Impact of Somatic Mutations on Survival Outcomes in Patients With Anaplastic Thyroid Carcinoma.

PURPOSE: Anaplastic thyroid carcinoma (ATC) uniformly present with aggressive disease, but the mutational landscape of tumors varies. We aimed to determine whether tumor mutations affect survival outcomes in ATC. MATERIALS AND METHODS: Patients who underwent mutation sequencing using targeted gene panels between 2005 and 2019 at a tertiary referral center were included. Associations between mutation status and survival outcomes were assessed using Cox proportional hazards models. RESULTS: A total of 202 patients were included, where 122 died of ATC (60%). The median follow-up was 31 months (interquartile range, 18-45 months). The most common mutations were in TP53 (59%), BRAF (41%), TERT promoter (37%), and the RAS gene family (22%). Clinicopathologic characteristics and overall survival (OS) significantly correlated with mutations in BRAFV600E and RAS, which were mutually exclusive. The BRAFV600E mutation was associated with the presence of a papillary thyroid carcinoma precursor and significantly better OS (median OS: 24 months). RAS-mutated patients more commonly presented without cervical lymph node involvement but had the worst OS (median OS: 6 months). Tumors that were wild-type for both BRAF and RAS were enriched for NF1 mutations and harbored intermediate prognosis (median OS: 15 months). In multivariate analyses, RAS mutations were associated with a more than 2.5-fold higher risk of death (adjusted hazard ratio, 2.64; 95% CI, 1.66 to 4.20) compared with BRAFV600E. In patients treated with BRAF-directed therapy (n = 60), disease progression occurred in 48% of patients (n = 29). The median progression-free survival was 14 months. The presence of a TP53 mutation was independently associated with reduced progression-free survival in BRAFV600E-mutated patients treated with BRAF-directed therapy (adjusted hazard ratio, 2.89; 95% CI, 1.35 to 6.21). CONCLUSION: Mutation analysis provides prognostic information in ATC and should be incorporated into routine clinical care.

Hallmarks of RET and Co-occuring Genomic Alterations in RET-aberrant Cancers.

Activating receptor-tyrosine kinase rearranged during transfection (RET) mutations and fusions are potent drivers of oncogenesis. The recent FDA approvals of highly potent and selective RET inhibitors, selpercatinib and pralsetinib, has altered the therapeutic management of RET aberrant tumors. There is ample evidence of the role of RET signaling in certain cancers. RET aberrations as fusions or mutations occur in multiple cancers, however, there is considerable phenotypic diversity. There is emerging data on the lack of responsiveness of immunotherapy in RET-altered cancers. Herein, we review the registrational data from the selective RET-inhibitor trials, and comprehensively explore RET alterations in pan-cancer adult malignancies and their co-alterations. These co-occuring alterations may define the future of RET inhibition from specific selective targeting to customized combination therapies as data are rapidly emerging on both on-target and off-target acquired resistance mechanisms. Fascinatingly, oncogenic RET fusions have been reported to mediate resistance to EGFR inhibition and KRASG12C inhibition.

Clinical Utility of Circulating Cell-Free DNA Mutations in Anaplastic Thyroid Carcinoma.

Background: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid cancer that requires a rapid diagnosis and treatment to achieve disease control. Gene mutation profiling of circulating cell-free DNA (cfDNA) in peripheral blood may help to facilitate early diagnosis and treatment selection. The relatively rapid turnaround time compared with conventional tumor mutation testing is a major advantage. The objectives of this study were to examine the concordance of ATC-related mutations detected in cfDNA with those detected in the corresponding tumor tissue, and to determine the prognostic significance of cfDNA mutations in ATC patients. Methods: The ATC patients who were diagnosed and treated at The University of Texas MD Anderson Cancer Center between January 2015 and February 2018 and who had cfDNA testing were included in this study. cfDNA was collected by blood draw and was analyzed by next-generation sequencing (NGS) using the Guardant360-73 gene platform. Results: A total of 87 patients were included in the study. The most frequently mutated genes detected in cfDNA were TP53, BRAF, and PIK3CA. In 28 treatment naive ATC patients, the concordance rate of detected mutations in TP53, BRAFV600E, and PIK3CA between cfDNA and matched tissue NGS was 82.1%, 92.9%, and 92.9%, respectively. Patients with a PIK3CA mutation detected on cfDNA had worse overall survival (OS) (p = 0.03). This association was observed across various treatment modalities, including surgery, cytotoxic chemotherapy, radiation, and BRAF inhibitor (BRAFi) therapy. With regard to treatment, BRAFi therapy significantly improved ATC OS (p = 0.003). Conclusions: cfDNA is a valuable tool to evaluate a tumor's molecular profile in ATC patients. We identified high concordance rates between the gene mutations identified via cfDNA analysis and those identified from the NGS of the corresponding tumor tissue sequencing. Identified mutations in cfDNA can potentially provide timely information to guide treatment selection and evaluate the prognosis in patients with ATC.

Evaluation of Overall Survival in Patients With Anaplastic Thyroid Carcinoma, 2000-2019.

Importance: Anaplastic thyroid carcinoma (ATC) historically has a 4-month median overall survival (OS) from time of diagnosis, with disease-specific mortality approaching 100%. The association between recent major advancements in treatment and OS has yet to be evaluated. Objective: To evaluate rates of OS in patients with ATC over the last 2 decades. Design, Setting, and Participants: Retrospective cohort study in a single tertiary care institution. Patients with histopathological confirmation of ATC from January 2000 to October 2019 were included and divided into 3 groups according to date of presentation: 2000-2013, 2014-2016, and 2017-2019. Main Outcomes and Measures: Overall survival compared among different treatment eras and differing therapies, including targeted therapy, immunotherapy, and surgery. Results: Of 479 patients (246 men [51%]; median age, 65.0 [range, 21.1-92.6] years) with ATC evaluated, 52 (11%) were stage IVA, 172 (36%) stage IVB, and 255 (53%) stage IVC at presentation. The median OS of the entire cohort was 0.79 years (9.5 months), ranging from 0.01 to 16.63. The OS at 1 and 2 years was 35% (95% CI, 29%-42%) and 18% (95% CI, 13%-23%) in the 2000-2013 group (n = 227), 47% (95% CI, 36%-56%) and 25% (95% CI, 17%-34%) in the 2014-2016 group (n = 100), and 59% (95% CI, 49%-67%) and 42% (95% CI, 30%-53%) in the 2017-2019 group (n = 152), respectively (P < .001). The hazard ratio was 0.50 (95% CI, 0.38-0.67) for the 2017-2019 group compared with the 2000-2013 patients (P < .001). Factors associated with improved OS included targeted therapy (hazard ratio, 0.49; 95% CI, 0.39-0.63; P < .001), the addition of immunotherapy to targeted therapy (hazard ratio, 0.58; 95% CI, 0.36-0.94; P = .03), and surgery following neoadjuvant BRAF-directed therapy (hazard ratio, 0.29; 95% CI, 0.10-0.78; P = .02). Patients undergoing surgery following neoadjuvant BRAF-directed therapy (n = 20) had a 94% 1-year survival with a median follow-up of 1.21 years. Conclusion and Relevance: In this large single-institution cohort study spanning nearly 20 years, changes in patient management appear to be associated with significant increase in survival. The era of untreatable ATC is progressively being replaced by molecular-based personalized therapies, with integration of multidisciplinary therapies including surgery and radiation therapy.

HEREDITARY ENDOCRINE TUMOURS: CURRENT STATE-OF-THE-ART AND RESEARCH OPPORTUNITIES: The state of science in medullary thyroid carcinoma: current challenges and unmet needs.

The 16th International Multiple Endocrine Neoplasia Workshop (MEN2019) held in Houston, TX, USA, focused on emerging topics in the pathogenesis and therapy of malignant endocrine tumors associated with MEN syndromes. With MEN-2 syndromes, the most common malignancy is medullary thyroid carcinoma (MTC). In the spirit of the original MEN meeting workshop model, the conference included didactic lectures and interactive working groups of clinicians and researchers focused on the state of science in MTC and ongoing challenges or unmet needs in the understanding of MTC and to develop strategies to address these issues.

HEREDITARY ENDOCRINE TUMOURS: CURRENT STATE-OF-THE-ART AND RESEARCH OPPORTUNITIES: Early thyroidectomy in multiple endocrine neoplasia: a four decade experience.

Forty years ago, physicians caring for the J-kindred, a 100+ member family with multiple endocrine neoplasia type 2A (MEN2A), hypothesized that early thyroidectomy based on measurement of the biomarker calcitonin could cure patients at risk for development of medullary thyroid carcinoma (MTC). We re-evaluated 22 family members with proven RET proto-oncogene mutations (C634G) who underwent thyroidectomy and central lymphadenectomy between 1972 and 1994 based on stimulated calcitonin abnormalities. Current disease status was evaluated by serum calcitonin measurement and neck ultrasound in 18 of the 22 prospectively screened patients. The median age of the cohort at thyroidectomy was 16.5 years (range 9-24). The median duration of follow-up at the time of examination was 40 years (range 21-43) with a median current age of 52 years (range 34-65). Fifteen of the 18 patients had no detectable serum calcitonin (<2 pg/mL). Three had detectable serum calcitonin measurements, inappropriately elevated following total thyroidectomy. None of the 16 patients imaged had an abnormal ultrasound. Survival analysis shows no MTC-related deaths in the prospectively screened patients, whereas there were many in prior generations. Early thyroidectomy based on biomarker testing has rendered 15 of 18 MEN2A patients (83%) calcitonin-free with a median follow-up period of 40 years. There have been no deaths in the prospectively screened and thyroidectomized group. We conclude that early thyroidectomy and central lymph node dissection is an effective prophylactic treatment for hereditary MTC.

Advances in Targeting RET-Dependent Cancers.

RET alterations have been characterized as oncogenic drivers in multiple cancers. The clinical validation of highly selective RET inhibitors demonstrates the utility of specific targeting of aberrantly activated RET in patients with cancers such as medullary thyroid cancer or non-small cell lung cancer. The remarkable responses observed have opened the field of RET-targeted inhibitors. In this review, we seek to focus on the impact of therapeutic RET targeting in cancers. SIGNIFICANCE: Successful clinical translation of selective RET inhibitors is poised to alter the therapeutic landscape of altered cancers. Questions that clearly need to be addressed relate to the ability to maintain long-term inhibition of tumor cell growth, how to prepare for the potential mechanisms of acquired resistance, and the development of next-generation selective RET inhibitors.

Novel use of a Clinical Laboratory Improvements Amendments (CLIA)-certified Cyclin-Dependent Kinase N2C (CDKN2C) loss assay in sporadic medullary thyroid carcinoma.

BACKGROUND: The cyclin-dependent-kinase inhibitor/retinoblastoma pathway has been implicated in sporadic medullary thyroid carcinoma tumorigenesis. Somatic CDKN2C loss has been associated with decreased overall survival in medullary thyroid carcinoma patients. We evaluated CDKN2C loss in a prospective clinical environment using a novel Clinical Laboratory Improvement Amendments-certified assay to confirm its association with aggressive disease and to interrogate response to targeted therapy. METHODS: Patients with advanced sporadic medullary thyroid carcinoma underwent tumor genotyping for the purpose of management of targeted therapy and prognostication. RESULTS: Of patients with informative CDKN2C assay results, 30 (51.8%) were haploinsufficient/1N and 28 (48.3%) were 2N. Forty patients (69.0%) had a somatic RET mutation, and 36.9% had alterations of both genes. Thirty patients (51.7%) were treated with systemic therapy. Presence of genetic alterations in CDKN2C or RET did not predict treatment response. Patients with 1N CDKN2C loss had significantly shorter time-to-distant-metastasis than patients with normal copy number (P = .03). CONCLUSION: This is the first evaluation in the clinical setting of CDKN2C haploinsufficiency in sporadic medullary thyroid carcinoma. Although a larger cohort and longer follow-up will be required, loss seems to be associated with more aggressive disease and may indicate patients that might receive benefit from treatment with a CDK inhibitor.

Risk Haplotypes Uniquely Associated with Radioiodine-Refractory Thyroid Cancer Patients of High African Ancestry.

BACKGROUND: Thyroid cancer patients with radioiodine-refractory (RAI-R) disease, resulting from insufficient RAI delivery and/or RAI resistance, have increased mortality and limited treatment options. To date, studies have largely focused on tumor mutations associated with different stages of disease, which could provide prognostic value for RAI-R disease. It was hypothesized that germline variants contributing to intrinsic differences in iodine metabolism, tumor microenvironment, and/or immune surveillance are associated with RAI-R disease. METHODS: Whole-genome genotyping data analysis was performed on 1145 Caucasian (CAU) patients, 244 of whom were RAI-R, and 55 African American (AA) patients, nine of whom were RAI-R. Germline-variant association studies were conducted using candidate genes involved in iodine metabolism or DNA-damage repair, as well as genome-wide association analysis. Initial data indicated several notable variants in a small number of patients (n = 7), who were later determined to be AA patients of >80% African ancestry (n = 37). This led to the study focusing on germline single nucleotide polymorphisms uniquely associated with RAI-R AA patients. Sanger sequencing was performed to validate risk alleles and identify the incidence of the common somatic mutations BRAFV600E, NRASQ61R, and HRASQ61R in AA patients whose primary tumor samples were available (28/55). RESULTS: TG, BRCA1, and NSMCE2 haplotypes were identified as being uniquely associated with RAI-R AA patients of >80% African ancestry. All patients with the TG haplotype (n = 4) had a biochemical incomplete response to RAI therapy. Patients with the NSMCE2 haplotype (n = 4) were diagnosed at a young age (13, 17, 17, and 26 years old) with distant metastatic disease at initial diagnosis. The BRCA1 haplotype co-occurred in three out of four patients with the NSMCE2 haplotype. The incidence of BRAFV600E appears lower in papillary thyroid carcinomas from AA patients of >80% African ancestry (3/14; 21%) than in AA patients of <80% African ancestry (6/9; 67%), albeit only just approaching statistical significance (p = 0.077). The tumors available from three RAI-R AA patients were negative for BRAFV600E, NRASQ61R, and HRASQ61R. CONCLUSIONS: The identification of candidate RAI-R risk haplotypes may allow early stratification of clinical manifestations of RAI-R disease followed by early intervention and personalized treatment strategies. Functional annotation of candidate RAI-R risk haplotypes may provide insights into the mechanisms underlying RAI-R disease.

Genetic characterization of medullary thyroid cancer in childhood survivors of the Chernobyl accident.

BACKGROUND: Radiation-associated fusion oncogenes play a direct role in papillary thyroid cancer development and pathogenic fusions have recently been reported in medullary thyroid cancer. To date, no studies have evaluated oncogenic events in medullary thyroid cancer in a radiation-exposed population. METHODS: Somatic and germline alterations, including RET fusions, were evaluated in paired medullary thyroid cancer tumor and normal samples from the Chernobyl Tissue Bank, a heavily screened population affected by the Chernobyl disaster. RESULTS: Tissue was available for 49 individuals. The median age of diagnosis was 26 years (range 9 to 43 years); 16 were radiation-exposed at a median age of 6 (range 2 days to 17 years). A total of 21 patients harbored germline RET mutations (codons 634[13], 918[5], 790[1], 609[1], and 620[1]); 4 had family history. Sporadic medullary thyroid cancer was identified in 27 patients (RET[18], KRAS[1], RET+KRAS[1], TP53[1], wild type [6]), with 1 RET fusion (1/49;2%). The age at operation for patients with hereditary medullary thyroid cancer was not different than sporadic medullary thyroid cancer (23.5 vs 28 years, P = .063). In sporadic medullary thyroid cancer, radiation was not associated with a difference in age at operation, tumor size, or tumor stage (P > .05). CONCLUSION: In a heavily screened cohort, genetic analysis revealed germline RET mutations in previously unrecognized probands and a remarkable number of sporadic medullary thyroid cancer cases with a young age at presentation.

Combinations of Tyrosine Kinase Inhibitor and ERAD Inhibitor Promote Oxidative Stress-Induced Apoptosis through ATF4 and KLF9 in Medullary Thyroid Cancer.

Medullary thyroid carcinoma (MTC) originates from the C cells of the thyroid gland, which secrete calcitonin. Lymph node and distant metastases are frequently present at diagnosis. Activating mutations of RET, a driver oncogene in MTC that encodes a tyrosine kinase receptor, prevents apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum (ER) stress. We hypothesized that the combination of a tyrosine kinase inhibitor (TKI) and an ATF4 inducer promotes cell death by triggering catastrophic oxidative stress and apoptotic cell death. Here, we report that the ER-associated protein degradation (ERAD) inhibitor eeyarestatin sensitized MTC cells to the TKIs, sunitinib and vandetanib, thereby leading to synergistic upregulation of ATF4 expression, accumulation of reactive oxygen species, and subsequent cell death. Genome-wide analysis of ATF4 interaction sites by chromatin immunoprecipitation (ChIP) sequencing revealed that among ATF4 target genes was KLF9 (Kruppel-like factor 9), which induces MTC apoptosis. ChIP assays revealed that ATF4 occupancy at the KLF9 promoter was increased in MTC cells treated with eeyarestatin or vandetanib alone and was further enhanced in cells treated with both drugs, leading to increased KLF9 transcription. Depletion of ATF4 by shRNA led to downregulation of KLF9 expression and prevented oxidative stress-induced cell death. Furthermore, we identified ATF4 target genes (LZTFL1, MKNK2, and SIAH1 with known tumor suppressor function) that were synergistically upregulated with the combination of TKI and ERAD inhibitor. IMPLICATIONS: These findings reveal a combination therapy that induces reactive oxygen species-dependent catastrophic cell death through induction of ATF4 and KLF9 transcriptional activity.

The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells.

The ETS family of transcription factors is involved in several normal remodeling events and pathological processes including tumor progression. ETS transcription factors are divided into subfamilies based on the sequence and location of the ETS domain. ETV5 (Ets variant gene 5; also known as ERM) is a member of the PEA3 subfamily. Our meta-analysis of normal, benign, and malignant thyroid samples demonstrated that ETV5 expression is upregulated in papillary thyroid cancer and was predominantly associated with BRAF V600E or RAS mutations. However, the precise role of ETV5 in these lesions is unknown. In this study, we used the KTC1 cell line as a model for human advanced papillary thyroid cancer (PTC) because the cells harbor the heterozygous BRAF (V600E) mutation together with the C250T TERT promoter mutation. The role of ETV5 in PTC proliferation was tested using RNAi followed by high-throughput screening. Signaling pathways driving ETV5 expression were identified using specific pharmacological inhibitors. To determine if ETV5 influences the expression of epithelial-to-mesenchymal (EMT) markers in these cells, an EMT PCR array was used, and data were confirmed by qPCR and ChIP-qPCR. We found that ETV5 is critical for PTC cell growth, is expressed downstream of the MAPK pathway, and directly upregulates the transcription factor TWIST1, a known marker of intravasation and metastasis. Increased ETV5 expression could therefore be considered as a marker for advanced PTCs and a possible future therapeutic target.

Transcriptional targeting of oncogene addiction in medullary thyroid cancer.

Metastatic medullary thyroid cancer (MTC) is incurable and FDA-approved kinase inhibitors that include oncogenic RET as a target do not result in complete responses. Association studies of human MTCs and murine models suggest that the CDK/RB pathway may be an alternative target. The objective of this study was to determine if CDKs represent therapeutic targets for MTC and to define mechanisms of activity. Using human MTC cells that are either sensitive or resistant to vandetanib, we demonstrate that palbociclib (CDK4/6 inhibitor) is not cytotoxic to MTC cells but that they are highly sensitive to dinaciclib (CDK1/2/5/9 inhibitor) accompanied by reduced CDK9 and RET protein and mRNA levels. CDK9 protein was highly expressed in 83 of 83 human MTCs and array-comparative genomic hybridization had copy number gain in 11 of 30 tumors. RNA sequencing demonstrated that RNA polymerase II-dependent transcription was markedly reduced by dinaciclib. The CDK7 inhibitor THZ1 also demonstrated high potency and reduced RET and CDK9 levels. ChIP-sequencing using H3K27Ac antibody identified a superenhancer in intron 1 of RET. Finally, combined inhibition of dinaciclib with a RET kinase inhibitor was synergistic. In summary, we have identified what we believe is a novel mechanism of RET transcription regulation that potentially can be exploited to improve RET therapeutic targeting.

Recontacting Patients with Updated Genetic Testing Recommendations for Medullary Thyroid Carcinoma and Pheochromocytoma or Paraganglioma.

BACKGROUND: No guidelines exist regarding physicians' duty to inform former patients about novel genetic tests that may be medically beneficial. Research on the feasibility and efficacy of disseminating information and patient opinions on this topic is limited. METHODS: Adult patients treated at our institution from 1950 to 2010 for medullary thyroid cancer, pheochromocytoma, or paraganglioma were included if their history suggested being at-risk for a hereditary syndrome but genetic risk assessment would be incomplete by current standards. A questionnaire assessing behaviors and attitudes was mailed 6 weeks after an information letter describing new genetic tests, benefits, and risks was mailed. RESULTS: Ninety-seven of 312 (31.1%) eligible patients with an identified mailing address returned the questionnaire. After receiving the letter, 29.2% patients discussed genetic testing with their doctor, 39.3% considered pursuing genetic testing, and 8.5% underwent testing. Nearly all respondents (97%) indicated that physicians should inform patients about new developments that may improve their or their family's health, and 71% thought patients shared this responsibility. Most patients understood the letter (84%) and were pleased it was sent (84%), although 11% found it upsetting. CONCLUSIONS: Patients believe it is important for physicians to inform them of potentially beneficial developments in genetic testing. However, physician-initiated letters to introduce new information appear inadequate alone in motivating patients to seek additional genetic counseling and testing. Further research is needed regarding optimal methods to notify former patients about new genetic tests and corresponding clinical and ethical implications.

A Homozygous RET K666N Genotype With an MEN2A Phenotype.

Context: Germline RET K666N mutation has been described as a pathogenic mutation with low disease penetrance for medullary thyroid cancer (MTC) without other features of multiple endocrine neoplasia type 2A. We describe a patient with homozygous RET K666N mutation with MTC and bilateral pheochromocytoma (PHEO). Case Description: A 59-year-old woman received a diagnosis of MTC after biopsy of two thyroid nodules. Coincident biochemical and radiologic testing was suspicious for bilateral PHEO, confirmed after bilateral adrenalectomy. There was no evidence of primary hyperparathyroidism (PHPT). She had a total thyroidectomy with neck dissection revealing bilateral MTC with lymph node metastases. Germline RET testing identified homozygous K666N mutations. Genetic testing of family members showed that both adult children harbor a heterozygous K666N mutation. Her 32-year-old son had an elevated calcitonin level and underwent thyroidectomy, which identified MTC. Her 30-year-old daughter had a normal calcitonin level. Prophylactic thyroidectomy showed C-cell hyperplasia only. Three of seven other family members were tested and found to carry the mutation. All had normal calcitonin levels, and none had biochemical evidence of PHEO or PHPT. Given the absence of PHEO in reported RET K666N families, our proband underwent genetic testing for causes of hereditary paragangliomas or PHEO. No additional mutations were identified. Conclusions: Here we report a case of a homozygous RET K666N mutation leading to coincident MTC and PHEO. Heterozygous presentations of RET K666N mutations have low penetrance for isolated MTC. We believe that the gene dosage associated with the homozygosity of this variant contributed to the occurrence of bilateral PHEO.

Recent advances and emerging therapies in anaplastic thyroid carcinoma.

Anaplastic thyroid cancer is a rare and aggressive thyroid cancer with an overall survival measured in months. Because of this poor prognosis and often advanced age at presentation, these patients have traditionally been treated palliatively and referred for hospice. However, recent progress using novel therapies has energized the field, and several promising clinical trials are now available for these patients. This review will highlight this progress and the potential treatments that could pave the way to improved outcomes and quality of life for patients with this disease.

Circulating BRAF V600E Cell-Free DNA as a Biomarker in the Management of Anaplastic Thyroid Carcinoma.

Purpose: Anaplastic thyroid cancer (ATC) is a deadly form of thyroid cancer. BRAF V600E is the only actionable mutation for which there is a Food and Drug Administration-approved drug combination. Rapid detection of BRAF V600E and initiation of therapy is critical. We explored the ability of droplet digital polymerase chain reaction (ddPCR) to identify this mutation in circulating cell-free DNA (cfDNA) in plasma. Materials and Methods: The ddPCR assay was evaluated for its sensitivity, specificity for detection of BRAF V600E cfDNA, and concordance with tumor tissue. The assay also was used to evaluate its potential role as a biomarker of response. Results: Forty-four patients with ATC who were tested for the BRAF mutation by tumor tissue DNA sequencing or immunohistochemistry were included. Sixteen BRAF V600E-positive patients had treatment samples to evaluate cfDNA levels as a biomarker of response in correlation with restaging scans. Concordance of ddPCR with tumor tissue was 93%, with a sensitivity of 85% and specificity of 100%. Area under the curve by Wilcoxon rank sum test was 0.9 (95% CI, 0.80 to 0.99; P < .001). As a biomarker of response to treatment, 94% of ddPCR samples were concordant with tumor shrinkage in restaging scans, and 47% were concordant with tumor growth (Fisher's exact test P = .0061). In addition, cfDNA levels by ddPCR were predictive of treatment response in 71% of samples. Conclusion: cfDNA detection by ddPCR is highly sensitive, specific, and concordant with mutation status on ATC tumors. ddPCR also can be used for monitoring cfDNA levels in conjunction with imaging scans in patients with ATC.

Genotype-phenotype pancreatic neuroendocrine tumor relationship in multiple endocrine neoplasia type 1 patients: A 23-year experience at a single institution.

BACKGROUND: The aim of this study was to investigate the genotype-phenotype relationship of pancreatic neuroendocrine tumors in patients with multiple endocrine neoplasia type 1 treated at our institution. METHODS: We conducted a retrospective chart review of all patients with multiple endocrine neoplasia type 1 treated at our center from January 1993 to December 2015. Presence of a pancreatic neuroendocrine tumor was determined based on imaging performed at any time from presentation to conclusion of follow-up. RESULTS: We reviewed 188 patients. The most common site of multiple endocrine neoplasia type 1 mutation was in exon 2 (34/188; 18%). Of 188 patients, 125 had a pancreatic neuroendocrine tumor (61%). Among all patients, 30 of 34 (88%) with an exon 2 mutation had a pancreatic neuroendocrine tumor compared with 95 of 154 (62%) with a mutation in exons 3-10 (P = .002). In the age group of 20 to 40 years, 8 of 9 patients with an exon 2 mutation had a pancreatic neuroendocrine tumor, compared with 24 of 52 patients (46%) with a mutation in exons 3-10 (P = .028). Patients with an exon 2 mutation had a greater frequency of pancreatic neuroendocrine tumor distant metastasis (53% vs 23%, P = .049). CONCLUSION: Young patients with multiple endocrine neoplasia type 1 and an exon 2 mutation appear to have a 2-fold greater risk for developing a pancreatic neuroendocrine tumor, and patients with an exon 2 mutation may be at greater risk for developing distant metastasis. Consideration should be given to more intensive screening and more liberal application of primary operative intervention in this potentially high-risk group.

Reduced Retinoblastoma Protein Expression Is Associated with Decreased Patient Survival in Medullary Thyroid Cancer.

BACKGROUND: The retinoblastoma (RB) transcriptional corepressor 1 protein functions to slow cell-cycle progression. Inactivation of RB by reduced expression and/or hyperphosphorylation allow for enhanced progression through the cell cycle. Murine models develop medullary thyroid carcinoma (MTC) after generalized loss of RB. However, RB expression in MTC has only been evaluated in a small number of tumors, with differing results. The objective of this study was to determine whether reduced expression of RB and/or overexpression of hyperphosphorylated RB predict MTC aggressive behavior. METHODS: Formalin-fixed, paraffin-embedded primary thyroid tumors and lymph node metastases from MTC patients were evaluated for calcitonin, RB, and phosphorylated RB (pRB) expression by immunohistochemistry. Two expert pathologists evaluated the slides in a blinded manner, and the immunohistochemistry results were compared to disease-specific survival as a primary endpoint. RESULTS: Seventy-four MTC samples from 56 patients were analyzed in this study, including 51 primary tumors and 23 lymph node metastases. The median follow-up time was 6.75 years after surgery (range 0.64-24.30 years), and the median primary tumor size was 30 mm (range 6-96 mm). Sixty-six percent of cases were classified as stage IV. RB nuclear expression was diffusely present in 88% of primary tumors and 78% of lymph node metastases. Nuclear pRB expression was present in 22% of primary tumors and 22% of lymph node metastases. On univariate analysis, reduced RB (<75% tumor cell staining) trended with lower MTC-specific survival for primary tumor and metastatic nodes (primary tumor hazard ratio = 3.54 [confidence interval 0.81-15.47], p = 0.08; and lymph node hazard ratio = 4.35 [confidence interval 0.87-21.83], p = 0.05). For primary tumors, multivariable analysis showed that low nuclear RB expression was independently associated with worse disease-specific (p = 0.01) and overall (p = 0.02) survival. pRB levels were not associated with survival for either primary tumor or lymph node metastases. CONCLUSIONS: Reduced RB expression is associated with decreased patient survival in univariate and multivariable analyses, independent from patient age at surgery or advanced TNM stage. Future studies involving larger MTC patient populations are warranted to determine if lower RB expression levels may serve as a biomarker for aggressive disease in patients with MTC.