Joshua Klopper

Molecular Testing for Mutations in Improving the Fine-Needle Aspiration Diagnosis of Thyroid Nodules

CONTEXT Thyroid nodules are common in adults, but only a small fraction of them are malignant. Fine-needle aspiration (FNA) with cytological evaluation is the most reliable tool for cancer diagnosis in thyroid nodules. However, 10-40% of nodules are diagnosed as indeterminate by cytology, making it difficult to optimally manage these patients. OBJECTIVE The aim of this study was to establish the feasibility and role of testing for tumor-specific mutations in improving the FNA diagnosis of thyroid nodules. DESIGN The prospective study included 470 FNA samples of thyroid nodules from 328 patients. At the time of aspiration, a small portion of the material was collected and tested for BRAF, RAS, RET/PTC, and PAX8/PPARgamma mutations. The mutational status was correlated with cytology and either surgical pathology diagnosis or follow-up (mean, 34 months). RESULTS A sufficient amount of nucleic acids were isolated in 98% of samples. Thirty-two mutations were found, including 18 BRAF, eight RAS, five RET/PTC, and one PAX8/PPARgamma. The presence of any mutation was a strong indicator of cancer because 31 (97%) of mutation-positive nodules had a malignant diagnosis after surgery. A combination of cytology and molecular testing showed significant improvement in the diagnostic accuracy and allowed better prediction of malignancy in the nodules with indeterminate cytology. CONCLUSIONS These results indicate that molecular testing of thyroid nodules for a panel of mutations can be effectively performed in a clinical setting. It enhances the accuracy of FNA cytology and is of particular value for thyroid nodules with indeterminate cytology.

Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification

CONTEXT Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. OBJECTIVE We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. RESULTS Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

Multicenter Clinical Experience with the Afirma Gene Expression Classifier

BACKGROUND Increasingly, patients with thyroid nodule cytology labeled atypical (or follicular lesion) of undetermined significance (AUS/FLUS) or follicular neoplasm (FN) undergo diagnostic analysis with the Afirma gene expression classifier (GEC). No long-term, multisite analysis of Afirma GEC performance has yet been performed. METHODS We analyzed all patients who had received Afirma GEC testing at five academic medical centers between 2010 and 2013. Nodule and patient characteristics, fine needle aspiration cytology, Afirma GEC results, and subsequent clinical or surgical follow-up were obtained for 339 patients. Results were analyzed for pooled test performance, impact on clinical care, and site-to-site variation. RESULTS Three hundred thirty-nine patients underwent Afirma GEC testing of cytologically indeterminate nodules (165 AUS/FLUS; 161 FN; 13 suspicious for malignancy) and 174 of 339 (51%) indeterminate nodules were GEC benign, whereas 148 GEC were suspicious (44%). GEC results significantly altered care recommendations, as 4 of 175 GEC benign were recommended for surgery in comparison to 141 of 149 GEC suspicious (P<.01). Of 121 Cyto Indeterminate/GEC Suspicious nodules surgically removed, 53 (44%) were malignant. Variability in site-to-site GEC performance was confirmed, as the proportion of GEC benign varied up to 29% (P=.58), whereas the malignancy rate in nodules cytologically indeterminate/GEC suspicious varied up to 47% (P=.11). Seventy-one of 174 GEC benign nodules had documented clinical follow-up for an average of 8.5 months, in which 1 of 71 nodules proved cancerous. CONCLUSIONS These multicenter, clinical experience data confirm originally published Afirma GEC test performance and demonstrate its substantial impact on clinical care recommendations. Although nonsignificant site-to-site variation exists, such differences should be anticipated by the practicing clinician. Follow-up of GEC benign nodules thus far confirm the clinical utility of this diagnostic test.

The Impact of Benign Gene Expression Classifier Test Results on the Endocrinologist–Patient Decision to Operate on Patients with Thyroid Nodules with Indeterminate Fine-Needle Aspiration Cytopathology

BACKGROUND Seventy-five percent of thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology are found to be benign postoperatively. A novel genomic test, the Afirma gene expression classifier (AGEC), has been available for clinical use in the United States, since late 2010. In 2010, two modest-sized validation studies showed that the AGEC could identify a benign gene expression signature in indeterminate cytology thyroid FNA samples with a negative predictive value >95%. The objective of this study was to evaluate how the AGEC impacted the joint decision of the endocrinologist and patient to operate when FNA cytology was indeterminate, but the AGEC reading of the nodule was benign. METHODS In this cross-sectional cohort study, data were contributed retrospectively by 51 endocrinologists at 21 practice sites that had previously obtained ≥3 benign AGEC readings in ≥1 cm nodules with indeterminate FNA cytology readings. Information regarding demographic data, nodule size and location, decision to operate, surgery type (hemithyroidectomy [HT] or total thyroidectomy [TT]), and reason for recommending surgery was retrospectively collected. RESULTS Compared to a 74% previous historical rate of surgery for cytologically indeterminate nodules, the operative rate fell to 7.6% during the period that AGEC were obtained in the clinical practices, a highly significant reduction in the decision to operate (p<0.001). The rate of surgery on cytologically indeterminate nodules that were benign by the AGEC reading did not differ from the historically reported rate of operation on cytologically benign nodules (p=0.41). The four primary reasons reported by the physicians for operating on nodules with a benign AGEC reading, in descending order: large nodule size (46.4%), symptomatic nodules (25.0%), rapidly growing nodules (10.7%), or a second suspicious or malignant nodule in the same patient (10.7%). These reasons are concordant with those typically given for operation on cytologically benign nodules. CONCLUSIONS In a substantial group of medical practices, obtaining an AGEC test in patients with cytologically indeterminate nodules was associated with a striking reduction in the rate of diagnostic thyroidectomy. Approximately, one surgery was avoided for every two AGEC tests run on thyroid FNAs with indeterminate cytology.

Tumor-Associated Lymphocytes and Increased FoxP3+ Regulatory T Cell Frequency Correlate with More Aggressive Papillary Thyroid Cancer

CONTEXT Ten to 30% of patients with papillary thyroid cancer (PTC) develop recurrent disease and may benefit from innovative adjuvant therapies. Immune-based therapies are under investigation to treat many types of cancer. The role of the immune system in PTC is poorly understood. OBJECTIVE We investigated whether tumor-associated lymphocytes (TAL), in the absence of background thyroiditis (LT), contribute to disease severity. We hypothesized that the type of lymphocytes associated with PTC would correlate with parameters of disease. DESIGN This retrospective study analyzed archived PTC samples for the presence of TAL and/or LT. A group of patients with TAL was evaluated for lymphocyte subsets by immunohistofluorescence. PATIENTS AND SETTING One hundred PTC patients were analyzed for LT and TAL, and 10 PTC patients with TAL were assessed for lymphocyte subsets at University of Colorado Hospital. MAIN OUTCOME We assessed correlations between disease and the presence of TAL, LT, and lymphocyte subset frequency. RESULTS Patients with TAL exhibited higher disease stage and increased incidence of invasion and lymph node metastasis compared with patients without lymphocytes or with LT. CD4(+) T cell frequency correlated with tumor size (r = 0.742; P = 0.017). FoxP3(+) regulatory T cell (Treg) frequency correlated with lymph node metastases (r = 0.858; P = 0.002), and CD8 to Treg ratio correlated inversely with tumor size (r = -0.804; P = 0.007). CONCLUSIONS TAL and high Treg frequency in primary thyroid tumors correlates with more aggressive disease. Future prospective studies may identify Treg frequency as a predictive factor in PTC, and the suppressive effects of Treg should be considered in the design of immune-based therapies.

Efatutazone, an Oral PPAR-γ Agonist, in Combination With Paclitaxel in Anaplastic Thyroid Cancer: Results of a Multicenter Phase 1 Trial

PURPOSE A phase 1 study was initiated to determine the safety, potential effectiveness, and maximal tolerated dose and recommended phase 2 dose of efatutazone and paclitaxel in anaplastic thyroid cancer. EXPERIMENTAL DESIGN Patients received efatutazone (0.15, 0.3, or 0.5 mg) orally twice daily and then paclitaxel every 3 weeks. Patient tolerance and outcomes were assessed, as were serum efatutazone pharmacokinetics. RESULTS Ten of 15 patients were women. Median age was 59 years. Seven patients received 0.15 mg of efatutazone, 6 patients received 0.3 mg, and 2 patients received 0.5 mg. One patient receiving 0.3 mg of efatutazone had a partial response from day 69 to day 175; 7 patients attained stable disease. Median times to progression were 48 and 68 days in patients receiving 0.15 mg of efatutazone and 0.3 mg of efatutazone, respectively; corresponding median survival was 98 vs 138 days. The median peak efatutazone blood level was 8.6 ng/mL for 0.15-mg dosing vs 22.0 ng/mL for 0.3-mg twice daily dosing. Ten patients had grade 3 or greater adverse events (Common Terminology Criteria for Adverse Events), with 2 of these (anemia and edema) related to efatutazone. Thirteen events of edema were reported in 8 patients, with 2 of grade 3 or greater. Eight patients had ≥1 serious adverse event, with 1 of these (anemia) attributed to efatutazone and 1 (anaphylactic reaction) related to paclitaxel. The maximal tolerated dose was not achieved. Angiopoietin-like 4 was induced by efatutazone in tissue biopsy samples of 2 patients. CONCLUSIONS Efatutazone and paclitaxel in combination were safe and tolerated and had biologic activity.

Programmed Death-1+ T Cells and Regulatory T Cells Are Enriched in Tumor-Involved Lymph Nodes and Associated with Aggressive Features in Papillary Thyroid Cancer

CONTEXT Recurrent metastatic lymph node (LN) disease is common in patients with papillary thyroid cancer (PTC). Novel prognostic markers may be helpful in guiding a therapeutic approach. Our previous studies revealed that immune suppression is evident in PTC and associated with more severe disease. OBJECTIVE To characterize the immune response to metastatic PTC, we assessed CD4(+) T cell polarization in LN. In addition, we investigated the role of programmed death-1 (PD-1) and T cell exhaustion. DESIGN Uninvolved (UILN) and tumor-involved lymph nodes (TILN) were sampled ex vivo by fine-needle biopsy. T cell subsets were identified by flow cytometry. In parallel, archived TILN specimens were characterized by immunofluorescence. SETTING The study was conducted at the University of Colorado Hospital. PATIENTS Data were collected on 94 LN from 19 patients with PTC undergoing neck dissection. MAIN OUTCOME T cell subset frequencies were compared in UILN and TILN and assessed for correlation with recurrent disease and extranodal invasion. RESULTS Regulatory CD4(+) T cells (Treg) were enriched in TILN compared with UILN and further elevated in TILN from patients with recurrent disease. PD-1(+) T cells were present at high frequency in TILN and markedly enriched in TILN that showed evidence of extranodal invasion. In TILN, Treg frequency correlated with PD-1(+) T cell frequencies. Although PD-1(+) T cells produced interferon-γ, they failed to fully down-regulate CD27 and were not actively proliferating. CONCLUSIONS Increased Treg and PD-1(+) T cell frequencies in LN may be indicative of aggressive recurrent PTC. Future prospective studies are necessary to determine the prognostic and therapeutic value of these findings in PTC.

Management of recurrent and persistent metastatic lymph nodes in well-differentiated thyroid cancer: A multifactorial decision-making guide for the thyroid cancer care collaborative

Well‐differentiated thyroid cancer (WDTC) recurs in up to 30% of patients. Guidelines from the American Thyroid Association (ATA) and the National Comprehensive Cancer Network (NCCN) provide valuable parameters for the management of recurrent disease, but fail to guide the clinician as to the multitude of factors that should be taken into account. The Thyroid Cancer Care Collaborative (TCCC) is a web‐based repository of a patients clinical information. Ten clinical decision‐making modules (CDMMs) process this information and display individualized treatment recommendations.

ACTIVE SURVEILLANCE FOR PAPILLARY THYROID MICROCARCINOMA: NEW CHALLENGES AND OPPORTUNITIES FOR THE HEALTH CARE SYSTEM.

OBJECTIVE The dramatic increase in papillary thyroid carcinoma (PTC) is primarily a result of early diagnosis of small cancers. Active surveillance is a promising management strategy for papillary thyroid microcarcinomas (PTMCs). However, as this management strategy gains traction in the U.S., it is imperative that patients and clinicians be properly educated, patients be followed for life, and appropriate tools be identified to implement the strategy. METHODS We review previous active surveillance studies and the parameters used to identify patients who are good candidates for active surveillance. We also review some of the challenges to implementing active surveillance protocols in the U.S. and discuss how these might be addressed. RESULTS Trials of active surveillance support nonsurgical management as a viable and safe management strategy. However, numerous challenges exist, including the need for adherence to protocols, education of patients and physicians, and awareness of the impact of this strategy on patient psychology and quality of life. The Thyroid Cancer Care Collaborative (TCCC) is a portable record keeping system that can manage a mobile patient population undergoing active surveillance. CONCLUSION With proper patient selection, organization, and patient support, active surveillance has the potential to be a long-term management strategy for select patients with PTMC. In order to address the challenges and opportunities for this approach to be successfully implemented in the U.S., it will be necessary to consider psychological and quality of life, cultural differences, and the patients clinical status.

Consumptive Hypothyroidism Resulting from Hepatic Vascular Tumors in an Athyreotic Adult

CONTEXT Consumptive hypothyroidism is a rare syndrome resulting from increased catabolism of T(4) and T(3) by increased type 3 iodothyronine deiodinase (D3) activity. Consumptive hypothyroidism has primarily been described as a paraneoplastic syndrome in infants as well as in two adults with D3-expressing tumors. OBJECTIVE The aim of the study was to report the third case of consumptive hypothyroidism in an adult and the first in an athyreotic patient. DESIGN, SETTING, AND PATIENT We present a 38-yr-old athyreotic female who was euthyroid on a stable therapeutic dose of thyroid hormone for many years and then developed marked hyperthyrotropinemia, coincident with the discovery of large D3-expressing hepatic vascular tumors. The patient also had low serum T(3) and elevated serum rT(3). Hyperthyrotropinemia transiently worsened after surgical resection of the vascular tumors and then persisted for 3 wk after the operation, despite further increases in levothyroxine therapy. INTERVENTION The patients vascular tumor and adjacent normal liver parenchyma were probed with a polyclonal antibody directed against D3. MAIN OUTCOME MEASURES AND RESULTS D3 immunostaining of the patients vascular tumor was positive, with no significant immunoreactivity in the adjacent normal hepatic tissue. CONCLUSIONS This is the third case report of consumptive hypothyroidism in an adult and the first in an athyreotic individual. This case demonstrates that hyperthyrotropinemia may persist after partial liver resection, possibly from the hepatic resection itself.