, many challenges remain. Future work will need to unravel the complexity of thyroid cancer ecosystems, which is likely to be a major determinant of their biological behavior and on how they respond to therapy.

Prognostic Value of Preoperative Molecular Testing and Implications for Initial Surgical Management in Thyroid Nodules Harboring Suspected (Bethesda V) or Known (Bethesda VI) Papillary Thyroid Cancer.

Importance: Molecular testing is commonly used in the diagnosis of thyroid nodules with indeterminate cytology. The role of molecular testing in prognosticating oncologic outcomes in thyroid nodules with suspicious or malignant cytology is unclear. Objective: To determine whether molecular profiling of Bethesda V (suspicious for thyroid cancer) and VI (thyroid cancer) nodules is associated with improved prognostication and whether it may inform initial treatment. Design, Setting, and Participants: This retrospective cohort study included consecutive patients with Bethesda V or VI nodules who underwent surgery, with histopathology showing differentiated thyroid cancer, between May 1, 2016, and July 31, 2019 in the University of California, Los Angeles health system. Data were analyzed between April 2, 2021, and January 18, 2023. Exposures: Masked ThyroSeq, version 3 molecular analysis after completion of initial treatment and acquisition of follow-up data. Main Outcomes and Measures: Structural disease persistence or recurrence, distant metastasis, and recurrence-free survival were assessed using ThyroSeq Cancer Risk Classifier (CRC) molecular risk groups (low, RAS-like; intermediate, BRAF-like; high, combination of BRAF/RAS plus TERT or other high-risk alterations) using Cox proportional hazards regression models. Results: In 105 patients with papillary thyroid cancer (median [IQR] follow-up, 3.8 [3.0-4.7] years), ThyroSeq identified genomic alterations in 100 (95%) samples (6 [6%] low risk, 88 [88%] intermediate risk, and 6 [6%] high risk; median [IQR] age, 44 [34-56] years; 68 [68%] female and 32 [32%] male). No patients with low-risk or negative results experienced recurrence. Of the 88 patients with intermediate risk, 6 (7%) experienced local recurrence, with 1 of them also developing distant metastasis. The 6 patients with high risk (all with BRAF V600E plus TERT mutation) underwent total thyroidectomy followed by radioactive iodine (RAI) ablation. Four patients with high risk (67%) experienced local recurrence, with 3 of them also developing distant metastasis. Thus, patients with high-risk alterations were more likely to experience persistence or recurrence and distant metastasis than patients with intermediate risk. In a multivariable analysis incorporating patient age, sex, cancer size, ThyroSeq molecular risk group, extrathyroidal extension, lymph node positivity, American Thyroid Association risk, and RAI ablation, only cancer size (hazard ratio, 1.36; 95% CI, 1.02-1.80) and ThyroSeq CRC molecular risk group (high vs intermediate and low: hazard ratio, 6.22; 95% CI, 1.04-37.36) were associated with structural recurrence. Conclusions and Relevance: Among the 6% of patients with high-risk ThyroSeq CRC alterations in this cohort study, the majority experienced recurrence or distant metastasis despite initial treatment with total thyroidectomy and RAI ablation. In contrast, patients with low- and intermediate-risk alterations had a low recurrence rate. Preoperative knowledge of molecular alteration status at diagnosis may allow for deescalation of initial surgery and refining of the intensity of postoperative surveillance in patients presenting with Bethesda V and VI thyroid nodules.

Molecular Profiling of 50 734 Bethesda III-VI Thyroid Nodules by ThyroSeq v3: Implications for Personalized Management.

CONTEXT: Comprehensive genomic analysis of thyroid nodules for multiple classes of molecular alterations detected in a large series of fine needle aspiration (FNA) samples has not been reported. OBJECTIVE: To determine the prevalence of clinically relevant molecular alterations in Bethesda categories III-VI (BCIII-VI) thyroid nodules. METHODS: This retrospective analysis of FNA samples, tested by ThyroSeq v3 using Genomic Classifier and Cancer Risk Classifier at UPMC Molecular and Genomic Pathology laboratory, analyzed the prevalence of diagnostic, prognostic, and targetable genetic alterations in a total of 50 734 BCIII-VI nodules from 48 225 patients. RESULTS: Among 50 734 informative FNA samples, 65.3% were test-negative, 33.9% positive, 0.2% positive for medullary carcinoma, and 0.6% positive for parathyroid. The benign call rate in BCIII-IV nodules was 68%. Among test-positive samples, 73.3% had mutations, 11.3% gene fusions, and 10.8% isolated copy number alterations. Comparing BCIII-IV nodules with BCV-VI nodules revealed a shift from predominantly RAS-like alterations to BRAF V600E-like alterations and fusions involving receptor tyrosine kinases (RTK). Using ThyroSeq Cancer Risk Classifier, a high-risk profile, which typically included TERT or TP53 mutations, was found in 6% of samples, more frequently BCV-VI. RNA-Seq confirmed ThyroSeq detection of novel RTK fusions in 98.9% of cases. CONCLUSION: In this series, 68% of BCIII-IV nodules were classified as negative by ThyroSeq, potentially preventing diagnostic surgery in this subset of patients. Specific genetic alterations were detected in most BCV-VI nodules, with a higher prevalence of BRAF and TERT mutations and targetable gene fusions compared to BCIII-IV nodules, offering prognostic and therapeutic information for patient management.

Unraveling the significance of TSHR mutations in indeterminate thyroid cytology specimens.

OBJECTIVES: We investigated the clinical significance of thyroid-stimulating hormone receptor (TSHR) mutations detected in thyroid fine needle aspiration (FNA) specimens. METHODS: The pathology archives at our institution were reviewed between 2018 and 2021 for indeterminate (Bethesda category III and IV) specimens with Thyroseq® analysis showing TSHR mutations. RESULTS: A total of 2184 cases diagnosed as atypia/follicular lesion of undetermined significance (AUS/FLUS), and 2625 diagnosed as follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) were identified. A total of 1735 AUS/FLUS and 2339 SFN/FN underwent Thyroseq® analysis; 69 showed TSHR mutations (1.6%, 59 female, 10 male, average age: 55 years). Ten cases showed oncocytic features. Twelve patients underwent radionuclide scans within 1 year of FNA:11 were hyperfunctioning. Nodule size and TSH levels were weakly correlated. Twenty-two different TSHR mutations were identified (most common: M453T). A second mutation was found in five cases (EZH1 n = 2, and EIF1AX n = 3). The expression of sodium-iodide transporter (NIS) mRNA was in the range of 0.01%-62.43% out of all sequencing reads, and was elevated in 49 (71%) cases. Surgical pathology follow-up was available in five cases (all benign except one). On follow-up available for 38 cases (mean: 24 months; range: 7-47 months), 34 (89.5%) nodules remained stable and 3 (8%) increased in size. CONCLUSIONS: TSHR mutations in thyroid FNA samples classified as indeterminate are rare, generally benign, and commonly associated with autonomy on scan if performed.

Molecular Profiles of Noninvasive, Minimally Invasive, and Invasive Follicular Patterned Thyroid Neoplasms with Papillary Nuclear Features.

Background: An increasing amount of data is being published, which risk-stratify thyroid tumors according to genetic signatures and histological morphology. Typically, follicular patterned lesions have been shown to harbor RAS-like mutations with more indolent behaviors. Our study aims to examine the extent of similarity among three groups of follicular patterned lesions with papillary nuclear features-noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) with capsular invasion and/or angioinvasion, and infiltrative follicular variant of papillary thyroid carcinoma (iFVPTC)-to help clarify whether NIFTP and EFVPTC represent a histological continuum and the degree to which the genomic landscape further separates higher risk follicular patterned tumors such as iFVPTC from more indolent ones (EFVPTC and NIFTP). Methods: ThyroSeq test results were compared for cases with histological NIFTP, EFVPTC, and iFVPTC in this retrospective study. Genetic drivers were subcategorized by level of aggressiveness. Gene expression alterations (GEAs) and copy number alterations (CNAs) were compared among the three histological groups. Results: NIFTP and EFVPTC cases displayed predominantly RAS-like alterations (100% and 75%, respectively) and RAS-like GEAs (55.2% and 47.2%, respectively), and many showed CNAs with 22q-loss. Despite a predominance of RAS-like alterations, EFVPTC cases showed molecular heterogeneity with significantly more intermediate and aggressive drivers (22.3% of cases) than NIFTP (0%) (p = 0.0068). iFVPTC cases displayed molecular profiles in between that of traditional follicular patterned lesions and classical papillary thyroid carcinoma, predominantly displaying intermediate and aggressive drivers (61.6%), which was significantly higher than that of EFVPTC (22.3%, p = 0.0158) and NIFTP (0%, p < 0.0001), illustrating the higher MAP kinase activity of iFVPTC. There was no significant difference, however, in comparing GEAs among the three histological groups. Conclusions: While follicular patterned lesions with papillary nuclear features overall tend to display RAS-like alterations, EFVPTC cases, followed by iFVPTC in this series, showed increasing proportions of more aggressive drivers. EFVPTC and NIFTP show much molecular overlap, with predominance of RAS-like alterations, suggesting that these tumors are part of a genetic continuum, while still ranked differentially. Preoperative molecular testing can potentially distinguish EFVPTC and iFVTPC from NIFTP based on a particular molecular signature, optimizing patient management.

Molecular Testing Predicts Incomplete Response to Initial Therapy in Differentiated Thyroid Carcinoma Without Lateral Neck or Distant Metastasis at Presentation: Retrospective Cohort Study.

Background: Molecular testing (MT) is emerging as a potential prognostic factor that can be available before treatment of differentiated thyroid carcinoma begins. Among patients eligible for either lobectomy or total thyroidectomy as their initial therapy, our study aims were to assess (1) if conventionally available preoperative factors are associated with incomplete response to initial therapy, and (2) if MT results can be a surrogate for the ATA Risk Stratification System (RSS) to estimate risk of recurrence. Methods: The data of consecutive thyroid cancer patients without preoperative lateral neck disease or distant metastasis who underwent index thyroidectomy between November 1, 2017 and October 31, 2021 were reviewed. Logistic regression models including preoperative variables such as MT and/or the postoperatively available RSS were constructed to predict disease recurrence, either structural or biochemical. Model discrimination using the c-statistic and goodness-of-fit test were compared. Results: Among 945 patients studied, 50 (5.2%) recurred with 18-month median follow-up. Recurrences were detected in 17 (2.9%), 20 (6.7%), and 13 (22.8%) patients with RSS-low, -intermediate, and -high cancers, respectively (p < 0.001). In multivariable analysis, only tumor size was associated with recurrence (odds ratio [OR] 1.3, 95% confidence interval [CI] 1.1-1.5). In a different model analyzing 440 (46.6%) patients with available MT results, recurrence was associated with both larger tumor size (OR 1.4 [95% CI 1.1-1.8]) and MT results (p < 0.001). Including MT improved the c-statistic by 27%, which was statistically no different than the model incorporating only the RSS (p = 0.15). Conclusions: Disease recurrence was observed across all ATA RSS categories in short-term follow-up, and tumor size was the only conventional preoperative factor associated with recurrence. When MT results were incorporated, they not only improved predictive ability beyond tumor size alone, but also yielded similar ability as the gold standard ATA RSS. Thus, MT results might aid the development of novel preoperative risk stratification algorithms.

Inhibition of ALK-Signaling Overcomes STRN-ALK-Induced Downregulation of the Sodium Iodine Symporter and Restores Radioiodine Uptake in Thyroid Cells.

Background: Radioiodine (RAI) is commonly used for thyroid cancer treatment, although its therapeutic benefits are restricted to iodine-avid tumors. The RAI-refractory disease develops with tumor dedifferentiation involving loss of sodium-iodine symporter (NIS). Thyroid cancers driven by ALK fusions are prone to dedifferentiation, and whether targeted ALK inhibition may enhance RAI uptake in these tumors remains unknown. The aim of this study was to determine the levels of NIS expression during the progression of ALK fusion-driven thyroid cancer, assess the effects of ALK activation on NIS-mediated RAI uptake, and test pharmacological options for its modulation. Methods: The expression of NIS at different stages of ALK-driven carcinogenesis was analyzed using a mouse model of STRN-ALK-driven thyroid cancer. For in vitro experiments, a system of doxycycline-inducible expression of STRN-ALK was generated using PCCL3 normal thyroid cells. The STRN-ALK-induced effects were evaluated with quantitative reverse transcription polymerase chain reaction, Western blot, immunofluorescence, RNA sequencing, and gene sets pathways analyses. RAI uptake was measured using 131I. Treatment experiments were done with FDA-approved ALK inhibitors (crizotinib and ceritinib), MEK inhibitor selumetinib, and JAK1/2 inhibitor ruxolitinib. Results: We found that Nis downregulation occurred early in ALK-driven thyroid carcinogenesis, even at the stage of well-differentiated cancer, with a complete loss in poorly differentiated thyroid carcinomas. Acute STRN-ALK expression in thyroid cells resulted in increased MAPK, JAK/STAT3, and PI3K/AKT/mTOR signaling outputs associated with significant ALK-dependent downregulation of the majority of thyroid differentiation and iodine metabolism/transport genes, including Slc5a5 (Nis), Foxe1, Dio1, Duox1/2, Duoxa2, Glis3, Slc5a8, and Tg. Moreover, STRN-ALK expression in thyroid cells induced a significant loss of membranous NIS and a fourfold decrease of the NIS-mediated RAI uptake, which were reversed by ALK inhibitors crizotinib and ceritinib. In addition, a strong dose-dependent restoration of NIS with its membranous redistribution in STRN-ALK-expressing thyroid cells was observed after inhibition of MAPK signaling with selumetinib, which exhibited a cumulative effect with JAK1/2 inhibitor ruxolitinib. Conclusions: The findings of this preclinical study showed that ALK fusion-induced downregulation of NIS, the prerequisite of RAI refractoriness, could be reversed in thyroid cells by either direct inhibition of ALK or its downstream signaling pathways.

A thyroid EIF1AX story: how clinical, cytologic, and molecular surveillance led to appropriate management.

INTRODUCTION: Indeterminate thyroid cytology diagnoses are associated with intermediate risks of malignancy. Application of molecular testing (MT) to indeterminate specimens provides additional diagnostic and prognostic information. While a positive or suspicious MT result may prompt surgery, a negative MT result is associated with a low probability of cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear features and approximates that of a benign cytology diagnosis. Furthermore, ThyroSeq v3 MT has a "currently negative" result for findings with the probability of cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear feature that is slightly greater than that for the negative ThyroSeq v3 MT result but less than 10%, suggesting active surveillance. In this report, we discuss a case of a patient for whom clinical, cytologic, and molecular surveillance led to timely surgery and management. CLINICAL DETAILS: A 53-year-old man with a thyroid isthmus nodule had a fine-needle aspiration cytology diagnosis of atypia of undetermined significance and a subsequent ThyroSeq v3 MT, which revealed an EIF1AX mutation and a "currently negative" MT result. Surveillance with additional fine-needle aspiration samples demonstrated concerning genomic alterations (fluctuating EIF1AX allelic frequency and a non-V600E BRAF mutation), culminating in the conversion to a positive MT result 3 years later. Resection revealed an encapsulated noninvasive, oncocytic solid subtype of papillary thyroid carcinoma with increased mitotic activity. CONCLUSION: The case is notable for clinical, cytologic, and molecular surveillance demonstrating sequential pathologic alterations in an indeterminate thyroid nodule with EIF1AX mutation, leading to timely resection of the neoplasm before invasion manifested.

Association of comprehensive thyroid cancer molecular profiling with tumor phenotype and cancer-specific outcomes.

BACKGROUND: Molecular testing improves the diagnostic accuracy of thyroid cancer. Whether specific molecular testing results are associated with tumor phenotype or provide prognostic information needs further delineation. METHODS: Consecutive thyroid cancer patients after index thyroidectomy with ThyroSeq version 3 (Rye Brook, NY) molecular testing obtained on preoperative fine-needle aspiration or thyroidectomy specimens from patients with thyroid cancer were categorized into 3 molecular risk groups based on detected mutations, fusions, copy number alterations, and/or gene expression alterations and correlated with histopathology and recurrence, defined as biochemical or structural. RESULTS: Of 578 patients, 49.9%, 37.5%, and 12.6% had molecular risk group-low, molecular risk group-intermediate, and molecular risk group-high cancers, respectively. With a median 19-month follow-up, 9.1% patients recurred. Compared with molecular risk group-low, molecular risk group-intermediate cancers were diagnosed in younger patients and more often had microscopic extrathyroidal extension, involved margins, and nodal disease. Compared with molecular risk group-intermediate, molecular risk group-high cancers were diagnosed in older patients and more often had gross extrathyroidal extension and vascular invasion. In multivariable analysis, recurrence was more likely in molecular risk group-high cancers than in molecular risk group-intermediate (hazard ratio = 4.0; 95% confidence interval, 1.9-8.6; P < .001) and more likely in molecular risk group-intermediate than in molecular risk group-low (hazard ratio = 5.0; 95% confidence interval, 2.0-12.5; P < .001). CONCLUSION: Using modern comprehensive genotyping, the genetic profile of thyroid cancers can be categorized into 3 novel molecular risk groups that were associated with histopathologic phenotype and recurrence in short-term follow-up.

Identifying metastatic renal cell carcinoma in thyroid fine-needle aspirates by molecular testing.

Renal cell carcinoma (RCC) is the most common type of cancer found to metastasize to the thyroid gland. These tumors may represent a diagnostic challenge in cytology. However, most RCC tumors carry VHL alterations, which are rare in primary thyroid tumors. The aim of this study was to evaluate the utility of molecular testing in detecting metastatic RCC in thyroid fine-needle aspiration (FNA) samples. From November 2017 until March 2022, thyroid FNA samples with ThyroSeq v3 results showing both VHL alterations and low/absent expression of thyroid cell markers were analyzed. Eighteen samples from 15 patients met the inclusion criteria. On molecular analysis, deleterious VHL mutations were found in nine (50%) nodules, VHL copy number alteration (CNA) in two (11%), and both mutations and CNA in seven (39%). None of the cases showed mutations commonly found in thyroid tumors. The mean age of these patients was 68 (range, 49-89) years with a male to female ratio of 2:1. Eight (53%) patients had multiple thyroid nodules on ultrasound. On cytology, 14 (78%) nodules were diagnosed as Bethesda III, 2 (11%) as Bethesda IV, and 2 (11%) as Bethesda V. At the time of cytology review, the history of RCC, sometimes remote, was available for ten patients. Of the 14 patients with medical history or surgical follow-up available, all had history of RCC or renal mass or revealed metastatic RCC on thyroidectomy. This study demonstrates that molecular testing can reliably identify metastatic RCC in thyroid nodules with indeterminate cytology, which could improve patient management.

Prognostic of recurrence and survival in poorly differentiated thyroid cancer.

The prognosis of poorly differentiated thyroid carcinomas (PDTC) defined by the Turin criteria is variable. The aim of this study on 51 PDTC patients was to determine clinical, histological and molecular prognostic factors associated with recurrence in patients with localized disease at initial treatment and with overall survival in patients with distant metastases. Of 40 patients for whom next-generation sequencing (NGS) by ThyroSeq v3 was able to be performed on historical samples, we identified high-risk molecular signature (TERT, TP53 mutations) in 24 (60%) cases, intermediate risk signature in 9 (22.5%) cases and low-risk signature in 7 (17.5%) cases. Potentially actionable mutations were identified in 10% of cases. After a median follow-up of 57.5 months, recurrence occurred in 11 (39%) of the 28 patients with localized disease. The American Thyroid Association (ATA) high risk of relapse, high mitotic count, high molecular risk signature and CD163 expression were associated with recurrence (P = 0.009, 0.01, 0.049, 0.03 respectively). After a median follow-up of 49.5 months, thyroid cancer-related death occurred in 53% of the patients with distant metastases. There was no significant prognostic factor associated with death in univariate analysis. However, none of the patients with intermediate ATA risk of recurrence and none of the patients with low-risk molecular signature died from the disease. In addition, high molecular-risk signature was associated with the presence of synchronous or metachronous distant metastasis (P = 0.007) and with poor overall survival (P = 0.01). In conclusion, ATA risk of relapse and high mitotic count was associated with higher rate of recurrence in localized PDTC. High molecular-risk signature was associated with the presence of distant metastasis and poor overall survival. Further studies are needed to determine if molecular testing adds to ATA risk stratification or response to therapy in predicting outcomes.

Performance of a Multigene Genomic Classifier in Thyroid Nodules with Suspicious for Malignancy Cytology.

Background: Molecular testing is increasingly used to refine the probability of cancer and assess recurrence risk in thyroid nodules with Bethesda III/IV fine needle aspiration (FNA) cytology. However, limited data exist for Bethesda V (suspicious for malignancy [SFM]) samples. This study evaluated the performance of ThyroSeq v3 (TSv3) in thyroid nodules with SFM cytology. Methods: In this single-institution retrospective cohort study, consecutive thyroid FNA samples diagnosed as SFM with TSv3 testing and known surgical outcome were identified. Clinical, pathology, and molecular findings were reviewed. The TSv3 Cancer Risk Classifier was used to determine molecular risk groups (MRGs). For test-negative cases diagnosed as cancer/noninvasive follicular thyroid neoplasm with papillary-like nuclear features, TSv3 was performed on the resected tumors. Results: Among 128 SFM samples studied, 100 (78.1%) were TSv3 positive, and 28 (21.9%) were negative. The cancer prevalence on surgery was 82.8%. Among test-positive samples, 95% were malignant and 5% benign. Among test-negative samples, 17 (60.7%) were benign and 11 (39.3%) malignant. Overall, TSv3 had a sensitivity of 89.6% (confidence interval; CI 82.4-94.1) and a specificity of 77.3% (CI 56.6-89.9). For a cancer prevalence of 50-75% expected in SFM cytology by the Bethesda system, the negative predictive value was expected to range from 71.2% to 88.1% and the positive predictive value from 79.8% to 92.2%. Among test-positive nodules, 20% were MRG-Low (mostly RAS-like alterations), 66% MRG-Intermediate (mostly BRAF-like alterations), and 14% MRG-High. Among patients with cancer, 65 (61.3%) were American Thyroid Association low risk, 25 (23.6%) intermediate risk, and 6 (5.7%) high risk. During the mean follow-up of 51.2 months (range: <1 to 470 months), 12 (13.0%) patients had disease recurrence, which was more common in MRG-High (54.6%) compared with MRG-Intermediate (9.5%) and MRG-Low (0%) cancers (p < 0.001). Upon reexamining tumors with false-negative results, half of evaluable cases had alterations likely missed due to limiting FNA sampling, and the remainder represented low-risk tumors. Potentially targetable alterations were identified in 10 samples. Conclusions: In this large series of SFM thyroid nodules, TSv3 further improved cancer prediction and detected RAS-like, BRAF-like, high-risk, and potentially targetable alterations, all of which may inform more optimal patient management. MRGs were associated with recurrence-free survival, offering potential preoperative cancer risk stratification.