Martha A. Zeiger

Association Between BRAF V600E Mutation and Mortality in Patients With Papillary Thyroid Cancer

IMPORTANCE BRAF V600E is a prominent oncogene in papillary thyroid cancer (PTC), but its role in PTC-related patient mortality has not been established. OBJECTIVE To investigate the relationship between BRAF V600E mutation and PTC-related mortality. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of 1849 patients (1411 women and 438 men) with a median age of 46 years (interquartile range, 34-58 years) and an overall median follow-up time of 33 months (interquartile range, 13-67 months) after initial treatment at 13 centers in 7 countries between 1978 and 2011. MAIN OUTCOMES AND MEASURES Patient deaths specifically caused by PTC. RESULTS Overall, mortality was 5.3% (45/845; 95% CI, 3.9%-7.1%) vs 1.1% (11/1004; 95% CI, 0.5%-2.0%) (P < .001) in BRAF V600E-positive vs mutation-negative patients. Deaths per 1000 person-years in the analysis of all PTC were 12.87 (95% CI, 9.61-17.24) vs 2.52 (95% CI, 1.40-4.55) in BRAF V600E-positive vs mutation-negative patients; the hazard ratio (HR) was 2.66 (95% CI, 1.30-5.43) after adjustment for age at diagnosis, sex, and medical center. Deaths per 1000 person-years in the analysis of the conventional variant of PTC were 11.80 (95% CI, 8.39-16.60) vs 2.25 (95% CI, 1.01-5.00) in BRAF V600E-positive vs mutation-negative patients; the adjusted HR was 3.53 (95% CI, 1.25-9.98). When lymph node metastasis, extrathyroidal invasion, and distant metastasis were also included in the model, the association of BRAF V600E with mortality for all PTC was no longer significant (HR, 1.21; 95% CI, 0.53-2.76). A higher BRAF V600E-associated patient mortality was also observed in several clinicopathological subcategories, but statistical significance was lost with adjustment for patient age, sex, and medical center. For example, in patients with lymph node metastasis, the deaths per 1000 person-years were 26.26 (95% CI, 19.18-35.94) vs 5.93 (95% CI, 2.96-11.86) in BRAF V600E-positive vs mutation-negative patients (unadjusted HR, 4.43 [95% CI, 2.06-9.51]; adjusted HR, 1.46 [95% CI, 0.62-3.47]). In patients with distant tumor metastasis, deaths per 1000 person-years were 87.72 (95% CI, 62.68-122.77) vs 32.28 (95% CI, 16.14-64.55) in BRAF V600E-positive vs mutation-negative patients (unadjusted HR, 2.63 [95% CI, 1.21-5.72]; adjusted HR, 0.84 [95% CI, 0.27-2.62]). CONCLUSIONS AND RELEVANCE In this retrospective multicenter study, the presence of the BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Because overall mortality in PTC is low and the association was not independent of tumor features, how to use BRAF V600E to manage mortality risk in patients with PTC is unclear. These findings support further investigation of the prognostic and therapeutic implications of BRAF V600E status in PTC.

BRAF Mutation Testing of Thyroid Fine-Needle Aspiration Biopsy Specimens for Preoperative Risk Stratification in Papillary Thyroid Cancer

PURPOSE This study investigated the utility of BRAF mutation testing of thyroid fine-needle aspiration biopsy (FNAB) specimens for preoperative risk stratification in papillary thyroid cancer (PTC). PATIENTS AND METHODS We assessed the T1799A BRAF mutation status in thyroid FNAB specimens obtained from 190 patients before thyroidectomy for PTC and its association with clinicopathologic characteristics of the tumor revealed postoperatively. RESULTS We observed a significant association of BRAF mutation in preoperative FNAB specimens with poorer clinicopathologic outcomes of PTC. In comparison with the wild-type allele, BRAF mutation strongly predicted extrathyroidal extension (23% v 11%; P = .039), thyroid capsular invasion (29% v 16%; P = .045), and lymph node metastasis (38% v 18%; P = .002). During a median follow-up of 3 years (range, 0.6 to 10 years), PTC persistence/recurrence was seen in 36% of BRAF mutation-positive patients versus 12% of BRAF mutation-negative patients, with an odds ratio of 4.16 (95% CI, 1.70 to 10.17; P = .002). The positive and negative predictive values for preoperative FNAB-detected BRAF mutation to predict PTC persistence/recurrence were 36% and 88% for overall PTC and 34% and 92% for conventional PTC, respectively. CONCLUSION Preoperative BRAF mutation testing of FNAB specimens provides a novel tool to preoperatively identify PTC patients at higher risk for extensive disease (extrathyroidal extension and lymph node metastases) and those who are more likely to manifest disease persistence/recurrence. BRAF mutation, as a powerful risk prognostic marker, may therefore be useful in appropriately tailoring the initial surgical extent for patients with PTC.

BRAF V600E and TERT Promoter Mutations Cooperatively Identify the Most Aggressive Papillary Thyroid Cancer With Highest Recurrence

PURPOSE To investigate the prognostic value of the BRAF V600E mutation and the recently identified TERT promoter mutation chr5:1,295,228C>T (C228T), individually and in their coexistence, in papillary thyroid cancer (PTC). PATIENTS AND METHODS We performed a retrospective study of the relationship of BRAF and TERT C228T mutations with clinicopathologic outcomes of PTC in 507 patients (365 women and 142 men) age 45.9 ± 14.0 years (mean ± SD) with a median follow-up of 24 months (interquartile range, 8 to 78 months). RESULTS Coexisting BRAF V600E and TERT C228T mutations were more commonly associated with high-risk clinicopathologic characteristics of PTC than they were individually. Tumor recurrence rates were 25.8% (50 of 194;77.60 recurrences per 1,000 person-years; 95% CI, 58.81 to 102.38) versus 9.6% (30 of 313; 22.88 recurrences per 1,000 person-years; 95% CI, 16.00 to 32.72) in BRAF mutation-positive versus -negative patients (hazard ratio [HR], 3.22; 95% CI, 2.05 to 5.07) and 47.5% (29 of 61; 108.55 recurrences per 1,000 person-years; 95% CI, 75.43 to 156.20) versus 11.4% (51 of 446; 30.21 recurrences per 1,000 person-years; 95% CI, 22.96 to 39.74) in TERT mutation-positive versus -negative patients (HR, 3.46; 95% CI, 2.19 to 5.45). Recurrence rates were 68.6% (24 of 35; 211.76 recurrences per 1,000 person-years; 95% CI, 141.94 to 315.94) versus 8.7% (25 of 287; 21.60 recurrences per 1,000 person-years; 95% CI, 14.59 to 31.97) in patients harboring both mutations versus patients harboring neither mutation (HR, 8.51; 95% CI, 4.84 to 14.97), which remained significant after clinicopathologic cofactor adjustments. Disease-free patient survival curves displayed a moderate decline with BRAF V600E or TERT C228T alone but a sharp decline with two coexisting mutations. CONCLUSION Coexisting BRAF V600E and TERT C228T mutations form a novel genetic background that defines PTC with the worst clinicopathologic outcomes, providing unique prognostic and therapeutic implications.

A Large Multicenter Correlation Study of Thyroid Nodule Cytopathology and Histopathology

BACKGROUND Fine-needle aspiration (FNA) biopsies are the cornerstone of preoperative evaluation of thyroid nodules, but FNA diagnostic performance has varied across different studies. In the course of collecting thyroid FNA specimens for the development of a molecular diagnostic test, local cytology and both local and expert panel surgical pathology results were reviewed. METHODS Prospective FNAs were collected at 21 clinical sites. Banked FNAs were collected from two academic centers. Cytology and corresponding local and expert panel surgical pathology results were compared to each other and to a meta-review of 11 recently published U.S.-based thyroid FNA studies. RESULTS FNA diagnostic performance was comparable between the study specimens and the meta-review. Histopathology malignancy rates for prospective clinic FNAs were 34% for cytology indeterminate cases and 98% for cytology malignant cases, comparable to the figures found in the meta-review (34% and 97%, respectively). However, histopathology malignancy rates were higher for cytology benign cases in the prospective clinic FNA subcohort (11%) than in the meta-review (6%, with meta-review rates of 10% at community sites and 2% at academic centers, p < 0.0001). Resection rates for prospective clinic FNAs were also comparable to the meta-review for both cytology indeterminate cases (62% vs. 59%, respectively) and cytology malignant cases (82% vs. 81%, respectively). Surgical pathology categorical disagreement (benign vs. malignant diagnosis) was higher between local pathology and a consensus of the two expert panelists (11%) than between the two expert panelists both pre- (8%) and postconferral (3%). CONCLUSIONS Although recent guidelines for FNA biopsy and interpretation have been published, the rates of false-positive and false-negative results remain a challenge. Two-thirds of cytology indeterminate cases were benign postoperatively and may decrease with the development of an accurate molecular diagnostic test. High disagreement rates between local and expert panel histopathology diagnosis suggests that central review for surgical diagnoses should be used when developing diagnostic tests based on resected thyroid specimens.

Mutational analysis of BRAF in Fine needle aspiration biopsies of the thyroid: A potential application for the preoperative assessment of thyroid nodules

Background: Fine needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules, but subsequent patient management is often complicated by the inability to decisively recognize malignancy on cytologic grounds alone. Activating mutations of the BRAF oncogene commonly occur in papillary thyroid carcinomas (PTCs) but not in other types of benign and malignant thyroid lesions. Mutational analysis of FNAs could enhance selection of thyroid nodules for surgical removal. Methods: Ninety-five excised PTCs along with 49 corresponding FNAs were evaluated for BRAF mutations by a newly developed assay that uses a novel primer extension method (MutectorR assay) and by direct sequencing. An additional 42 FNAs from thyroid nodules that were excised based on a suspicion of malignancy were also evaluated. Results:BRAF mutations were identified in 36 of the 95 (38%) excised PTCs. By histological subtype, BRAF mutations were more common in conventional PTCs than in the follicular variant (67% versus 12%; P < 0.0001; χ2). Analysis of the preoperative FNAs accurately reflected BRAF status of the resected PTC in 46 of the 49 paired samples (94% concordance). In FNA samples grouped according to the preoperative cytologic findings (malignant, n = 25; benign, n = 11; and indeterminate, n = 55), a BRAF mutation confirmed the diagnosis of PTC in 72% of carcinomas within the malignant group, and it established the diagnosis of PTC in 16% of carcinomas within the indeterminate group. BRAF mutations were not detected in FNAs from 32 benign thyroid lesions. Direct sequencing and the MutectorR assay yielded completely concordant results. Conclusions:BRAF mutations are common in conventional PTCs, and they are specific for PTC. A BRAF mutation can be reliably detected in cells aspirated from a thyroid nodule suggesting a role for this marker in the preoperative evaluation of thyroid nodules.

Molecular Classification of Thyroid Nodules Using High-Dimensionality Genomic Data

OBJECTIVE We set out to develop a molecular test that distinguishes benign and malignant thyroid nodules using fine-needle aspirates (FNA). DESIGN We used mRNA expression analysis to measure more than 247,186 transcripts in 315 thyroid nodules, comprising multiple subtypes. The data set consisted of 178 retrospective surgical tissues and 137 prospectively collected FNA samples. Two classifiers were trained separately on surgical tissues and FNAs. The performance was evaluated using an independent set of 48 prospective FNA samples, which included 50% with indeterminate cytopathology. RESULTS Performance of the tissue-trained classifier was markedly lower in FNAs than in tissue. Exploratory analysis pointed to differences in cellular heterogeneity between tissues and FNAs as the likely cause. The classifier trained on FNA samples resulted in increased performance, estimated using both 30-fold cross-validation and an independent test set. On the test set, negative predictive value and specificity were estimated to be 96 and 84%, respectively, suggesting clinical utility in the management of patients considering surgery. Using in silico and in vitro mixing experiments, we demonstrated that even in the presence of 80% dilution with benign background, the classifier can correctly recognize malignancy in the majority of FNA samples. CONCLUSIONS The FNA-trained classifier was able to classify an independent set of FNAs in which substantial RNA degradation had occurred and in the presence of blood. High tolerance to dilution makes the classifier useful in routine clinical settings where sampling error may be a concern. An ongoing multicenter clinical trial will allow us to validate molecular test performance on a larger independent test set of prospectively collected thyroid FNAs.

Comparison of SPECT/CT, SPECT, and Planar Imaging with Single- and Dual-Phase 99mTc-Sestamibi Parathyroid Scintigraphy

Various methodologies for 99mTc-sestamibi parathyroid scintigraphy are in clinical use. There are few direct comparisons between the different methods and even less evidence supporting the superiority of one over another. Some reports suggest that SPECT is superior to planar imaging. The addition of CT to SPECT may further improve parathyroid adenoma localization. The purpose of our investigation was to compare hybrid SPECT/CT, SPECT, and planar imaging and to determine whether dual-phase imaging is advantageous for the 3 methodologies. Methods: Scintigraphy was performed on 110 patients with primary hyperparathyroidism and no prior neck surgery. Of these, 98 had single adenomas and are the subject of this review. Planar imaging and SPECT/CT were performed at 15 min and 2 h after injection. Six image sets (early and delayed planar imaging, SPECT, and SPECT/CT) and combinations of the 2 image sets were reviewed for adenoma localization at 13 possible sites. Each review was scored for location and certainty of focus by 2 reviewer groups. Surgical location served as the standard. Sensitivity, specificity, area under the curve, positive predictive value, negative predictive value, and κ-values were determined for each method. Results: The overall κ-coefficient (certainty of adenoma focus) between reading groups was 0.68 (95% confidence interval, 0.66–0.70). The highest values were for dual-phase studies that included SPECT/CT. Dual-phase planar imaging, SPECT, and SPECT/CT were statistically significantly superior to single-phase early or delayed imaging in sensitivity, area under the curve, and positive predictive value. Neither single-phase nor dual-phase SPECT was statistically superior to dual-phase planar imaging. Early-phase SPECT/CT in combination with any delayed imaging method was superior to dual-phase planar imaging or SPECT for sensitivity, area under the curve, and positive predictive value. Conclusion: Early SPECT/CT in combination with any delayed imaging method was statistically significantly superior to any single- or dual-phase planar or SPECT study for parathyroid adenoma localization. Localization with dual-phase acquisition was more accurate than with single-phase 99mTc-sestamibi scintigraphy for planar imaging, SPECT, and SPECT/CT.

Using Gene Expression Profiling to Differentiate Benign versus Malignant Thyroid Tumors

DNA microarrays allow quick and complete evaluation of a cells transcriptional activity. Expression genomics is very powerful in that it can generate expression data for a large number of genes simultaneously across multiple samples. In cancer research, an intriguing application of expression arrays includes assessing the molecular components of the neoplastic process and utilizing the data for cancer classification (Miller LD, et al. Cancer Cell 2002;2:353-61). Classification of human cancers into distinct groups based on their molecular profile rather than their histological appearance may prove to be more relevant to specific cancer diagnoses and cancer treatment regimes. Several attempts to formulate a consensus about classification and treatment of thyroid carcinoma based on standard histopathological analysis have resulted in published guidelines for diagnosis and initial disease management (Sherman SI. Lancet 2003;361:501-11). In the past few decades, no improvement has been made in the differential diagnosis of thyroid tumors by fine needle aspiration biopsy, specifically suspicious or indeterminate thyroid lesions, suggesting that a new approach to this should be explored. Therefore, in this study, we developed a gene expression approach to diagnose benign versus malignant thyroid lesions in 73 patients with thyroid tumors. We successfully built a 10 and 6 gene model able to differentiate benign versus malignant thyroid tumors. Our results support the premise that a molecular classification system for thyroid tumors is possible, and this in turn may provide a more accurate diagnostic tool for the clinician managing patients with suspicious thyroid lesions.

Association of aberrant methylation of tumor suppressor genes with tumor aggressiveness and BRAF mutation in papillary thyroid cancer

The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation‐induced gene silencing in PTC‐derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase‐3 (TIMP3), SLC5A8, death‐associated protein kinase (DAPK) and retinoic acid receptor β2 (RARβ2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall‐cell PTC subtypes than in less aggressive follicular‐variant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARβ2, in association with BRAF mutation, may be an important step in PTC tumorigenesis and progression.

Parathyroidectomy in Maryland: Effects of an endocrine center

BACKGROUND Surgery for hyperparathyroidism is associated with high cure rates and low morbidity and mortality when performed by experienced surgeons. We wanted to determine whether referral of patients with hyperparathyroidism to an endocrine surgery center has an impact on patient outcomes and costs. METHODS Data from 901 patients who underwent parathyroidectomy recorded in the Maryland inpatient discharge database between 1990 and 1994 at 52 hospitals were compared with 169 consecutive patients who underwent surgical exploration by one surgeon (R.U.) at the Johns Hopkins Hospital. RESULTS Although in 47 of 52 hospitals fewer than 10 parathyroidectomies were performed each year, in these hospitals four of five related deaths occurred before patient discharge. The percentage of parathyroidectomies in Maryland performed by one endocrine surgeon has increased from 8% in 1990 to 21% in 1994 and is associated with a 97% cure rate and no mortality. Moreover, while hospital length of stay (LOS) in the state has decreased from 7 to 3.1 days, LOS for the high-volume provider has declined to a mean of 1.3 days. CONCLUSIONS Patients with hyperparathyroidism are increasingly referred to an endocrine surgery center, which results in a high cure rate, low morbidity, no mortality, and a shorter LOS. Improved surgical outcomes and lower costs depend on an experienced surgeon and argue for the referral of these patients to endocrine surgery centers.