Publications by authors named "Rengyun Liu"

31 Publications

Therapeutic targeting of FOS in mutant cancers through removing TERT suppression of apoptosis via regulating and .

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes, and Metabolism, The Johns Hopkins University School of Medicine, Baltimore, MD 21287

The telomerase reverse transcriptase (TERT) has long been pursued as a direct therapeutic target in human cancer, which is currently hindered by the lack of effective specific inhibitors of TERT. The FOS/GABPB/(mutant) cascade plays a critical role in the regulation of mutant , in which FOS acts as a transcriptional factor for to up-regulate the expression of GABPB, which in turn activates mutant but not wild-type promoter, driving TERT-promoted oncogenesis. In the present study, we demonstrated that inhibiting this cascade by targeting FOS using FOS inhibitor T-5224 suppressed mutant cancer cells and tumors by inducing robust cell apoptosis; these did not occur in wild-type cells and tumors. Mechanistically, among 35 apoptotic cascade-related proteins tested, the apoptosis induced in this process specifically involved the transcriptional activation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 () and inactivation of two key players in the apoptotic cascade, which normally initiate and suppress the apoptotic cascade, respectively. These findings with suppression of FOS were reproduced by direct knockdown of TERT and prevented by prior knockdown of TRAIL-R2. Further experiments demonstrated that TERT acted as a direct transcriptional factor of up-regulating its expression. Thus, this study identifies a therapeutic strategy for promoter mutation-driven cancers by targeting FOS in the FOS/GABPB/(mutant) cascade, circumventing the current challenge in pharmacologically directly targeting TERT itself. This study also uncovers a mechanism through which TERT controls cell apoptosis by transcriptionally regulating two key players in the apoptotic cascade.
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http://dx.doi.org/10.1073/pnas.2022779118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980366PMC
March 2021

Towards precision medicine in thyroid cancer.

Ann Transl Med 2020 Oct;8(19):1212

Department of Endocrinology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

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http://dx.doi.org/10.21037/atm-20-6450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607071PMC
October 2020

promoter mutation determines apoptotic and therapeutic responses of -mutant cancers to BRAF and MEK inhibitors: Achilles Heel.

Proc Natl Acad Sci U S A 2020 07 19;117(27):15846-15851. Epub 2020 Jun 19.

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21827;

Combination use of BRAF V600E inhibitor dabrafenib and MEK inhibitor trametinib has become a standard treatment for human cancers harboring  V600E. Its anticancer efficacies vary, however, with dramatic efficacy in some patients and drug resistance/tumor recurrence in others, which is poorly understood. Using thyroid cancer, melanoma, and colon cancer cell models, we showed that dabrafenib and trametinib induced robust apoptosis of cancer cells harboring both V600E and  promoter mutations but had little proapoptotic effect in cells harboring only  V600E. Correspondingly, the inhibitors nearly completely abolished the growth of in vivo tumors harboring both mutations but had little effect on tumors harboring only V600E. Upon drug withdrawal, tumors harboring both mutations remained hardly measurable but tumors harboring only  V600E regrew rapidly. BRAF V600E/MAP kinase pathway is known to robustly activate mutant promoter of TERT, a strong apoptosis suppressor. Thus, for survival, cancer cells harboring both mutations may have evolved to rely on  V600E-promoted and high-TERT expression-mediated suppression of apoptosis. As such, inhibition of BRAF/MEK can trigger strong apoptosis-induced cell death and hence tumor abolishment. This does not happen in cells harboring only  V600E as they have not developed reliance on TERT-mediated suppression of apoptosis due to the lack of mutant promoter-driven high- expression. promoter mutation governs BRAF-mutant cancer cells' apoptotic and hence therapeutic responses to BRAF/MEK inhibitors. Thus, the genetic duet of V600E and  promoter mutation represents an Achilles Heel for effective therapeutic targeting and response prediction in cancer.
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http://dx.doi.org/10.1073/pnas.2004707117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355024PMC
July 2020

The Genetic Duet of V600E and Promoter Mutations Robustly Predicts Loss of Radioiodine Avidity in Recurrent Papillary Thyroid Cancer.

J Nucl Med 2020 02 2;61(2):177-182. Epub 2019 Aug 2.

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and

V600E and promoter mutations, particularly their genetic duet, are well known to be associated with poor clinical outcomes of papillary thyroid cancer (PTC). Loss of radioactive iodine (RAI) avidity in recurrent PTC is a major cause of treatment failure and hence poor clinical outcomes. This study investigated the role of mutation patterns in loss of RAI avidity in recurrent PTC. This was a retrospective study of the relationship between loss of RAI avidity in structural recurrent PTC and the genotype patterns of V600E and promoter mutations in 164 patients (104 women and 60 men) with a median age of 50 y (interquartile range, 35-62 y). The overall prevalence of RAI avidity loss in recurrent PTC was 62.8% (103/164). When the cohort was divided into mutation and wild-type groups, the RAI avidity loss was 80.4% versus 33.9% ( < 0.001) in V600E versus wild-type patients, with an adjusted odds ratio of 7.11 (95% confidence interval [CI], 3.24-16.27), and 89.4% versus 52.1% ( < 0.001) in mutation versus wild-type patients, with an adjusted odds ratio of 6.89 (95% CI, 2.28-25.66). When the cohort was divided into 4 genotypes, the RAI avidity loss was 70.3% (45/64), 55.6% (5/9), and 97.4% (37/38) in patients with V600E alone, mutation alone, and the genetic duet of coexisting and mutations, versus 30.2% (16/53) in patients with neither mutation ( < 0.001, = 0.251, and < 0.001, respectively). These corresponded to odds ratios of 5.39 (95% CI, 2.31-13.13), 2.84 (95% CI, 0.53-16.32), and 81.04 (95% CI, 11.67-3559.83), respectively. The synergy index was 13.28 (95% CI, 1.54-114.46; = 0.019) between V600E and mutation in cooperatively affecting RAI avidity. A similar genotype-associated expression pattern was observed for thyroid iodide-handling genes. V600E alone and, particularly, coexisting V600E and promoter mutations are strongly associated with loss of RAI avidity and impairment of the iodide-metabolizing machinery in recurrent PTC, showing a robust predictive value for failure of RAI treatment of PTC.
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http://dx.doi.org/10.2967/jnumed.119.227652DOI Listing
February 2020

Stage II Differentiated Thyroid Cancer Is a High-Risk Disease in Patients <45/55 Years Old.

J Clin Endocrinol Metab 2019 11;104(11):4941-4948

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes & Metabolism, Department of Medicine; Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: The mortality risk of stage II differentiated thyroid cancer (DTC) based on the American Joint Committee on Cancer (AJCC) staging system requires further investigation.

Methods: Retrospective study of DTC in the US Surveillance, Epidemiology, and End Results program for disease-specific mortality risk in various AJCC stages, with patient age stratification of stage II disease.

Results: Using AJCC staging system 6.0, hazard ratios (HRs) of mortality for stage II DTC in patients <45 yo and patients ≥45 yo and stages III, IVA, IVB, and IVC compared with stage I were 46.95, 4.95, 9.82, 57.37, 222.10, and 468.68, respectively, showing a robustly higher mortality risk in stage II disease in patients <45 yo than in older patients (P < 0.001), comparable with stage IVA (P = 0.482). Similar results were obtained with AJCC 7.0. With AJCC 8.0, HRs of mortality for stage II in patients <55 yo and patients ≥55 yo and stages III, IVA, and IVB compared with stage I were 75.16, 11.23, 69.45, 134.94, and 235.70, respectively, showing a robustly higher risk in stage II disease in patients <55 yo than in older patients (P < 0.001), comparable with stage III (P = 0.57). Kaplan-Meier survival curves displayed a sharp decline with stage II disease in patients <45/55 yo compared with older patients.

Conclusions: The mortality risk of stage II DTC was sharply differentiated at patient age 45/55 years, being robustly high in younger patients and comparable with stage III/IVA. This emphasizes the importance of considering age when managing stage II DTC and not treating it as a uniformly low-risk disease.
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http://dx.doi.org/10.1210/jc.2018-02809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453036PMC
November 2019

Identification and characterization of two novel oncogenic mTOR mutations.

Oncogene 2019 06 27;38(26):5211-5226. Epub 2019 Mar 27.

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.

Mammalian target of rapamycin (mTOR) signaling is often aberrantly activated, particularly when genetically altered, in human cancers. mTOR inhibitors targeting the activated mTOR signaling are highly promising anti-cancer drugs. Knowing the activating genetic change in mTOR can help guide the use of mTOR inhibitors for cancer treatment. This study was conducted to identify and characterize novel oncogenic mTOR mutations that can potentially be therapeutic targets in human cancer. We sequenced 30 exons of the mTOR gene in 12 thyroid cancer cell lines, 3 melanoma cell lines, 20 anaplastic thyroid cancer (ATC) tumors, and 23 melanoma tumors and functionally characterized the identified novel mTOR mutations in vitro and in vivo. We identified a novel point mutation A1256G in ATC cell line and G7076A in melanoma tumor in exon 9 and exon 51 of the mTOR gene, respectively. Over-expression of the corresponding mTOR mutants H419R and G2359E created through induced mutagenesis showed markedly elevated protein kinase activities associated with the activation of mTOR/p70S6K signaling in HEK293T cells. Stable expression of the two mTOR mutants in NIH3T3 cells strongly activated the mTOR/p70S6K signaling pathway and induced morphologic transformation, cell focus formation, anchorage-independent cell growth, and invasion. Inoculation of these mutant-expressing cells in athymic nude mice induced rapid tumor development, showing their driving oncogenicity. We also demonstrated that transfection with the novel mutants conferred cells high sensitivities to the mTOR inhibitor temsirolimus. We speculate that human cancers harboring these mTOR mutations, such as ATC and melanoma, may be effectively treated with inhibitors targeting mTOR.
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http://dx.doi.org/10.1038/s41388-019-0787-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597304PMC
June 2019

BRAF V600E Confers Male Sex Disease-Specific Mortality Risk in Patients With Papillary Thyroid Cancer.

J Clin Oncol 2018 09 2;36(27):2787-2795. Epub 2018 Aug 2.

Fei Wang, Shihua Zhao, and Yangang Wang, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China; Fei Wang, Xiaopei Shen, Guangwu Zhu, Rengyun Liu, and Mingzhao Xing, Johns Hopkins University School of Medicine, Baltimore, MD; David Viola and Rossella Elisei, University of Pisa, Pisa; Efisio Puxeddu, University of Perugia, Perugia; Laura Fugazzola and Carla Colombo, Istituto Auxologico Italiano, Istituto di Recovero e Cura a Carattere Scientifico (IRCCS), and University of Milan, Milan; Caterina Mian, University of Padua; Federica Vianello, Veneto Institute of Oncology, IRCCS, Padua, Italy; Barbara Jarzab and Agnieszka Czarniecka, Maria Sklodowska-Curie Institute Oncology Center, Gliwice, Poland; Alfred K. Lam, Griffith University, Gold Coast, Queensland; Christine J. O'Neill, Mark S. Sywak, and Roderick Clifton-Bligh, University of Sydney, Sydney, New South Wales, Australia; Linwah Yip, University of Pittsburgh, Pittsburgh, PA; Garcilaso Riesco-Eizaguirre, Hospital Universitario La Paz and Hospital Universitario de Móstoles; Garcilaso Riesco-Eizaguirre and Pilar Santisteban, Biomedical Research Institute "Alberto Sols" and Health Institute Carlos III, Madrid, Spain; and Bela Bendlova and Vlasta Sýkorová, Institute of Endocrinology, Prague, Czech Republic.

Purpose To test whether the prognostic risk of male sex in papillary thyroid cancer (PTC) is determined by BRAF V600E and can thus be stratified by BRAF status. Patients and Methods We retrospectively investigated the relationship between male sex and clinicopathologic outcomes in PTC, particularly mortality, with respect to BRAF status in 2,638 patients (male, n = 623; female, n = 2,015) from 11 centers in six countries, with median age of 46 years (interquartile range, 35-58 years) at diagnosis and median follow-up time of 58 months (interquartile range, 26-107 months). Results Distant metastasis rates in men and women were not different in wild-type BRAF PTC but were different in BRAF V600E PTC: 8.9% (24 of 270) and 3.7% (30 of 817; P = .001), respectively. In wild-type BRAF PTC, mortality rates were 1.4% (five of 349) versus 0.9% (11 of 1175) in men versus women ( P = .384), with a hazard ratio (HR) of 1.59 (95% CI, 0.55 to 4.57), which remained insignificant at 0.70 (95% CI, 0.23 to 2.09) after clinicopathologic multivariable adjustment. In BRAF V600E PTC, mortality rates were 6.6% (18 of 272) versus 2.9% (24 of 822) in men versus women ( P = .006), with an HR of 2.43 (95% CI, 1.30 to 4.53), which remained significant at 2.74 (95% CI, 1.38 to 5.43) after multivariable adjustment. In conventional-variant PTC, male sex similarly had no effect in wild-type BRAF patients; mortality rates in BRAF V600E patients were 7.2% (16 of 221) versus 2.9% (19 of 662) in men versus women ( P = .004), with an HR of 2.86 (95% CI, 1.45 to 5.67), which remained significant at 3.51 (95% CI, 1.62 to 7.63) after multivariable adjustment. Conclusion Male sex is a robust independent risk factor for PTC-specific mortality in BRAF V600E patients but not in wild-type BRAF patients. The prognostic risk of male sex in PTC can thus be stratified by BRAF status in clinical application.
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http://dx.doi.org/10.1200/JCO.2018.78.5097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145834PMC
September 2018

Regulation of mutant TERT by BRAF V600E/MAP kinase pathway through FOS/GABP in human cancer.

Nat Commun 2018 02 8;9(1):579. Epub 2018 Feb 8.

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes, & Metabolism, Department of Medicine, John Hopkins University School of Medicine, Baltimore, MD, 21287, USA.

The unique oncogene duet of coexisting BRAF V600E and TERT promoter mutations are widely found to be a robust genetic background promoting human cancer aggressiveness, but the mechanism is unclear. Here, we demonstrate that the BRAF V600E/MAP kinase pathway phosphorylates and activates FOS, which in turn acts as a transcription factor to bind and activate the GABPB promoter, increasing GABPB expression and driving formation of GABPA-GABPB complex; the latter selectively binds and activates mutant TERT promoter, upregulating TERT expression. Elevated TERT functions as a strong oncoprotein, robustly promoting aggressive behaviors of cancer cells and tumor development. We thus identify a molecular mechanism for the activation of mutant TERT by the BRAF V600E/MAP kinase pathway, in which FOS as a transcriptional factor of GABPB promoter plays a key role in functionally bridging the two oncogenes in cooperatively promoting oncogenesis, providing important cancer biological and clinical implications.
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http://dx.doi.org/10.1038/s41467-018-03033-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805723PMC
February 2018

Patient Age-Associated Mortality Risk Is Differentiated by BRAF V600E Status in Papillary Thyroid Cancer.

J Clin Oncol 2018 02 14;36(5):438-445. Epub 2017 Dec 14.

Xiaopei Shen, Guangwu Zhu, Rengyun Liu, and Mingzhao Xing, Johns Hopkins University School of Medicine, Baltimore, MD; David Viola and Rossella Elisei, University of Pisa, Pisa; Efisio Puxeddu, University of Perugia, Perugia; Laura Fugazzola and Carla Colombo, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Auxologico Italiano and University of Milan, Milan; Caterina Mian, University of Padua; Federica Vianello, Veneto Institute of Oncology, IRCCS, Padua, Italy; Barbara Jarzab and Agnieszka Czarniecka, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland; Alfred K. Lam, Menzies Health Institute Queensland, Griffith University, Gold Coast, Queensland; Christine J. O'Neill, Mark S. Sywak, and Roderick Clifton-Bligh, The University of Sydney, Sydney, New South Wales, Australia; Linwah Yip, University of Pittsburgh School of Medicine, Pittsburgh, PA; Garcilaso Riesco-Eizaguirre, Hospital Universitario La Paz and Hospital Universitario de Móstoles; Garcilaso Riesco-Eizaguirre and Pilar Santisteban, Biomedical Research Institute "Alberto Sols," Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid; Garcilaso Riesco-Eizaguirre and Pilar Santisteban, Ciberonc, Health Institute Carlos III, Madrid, Spain; and Bela Bendlova and Vlasta Sýkorová, Institute of Endocrinology, Prague, Czech Republic.

Purpose For the past 65 years, patient age at diagnosis has been widely used as a major mortality risk factor in the risk stratification of papillary thyroid cancer (PTC), but whether this is generally applicable, particularly in patients with different BRAF genetic backgrounds, is unclear. The current study was designed to test whether patient age at diagnosis is a major mortality risk factor. Patients and Methods We conducted a comparative study of the relationship between patient age at diagnosis and PTC-specific mortality with respect to BRAF status in 2,638 patients (623 men and 2,015 women) with a median age of 46 years (interquartile range, 35 to 58 years) at diagnosis and a median follow-up time of 58 months (interquartile range, 26 to 107 months). Eleven medical centers from six countries participated in this study. Results There was a linear association between patient age and mortality in patients with BRAF V600E mutation, but not in patients with wild-type BRAF, in whom the mortality rate remained low and flat with increasing age. Kaplan-Meier survival curves rapidly declined with increasing age in patients with BRAF V600E mutation but did not decline in patients with wild-type BRAF, even beyond age 75 years. The association between mortality and age in patients with BRAF V600E was independent of clinicopathologic risk factors. Similar results were observed when only patients with the conventional variant of PTC were analyzed. Conclusion The long-observed age-associated mortality risk in PTC is dependent on BRAF status; age is a strong, continuous, and independent mortality risk factor in patients with BRAF V600E mutation but not in patients with wild-type BRAF. These results question the conventional general use of patient age as a high-risk factor in PTC and call for differentiation between patients with BRAF V600E and wild-type BRAF when applying age to risk stratification and management of PTC.
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http://dx.doi.org/10.1200/JCO.2017.74.5497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807010PMC
February 2018

BRAF V600E Mutation-Assisted Risk Stratification of Solitary Intrathyroidal Papillary Thyroid Cancer for Precision Treatment.

J Natl Cancer Inst 2018 04;110(4):362-370

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD.

Background: Precise risk stratification-based treatment of solitary intrathyroidal papillary thyroid cancer (SI-PTC) that is larger than 1.0 cm and 4.0 cm or less is undefined.

Methods: A genetic-clinical risk study was performed on BRAF V600E in 955 patients (768 women and 187 men) with SI-PTC, with median age of 46 years and median clinical follow-up time of 64 months at 11 medical centers in six countries. The chi-square test or, for analyses with small numbers, Fisher's exact test was performed to compare recurrence rates. Recurrence-free probability was estimated by Kaplan-Meier (KM) analysis, and the independent effect of BRAF mutation on the recurrence was analyzed by Cox regression and Cox proportional hazard analyses. All statistical tests were two-sided.

Results: Recurrence of SI-PTC larger than 1.0 cm and 4.0 cm or less was 9.5% (21/221) vs 3.4% (11/319) in BRAF mutation vs wild-type BRAF patients, with a hazard ratio (HR) of 3.03 (95% confidence interval [CI] = 1.46 to 6.30) and a patient age- and sex-adjusted hazard ratio of 3.10 (95% CI = 1.49 to 6.45, P = .002). Recurrence rates of SI-PTC larger than 2.0 cm and 4.0 cm or less were 16.5% (13/79) vs 3.6% (5/139) in mutation vs wild-type patients (HR = 5.44, 95% CI = 1.93 to 15.34; and adjusted HR = 5.58, 95% CI = 1.96 to 15.85, P = .001). Recurrence rates of SI-PTC larger than 3.0 cm and 4 cm or less were 30.0% (6/20) vs 1.9% (1/54) in mutation vs wild-type patients (HR = 18.40, 95% CI = 2.21 to 152.98; and adjusted HR = 14.73, 95% CI = 1.74 to 124.80, P = .01). Recurrences of mutation-positive SI-PTC were comparable with those of counterpart invasive solitary PTC, around 20% to 30%, in tumors larger than 2.0 cm to 3.0 cm. BRAF mutation was associated with a statistically significant decrease in recurrence-free patient survival on KM analysis, particularly in SI-PTC larger than 2.0 cm and 4.0 cm or less. Similar results were obtained in conventional SI-PTC. The negative predictive values of BRAF mutation for recurrence were 97.8% (95% CI = 96.3% to 98.8%) for general SI-PTC and 98.2% (95% CI = 96.3% to 99.3%) for conventional SI-PTC.

Conclusions: BRAF V600E identifies a subgroup of SI-PTC larger than 1.0 cm and 4.0 cm or less, particularly tumors larger than 2.0 cm and 4.0 cm or less, that has high risk for recurrence comparable with that of invasive solitary PTC, making more aggressive treatment reasonable.
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http://dx.doi.org/10.1093/jnci/djx227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658860PMC
April 2018

When Somatic Mutations Are Associated With a Higher Aggressive Behavior-Reply.

JAMA Oncol 2017 10;3(10):1428-1429

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes, & Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.

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http://dx.doi.org/10.1001/jamaoncol.2017.1513DOI Listing
October 2017

The Prognostic Value of Tumor Multifocality in Clinical Outcomes of Papillary Thyroid Cancer.

J Clin Endocrinol Metab 2017 09;102(9):3241-3250

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes & Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287.

Context: Multifocality is often treated as a risk factor for papillary thyroid cancer (PTC), prompting aggressive treatments, but its prognostic value remains unestablished.

Objective: To investigate the role of tumor multifocality in clinical outcomes of PTC.

Methods: Multicenter study of the relationship between multifocality and clinical outcomes of PTC in 2638 patients (623 men and 2015 women) with median [interquartile range (IQR)] age of 46 (35 to 58) years and median (IQR) follow-up time of 58 (26 to 107) months at 11 medical centers in six countries. Surveillance, Epidemiology and End Results (SEER) data were used for validation.

Results: Disease recurrence in multifocal and unifocal PTC was 198 of 1000 (19.8%) and 221 of 1624 (13.6%) (P < 0.001), with a hazard ratio of 1.55 [95% confidence interval (CI), 1.28 to 1.88], which became insignificant at 1.13 (95% CI, 0.93 to 1.37) on multivariate adjustment. Similar results were obtained in PTC variants: conventional PTC, follicular-variant PTC, tall-cell PTC, and papillary thyroid microcarcinoma. There was no association between multifocality and mortality in any of these PTC settings, whereas there was a strong association between classic risk factors and cancer recurrence or mortality, which remained significant after multivariate adjustment. In 1423 patients with intrathyroidal PTC, disease recurrence was 20 of 455 (4.4%) and 41 of 967 (4.2%) (P = 0.892) and mortality was 0 of 455 (0.0%) and 3 of 967 (0.3%) (P = 0.556) in multifocal and unifocal PTC, respectively. The results were reproduced in 89,680 patients with PTC in the SEER database.

Conclusions: Tumor multifocality has no independent risk prognostic value in clinical outcomes of PTC; its indiscriminate use as an independent risk factor, prompting overtreatments of patients, should be avoided.
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http://dx.doi.org/10.1210/jc.2017-00277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587077PMC
September 2017

A six-genotype genetic prognostic model for papillary thyroid cancer.

Endocr Relat Cancer 2017 01 14;24(1):41-52. Epub 2016 Nov 14.

Laboratory for Cellular and Molecular Thyroid ResearchDivision of Endocrinology, Diabetes & Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

A unique prognostic role of the genetic duet of BRAF V600E and TERT promoter mutations in papillary thyroid cancer (PTC) has been recently established, but the role of RAS mutation in this genetic interplay remains to be established. Using The Cancer Genome Atlas (TCGA) data of patients with PTC from 19 medical centers, we investigated the interactions among the three mutations in clinical outcomes of PTC. We found that BRAF and RAS mutations were mutually exclusive, but both were associated with TERT promoter mutations, with the genetic duet of BRAF/RAS and TERT mutations occurring in 34/388 (8.76%) patients. BRAF/RAS or TERT mutation had no or minimal effect alone, whereas coexisting BRAF/RAS and TERT mutations had a robust synergistic effect on poor clinicopathologic outcomes of PTC, including disease recurrence and patient mortality. For example, PTC recurrence rate was 52% with coexisting BRAF V600E/RAS and TERT promoter mutations vs 6.9% with no mutation, corresponding to a HR of 8.17 (95% CI 3.09-21.58), which remained significant at 14.71 (95% CI 2.79-77.61) after adjustment for clinicopathologic factors and institution. BRAF/RAS mutation or TERT mutation alone minimally affected Kaplan-Meier patient survival curves, whereas the genetic duet was associated with a sharp curve decline. Thus, by confirming and expanding previous findings in single-institution studies, this multicenter data analysis establishes a six-genotype genetic prognostic model for poor outcomes of PTC with a risk order of genetic duet of BRAF V600E/RAS mutation and TERT mutation >BRAF V600E = TERT mutation alone >RAS mutation alone = wild-type genes.
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http://dx.doi.org/10.1530/ERC-16-0402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132178PMC
January 2017

Epigenetically upregulated WIPF1 plays a major role in BRAF V600E-promoted papillary thyroid cancer aggressiveness.

Oncotarget 2017 Jan;8(1):900-914

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes & Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

How the BRAF V600E mutation promotes the pathogenesis and aggressiveness of papillary thyroid cancer (PTC) is not completely understood. Here we explored a novel mechanism involving WASP interacting protein family member 1 (WIPF1). In PTC tumors, compared with the wild-type BRAF, BRAF V600E was associated with over-expression and hypomethylation of the WIPF1 gene. In thyroid cancer cell lines with wild-type BRAF, WIPF1 expression was robustly upregulated upon introduced expression of BRAF V600E (P=0.03) whereas the opposite was seen upon BRAF knockdown or treatment with BRAF V600E or MEK inhibitors in cells harboring BRAF V600E. Methylation of a functionally critical region of the WIPF1 promoter was decreased by expressing BRAF V600E in cells harboring the wild-type BRAF and increased by BRAF knockdown or treatment with BRAF V600E or MEK inhibitors in cells harboring BRAF V600E mutation. Under-expression and hypermethylation of WIPF1 induced by stable BRAF knockdown was reversed by DNA demethylating agent 5'-azadeoxycytidine. Knockdown of WIPF1 robustly inhibited anchorage-independent colony formation, migration, and invasion of thyroid cancer cells and suppressed xenograft thyroid cancer tumor growth and vascular invasion, mimicking the effects of BRAF knockdown. In human PTC tumors, WIPF1 expression was associated with extrathyroidal invasion (P=0.01) and lymph node metastasis (P=2.64E-05). In summary, BRAF V600E-activated MAP kinase pathway causes hypomethylation and overexpression of WIPF1; WIPF1 then functions like an oncoprotein to robustly promote aggressive cellular and tumor behaviors of PTC. This represents a novel mechanism in BRAF V600E-promoted PTC aggressiveness and identifies WIPF1 as a novel therapeutic target for thyroid cancer.
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http://dx.doi.org/10.18632/oncotarget.13400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352205PMC
January 2017

Mortality Risk Stratification by Combining BRAF V600E and TERT Promoter Mutations in Papillary Thyroid Cancer: Genetic Duet of BRAF and TERT Promoter Mutations in Thyroid Cancer Mortality.

JAMA Oncol 2017 Feb;3(2):202-208

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes, & Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Importance: BRAF V600E and TERT promoter mutations can coexist in papillary thyroid cancer (PTC). This genetic duet was indicated to be involved in the aggressiveness of PTC, but its prognostic value in PTC-related mortality remains to be specifically established.

Objective: To establish the prognostic power of this genetic duet in PTC-specific mortality.

Design, Setting, And Participants: This genetic-clinical correlation study examined BRAF V600E and TERT promoter mutations (chr5:1,295,228C>T and chr5:1,295,250C>T) and PTC-specific mortality in 1051 patients (764 women and 287 men) with a median (interquartile range [IQR]) age of 46 (36-57) years, with a median (IQR) follow-up time of 89 (48-142) months (7.4 years).

Main Outcomes And Measures: BRAF V600E and TERT promoter mutation patterns and associated patient deaths caused by PTC.

Results: Papillary thyroid cancer-specific mortality occurred in 4 of 629 patients (0.6%) with neither mutation; 7 of 292 (2.4%) with BRAF V600E alone; 4 of 64 (6.3%) with TERT promoter mutation alone; and 15 of 66 (22.7%) with the genetic duet; and deaths per 1000-person years in patients harboring neither mutation, BRAF V600E alone, TERT mutation alone, or both mutations were 0.80 (95% CI, 0.30-2.13), 3.08 (95% CI, 1.47-6.46), 6.62 (95% CI, 2.48-17.64), and 29.86 (95% CI, 18.00-49.52), respectively. Compared with patients harboring neither mutation, HRs (95% CIs) for PTC-specific mortality were 3.08 (0.87-10.84) for BRAF V600E alone; 8.18 (2.04-32.75) with TERT mutation alone; and 37.77 (12.50-114.09) with both mutations. Papillary thyroid cancer-specific mortality for cases with both mutations remained significant (HR, 9.34; 95% CI, 2.53-34.48) after adjustment for clinicopathological factors, and the genetic duet showed a strong incremental and synergistic impact over either mutation alone. Kaplan-Meier analyses revealed a flat PTC-specific patient survival curve with neither mutation, a modest decline in the curve with either mutation alone, and a sharp decline in the curve with coexisting mutations. Even more robust mortality associations of the genetic duet were seen when only conventional-variant PTC (CPTC) was analyzed (HR, 54.46; 95% CI, 12.26-241.82), which remained strongly significant (HR, 18.56; 95% CI, 2.97-116.18) after adjustment for clinicopathological factors.

Conclusions And Relevance: These results demonstrate a simple 4-genotype classification of PTC, particularly CPTC, with a disease-specific mortality risk order of the genetic duet>BRAF V600E alone = TERT promoter mutation alone > wild-type for both genes, representing a powerful molecular prognostic system that can help pinpoint patients with the highest mortality risk.
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http://dx.doi.org/10.1001/jamaoncol.2016.3288DOI Listing
February 2017

Robust Thyroid Gene Expression and Radioiodine Uptake Induced by Simultaneous Suppression of BRAF V600E and Histone Deacetylase in Thyroid Cancer Cells.

J Clin Endocrinol Metab 2016 Mar 11;101(3):962-71. Epub 2016 Jan 11.

Laboratory for Cellular and Molecular Thyroid Research (W.C., R.L., G.Z., M.X.), Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; and Department of Nuclear Medicine (H.W.), Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.

Context: Use of BRAF V600E inhibitors to restore thyroid iodide-handling gene expression and radioactive iodine (RAI) avidity is an attractive therapeutic strategy for RAI-refractory thyroid cancer, but recent initial clinical responses were modest. Given histone deacetylation at the sodium/iodide symporter promoter by histone deacetylase (HDAC) as a mechanism, simultaneously targeting BRAF V600E and HDAC could be a more effective strategy.

Objectives: The objective of the study was to test whether suppressing both BRAF V600E and HDAC could more effectively induce thyroid gene expression and RAI uptake in thyroid cancer cells.

Research Design: We tested the BRAF V600E inhibitor PLX4032 (vemurafenib) and the HDAC inhibitor SAHA (vorinostat), two major anticancer drugs currently approved for clinical use, in inducing thyroid gene expression and RAI uptake in thyroid cancer cells.

Results: PLX4032 alone induced a modest expression of thyroid genes and RAI uptake preferentially in thyroid cancer cells harboring BRAF V600E. SAHA showed an effect in a genetic-independent manner in all the cells. A robust synergistic effect on thyroid gene expression and RAI uptake was observed in BRAF V600E-positive thyroid cancer cells when the two inhibitors were simultaneously used. This was dramatically enhanced further by TSH; triple combination of PLX4032, SAHA, and TSH showed the most robust effect on thyroid gene expression and RAI uptake in cells harboring BRAF V600E. Abundant sodium/iodide symporter protein expression in thyroid cancer cells under these conditions was confirmed by immunofluorescent microscopy.

Conclusions: Simultaneously suppressing BRAF V600E and HDAC, particularly when cotreated with TSH, induced a far more robust expression of thyroid genes and RAI uptake in thyroid cancer cells than suppressing BRAF V600E alone. Triple combination of PLX4032, SAHA, and TSH is a specific robust regimen to restore RAI avidity in RAI-refractory BRAF V600E-positive thyroid cancer, which warrants clinical trials to confirm.
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http://dx.doi.org/10.1210/jc.2015-3433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803151PMC
March 2016

TERT promoter mutations in thyroid cancer.

Endocr Relat Cancer 2016 Mar 5;23(3):R143-55. Epub 2016 Jan 5.

Laboratory for Cellular and Molecular Thyroid ResearchDivision of Endocrinology, Diabetes and Metabolism, The Johns Hopkins University School of Medicine, 1830 East Monument Street, Suite 333, Baltimore, Maryland 21287, USA

The 2013 discovery of Telomerase reverse transcriptase (TERT) promoter mutations chr5, 1,295,228 C>T (C228T) and 1,295,250 C>T (C250T) in thyroid cancer represents an important event in the thyroid cancer field and much progress has occurred since then. This article provides a comprehensive review of this exciting new thyroid cancer field. The oncogenic role of TERT promoter mutations involves their creation of consensus binding sites for E-twenty-six transcriptional factors. TERT C228T is far more common than TERT C250T and their collective prevalence is, on average, 0, 11.3, 17.1, 43.2 and 40.1% in benign thyroid tumors, papillary thyroid cancer (PTC), follicular thyroid cancer, poorly differentiated thyroid cancer and anaplastic thyroid cancer, respectively, displaying an association with aggressive types of thyroid cancer. TERT promoter mutations are associated with aggressive thyroid tumor characteristics, tumor recurrence and patient mortality as well as BRAF V600E mutation. Coexisting BRAF V600E and TERT promoter mutations have a robust synergistic impact on the aggressiveness of PTC, including a sharply increased tumor recurrence and patient mortality, while either mutation alone has a modest impact. Thus, TERT with promoter mutations represents a prominent new oncogene in thyroid cancer and the mutations are promising new diagnostic and prognostic genetic markers for thyroid cancer, which, in combination with BRAF V600E mutation or other genetic markers (e.g. RAS mutations), are proving to be clinically useful for the management of thyroid cancer. Future studies will specifically define such clinical utilities, elucidate the biological mechanisms and explore the potential as therapeutic targets of TERT promoter mutations in thyroid cancer.
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http://dx.doi.org/10.1530/ERC-15-0533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750651PMC
March 2016

Differential Clinicopathological Risk and Prognosis of Major Papillary Thyroid Cancer Variants.

J Clin Endocrinol Metab 2016 Jan 3;101(1):264-74. Epub 2015 Nov 3.

Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology, Diabetes & Metabolism, Department of Medicine (X.Shi., R.L., X.Shen., D.T., M.X.), Johns Hopkins University School of Medicine, Baltimore, MD 21287; Department of Surgery, Division of Pathology (F.B., R.G.), 56126 Pisa, Italy; The Endocrine Institute and The Liaoning Provincial Key Laboratory of Endocrine Diseases, Department of Endocrinology and Metabolism (H.G., Z.S., W.T.), The First Hospital of China Medical University, Shenyang, Liaoning Province 110001, China; Endocrine Surgery (T.J.M.), University Medical Center, Johannes Gutenberg University Mainz, 55101 Mainz, Germany; Human Cancer Genomic Research (K.A.K.), Research Centre, King Faisal Specialist Hospital and Research Centre, Riyadh 12713, Kingdom of Saudi Arabia; Fondazione Instituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca' Granda Policlinico, Milan, and Department of Pathophysiology and Transplantation (L.E., C.C.), University of Milan, 20122 Milan Italy; Endocrine Oncology Branch, Center for Cancer Research (E.K.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology (A.C., B.J.), 44-101 Gliwice, Poland; Department of Molecular Endocrinology (B.B., V.S.), Institute of Endocrinology, Prague 11694, Czech Republic; Institute of Molecular Pathology and Immunology (Manuel Sobrinho-Simões, M.S.S.; Paula Soares, P.So.), University of Porto (Ipatimup) and Medical Faculty of the University of Porto, 4200-319 Porto, Portugal; College of Medicine (Y.K.S., T.Y.K.), University of Ulsan College of Medicine, Seoul, South Korea; Department of Pathology (S.C.; S.L.A.), University Health Network, Toronto, ON M5G 2C4 Canada; Endocrine Unit, Department of Clinical and Experimental Medicine (D.V., R.E.), World Health Organization, Collaborating Center for the Study and Treatment of Thyroid Diseases and Other Endocrine and Meta

Context: Individualized management, incorporating papillary thyroid cancer (PTC) variant-specific risk, is conceivably a useful treatment strategy for PTC, which awaits comprehensive data demonstrating differential risks of PTC variants to support.

Objective: This study sought to establish the differential clinicopathological risk of major PTC variants: conventional PTC (CPTC), follicular-variant PTC (FVPTC), and tall-cell PTC (TCPTC).

Methods: This was a retrospective study of clinicopathological outcomes of 6282 PTC patients (4799 females and 1483 males) from 26 centers and The Cancer Genome Atlas in 14 countries with a median age of 44 years (interquartile range, 33-56 y) and median follow-up time of 37 months (interquartile range, 15-82 mo).

Results: The cohort consisted of 4702 (74.8%) patients with CPTC, 1126 (17.9%) with FVPTC, and 239 (3.8%) with TCPTC. The prevalence of high-risk parameters was significantly different among the three variants, including extrathyroidal invasion, lymph node metastasis, stages III/IV, disease recurrence, mortality, and the use (need) of radioiodine treatment (all P < .001), being highest in TCPTC, lowest in FVPTC, and intermediate in CPTC, following an order of TCPTC > CPTC ≫ FVPTC. Recurrence and mortality in TCPTC, CPTC, and FVPTC were 27.3 and 6.7%, 16.1 and 2.5%, and 9.1 and 0.6%, corresponding to events per 1000 person-years (95% confidence interval [CI]) of 92.47 (64.66-132.26) and 24.61 (12.31-49.21), 34.46 (30.71-38.66), and 5.87 (4.37-7.88), and 24.73 (18.34-33.35) and 1.68 (0.54-5.21), respectively. Mortality hazard ratios of CPTC and TCPTC over FVPTC were 3.44 (95% CI, 1.07-11.11) and 14.96 (95% CI, 3.93-56.89), respectively. Kaplan-Meier survival analyses showed the best prognosis in FVPTC, worst in TCPTC, and intermediate in CPTC in disease recurrence-free probability and disease-specific patient survival. This was particularly the case in patients at least 45 years old.

Conclusion: This large multicenter study demonstrates differential prognostic risks of the three major PTC variants and establishes a unique risk order of TCPTC > CPTC ≫ FVPTC, providing important clinical implications for specific variant-based management of PTC.
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http://dx.doi.org/10.1210/jc.2015-2917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701842PMC
January 2016

Association of TERT promoter mutation 1,295,228 C>T with BRAF V600E mutation, older patient age, and distant metastasis in anaplastic thyroid cancer.

J Clin Endocrinol Metab 2015 Apr 13;100(4):E632-7. Epub 2015 Jan 13.

Laboratory for Cellular and Molecular Thyroid Research (X.S., R.L., G.Z., X.L., M.X.), Division of Endocrinology, Diabetes & Metabolism, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; Department of Endocrinology & Metabolism (S.Q.), Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, China; Department of Pathology (J.B.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; Jilin Provincial Key Laboratory of Surgical Translational Medicine (H.S.), Department of Thyroid and Parathyroid Surgery, China-Japan Union Hospital, Jilin University, Changchun, Jilin Province 130033, China; Department of Pathology (E.W.), The First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province 110001, China; Department of Medical Image (Y.L.), Liaoning Cancer Hospital & Institute, Shenyang, Liaoning Province 110001, China; Department of Pathology (X.Y.), Shengjing Hospital of China Medical University, Shenyang, Liaoning Province 110004, China; Department of Endocrinology (J.Z.), Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong Province 250021, China; Department of Endocrinology (J.D.), The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province 116011, China; Department of Pathology (A.K.E.-N.), University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; and The Endocrine Institute and The Liaoning Provincial Key Laboratory of Endocrine Diseases (X.S., Z.S., W.T.), Department of Endocrinology and Metabolism, The First Hospital of China Medical University, Shenyang, Liaoning Province 110001, China.

Context: The aggressive role of TERT promoter mutations has been well established in differentiated thyroid cancer but has not been established in anaplastic thyroid cancer (ATC).

Research Design: We tested the mutation status by sequencing genomic tumor DNA and examined its relationship with clinicopathological characteristics of ATC.

Results: Among 106 American and Chinese ATC samples, TERT 1,295,228 C>T (termed TERT C228T) mutation was found in 37 (34.9%) cases, TERT promoter mutation 1,295,250 C>T was found in four cases (3.8%), and the two mutations were mutually exclusive and collectively found in 41 cases (38.7%). TERT C228T occurred in 28 of 90 (31.1%) wild-type BRAF cases vs nine of 16 (56.3%) BRAF V600E cases, with an odds ratio of 2.85 (95% confidence interval, 0.96-8.42; P = .05). Patient age was 67.6 ± 13.6 vs 61.6 ± 11.4 years in the TERT C228T vs wild-type TERT patients (P = .02), demonstrating an association between TERT C228T and older patient age. This association was also seen within the American cohort. In this cohort, which had more available clinicopathological data, TERT C228T was associated with distant metastasis of the tumor; specifically, distant metastasis occurred in 15 of 18 (83.3%) TERT C228T patients vs eight of 26 (30.8%) wild-type TERT patients, with an odds ratio of 11.25 (95% confidence interval, 2.53-50.08; P = .001). No association was found with patient sex, tumor size, lymph node metastasis, and extrathyroidal invasion of ATC.

Conclusions: This is the largest study on the aggressive role of TERT promoter mutations in ATC, demonstrating an association of TERT C228T with BRAF V600E, older patient age, and tumor distant metastasis in ATC.
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http://dx.doi.org/10.1210/jc.2014-3606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399285PMC
April 2015

Diagnostic and prognostic TERT promoter mutations in thyroid fine-needle aspiration biopsy.

Endocr Relat Cancer 2014 Oct 13;21(5):825-30. Epub 2014 Aug 13.

Laboratory for Cellular and Molecular Thyroid ResearchDivision of Endocrinology, Diabetes and Metabolism, The Johns Hopkins University School of Medicine, 1830 East Monument Street, Suite 333, Baltimore, Maryland 21287, USA

Two promoter mutations, chr5:1 295 228C>T and chr5:1 295 250C>T, in the gene for telomerase reverse transcriptase (TERT) have been recently identified in thyroid cancers and shown to be important in thyroid tumor pathogenesis. The diagnostic and prognostic potentials of testing for these mutations on thyroid fine-needle aspiration biopsy (FNAB) have not been investigated. Herein, we examined the two TERT promoter mutations along with the BRAF V600E mutation by direct DNA sequencing on 308 FNAB specimens preoperatively obtained from thyroid nodules with postoperatively confirmed pathological diagnoses. We found TERT promoter mutations in 0.0% (0/179) of benign thyroid nodules and 7.0% (9/129) of thyroid nodules of differentiated thyroid cancer, representing a 100% diagnostic specificity and 7.0% sensitivity, with the latter rising to 38.0% (49/129) when combined with BRAF V600E testing. Several TERT-promoter-mutation-positive thyroid nodules were cytologically indeterminate on FNAB. Approximately 80% of the TERT promoter mutation-positive thyroid nodules were thyroid cancers with aggressive clinicopathological behaviors, such as extrathyroidal invasion, lymph node metastases, distant metastases, disease recurrence or patient death. Thus, a positive TERT promoter mutation test not only definitively diagnoses a thyroid nodule as cancerous but also preoperatively identifies a cancer with aggressive potential. This is the first study, to our knowledge, of TERT promoter mutations on thyroid FNAB, demonstrating the value of this novel molecular testing in the diagnosis of thyroid nodules and preoperative risk stratification of thyroid cancer. Thus, testing of TERT promoter mutations on FNAB will enhance and improve the current molecular-based approaches to the management of thyroid nodules and thyroid cancer.
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http://dx.doi.org/10.1530/ERC-14-0359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175020PMC
October 2014

BRAF V600E and TERT promoter mutations cooperatively identify the most aggressive papillary thyroid cancer with highest recurrence.

J Clin Oncol 2014 Sep 14;32(25):2718-26. Epub 2014 Jul 14.

All authors: Johns Hopkins University School of Medicine, Baltimore, MD.

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.
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http://dx.doi.org/10.1200/JCO.2014.55.5094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145183PMC
September 2014

TERT promoter mutations and their association with BRAF V600E mutation and aggressive clinicopathological characteristics of thyroid cancer.

J Clin Endocrinol Metab 2014 Jun 11;99(6):E1130-6. Epub 2014 Mar 11.

Laboratory for Cellular and Molecular Thyroid Research (X.L., R.L., X.S., G.Z., A.K.M., M.X.), Division of Endocrinology, Diabetes & Metabolism, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; Department of Endocrinology & Metabolism (S.Q., C.S.), Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, China; Department of Endocrinology & Metabolism and Institute of Endocrinology (H.G., Z.S., W.T.), The First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province 110001, China; Department of Pathology (H.Y.), Changzheng Hospital, The Second Military Medical University, Shanghai 200003, China; Department of Endocrinology & Metabolism (Y.W.), The Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong Province 266003, China; and Jilin Provincial Key Laboratory of Surgical Translational Medicine (H.S.), Department of Thyroid and Parathyroid Surgery, China-Japan Union Hospital, Jilin University, Changchun, Jilin Province 130033, China.

Context: Promoter mutations chr5:1,295,228C>T and chr5:1,295,250C>T (termed C228T and C250T, respectively) in the gene for telomerase reverse transcriptase (TERT) have been reported in various cancers and need to be further investigated in thyroid cancer.

Objective: The aim of the study was to explore TERT promoter mutations in various thyroid tumors and examine their relationship with BRAF V600E mutation, iodine intake, and clinicopathological behaviors of thyroid cancer.

Design: TERT promoter and BRAF mutations were identified by sequencing genomic DNA of primary thyroid tumors from normal- and high-iodine regions in China, and clinicopathological correlation was analyzed.

Results: The C228T mutation was found in 9.6% (39 of 408) of papillary thyroid cancer (PTC), C250T was found in 1.7% (7 of 408) of PTC, and they were collectively found in 11.3% (46 of 408) of PTC. C228T was found in 31.8% (7 of 22) and C250T in 4.6% (1 of 22) of follicular thyroid cancer (FTC), and they were collectively found in 36.4% (8 of 22) of FTC. No TERT mutation was found in 44 benign thyroid tumors. The two mutations occurred in 3.8% (6 of 158) of BRAF mutation-negative PTC vs 16.0% (40 of 250) of BRAF mutation-positive PTC (P = 5.87 × 10(-4)), demonstrating their association. Unlike BRAF mutation, TERT promoter mutations were not associated with high iodine intake, but they were associated with older patient age, larger tumor size, extrathyroidal invasion, and advanced stages III/IV of PTC. Coexisting TERT and BRAF mutations were even more commonly and more significantly associated with clinicopathological aggressiveness.

Conclusions: In this large cohort, we found TERT promoter mutations to be common, particularly in FTC and BRAF mutation-positive PTC, and associated with aggressive clinicopathological characteristics.
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http://dx.doi.org/10.1210/jc.2013-4048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037723PMC
June 2014

[Meta-analysis of association between E-cadherin promoter methylation and lung cancer risk].

Zhongguo Fei Ai Za Zhi 2013 Jul;16(7):353-8

Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou Key Laboratory 
for Molecular Cancer Genetic, Suzhou 215123, China.

Background And Objective: As a member of the cadherin superfamily, E-cadherin plays a critical role in the maintenance of cell polarity and integrity. And E-cadherin was reported to be down-regulated and closely related to tumor initiation, invasion and metastasis. There have been a number of studies investigating the methylation of E-cadherin promoter in relation to various cancers. However, the association between E-cadherin promoter methylation and lung cancer risk is controversial. The aim of the present study is to evaluate the association of E-cadherin promoter methylation with risk of lung cancer by conducting a systematic meta-analysis.

Methods: Relevant studies were identified by searches of PubMed/MedLine and EMBASE databases before March 2013. Combined odds ratio (OR) and 95% confidence interval (CI) were used to assess the strength of the association between E-cadherin promoter methylation and lung cancer risk.

Results: A total of 1,288 samples from 13 independent studies were included in the meta-analysis. Overall, a significant association was observed between E-cadherin promoter methylation and lung cancer risk (OR=4.04, 95%CI: 2.00-8.13, P<0.001). Subgroup analyses by ethnic revealed that lung cancer risk was increased for individuals carrying the methylated E-cadherin in Asian and Caucasian populations (OR=3.28, 95%CI: 1.20-8.92; OR=5.72, 95%CI: 2.40-13.62).

Conclusions: The present meta-analysis indicates that the methylation of E-cadherin promoter is associated with risk of lung cancer.
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http://dx.doi.org/10.3779/j.issn.1009-3419.2013.07.04DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6000657PMC
July 2013

[Association of mutation and methylation in the promoter region of TIF1γ with non-small cell lung cancer].

Zhongguo Fei Ai Za Zhi 2013 May;16(5):227-32

Soochow University Laboratory of Cancer Molecular Genetics, Medical college of Soochow University, Suzhou Key Laboratory for Molecular Cancer Genetic, Suzhou 215123, China.

Background And Objective: TIF1γ (transcription intermediary factor 1 gamma), which belongs to transcription intermediary factor 1 family, is an inhibitor of the TGF-β/Smad signaling pathway and could inhibit the signal transduction mediated by TGF-β. The deficiency of TIF1γ expression in a variety of tumor cells suggests that TIF1γ may play as a tumor suppressor gene in cancer development. The aim of this study is to confirm the relationship between TIF1γ and non-small cell lung cancer (NSCLC) through exploring the expression of TIF1γ in NSCLC cells and tissues, and investigate the regulation mechanism of TIF1γ expression in NSCLC cells.

Methods: Thirteen NSCLC and the paired corresponding para-cancerous lung tissue samples and three cell lines (a normal bronchial epithelial cell line HBE and two NSCLC cell lines A549 and 95C) were selected. The quantitative real-time PCR and Western blotting were used to determine the expression of TIF1γ, and ImageJ was used to evaluate the relative expression of TIF1γ. Direct sequencing was performed to detect mutations within the promoter region of the TIF1γ gene and then Bisulfite-sequencing PCR (BSP) and cloning sequencing were carried out to test the methylation status of the promoter region of TIF1γ gene in the selected cell lines.

Results: Both mRNA and protein expression of TIF1γ were found significantly decreased in A549 and 95C compared with those in HBE (P<0.05). And in 9 pairs (69.2%) of tissues among the 13 pairs, the mRNA expression of TIF1γ gene was lower in the cancer tissues than that in the paired paracancerous lung tissues (P<0.05). No abnormal mutation was found in the -287 to -5 region of the promoter of TIF1γ gene in the three cell lines. Moreover, five CpG sites (including -214 bp,-128 bp,-124 bp, -65 bp and -55 bp) were detected in the promoter of TIF1γ gene by using BSP, and the methylation profiles in these CpG sites showed similar pattern between NSCLC cells and HBE cells.

Conclusions: TIF1γ may play a tumor suppressor role in the progression of NSCLC. No mutation was found in the -287- -5 region of the promoter of TIF1γ gene in a normal bronchial epithelial cell line and two NSCLC cell lines. But there are five CpG sites which can be methylated located in this region.
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http://dx.doi.org/10.3779/j.issn.1009-3419.2013.05.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6000610PMC
May 2013

[Meta analysis of association between Ser326Cys polymorphism of hOGG1 gene and risk of lung cancer].

Zhongguo Fei Ai Za Zhi 2011 Mar;14(3):205-10

Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou, China.

Background And Objective: Human 8-hydroxyguanine glycosylase (hOGG1) is one of the DNA repair genes, which can repair damaged DNA by specifically excising 8-dihydro-8-oxoguanine (8-OH-G). A considerable number of studies investigating hOGG1 Ser326Cys polymorphism were in relation to various cancers. However, the association of hOGG1 Ser326Cys polymorphism with risk of lung cancer is inconsistency. The aim of this study is to assess the association of hOGG1 Ser326Cys polymorphism with risk of lung cancer by conducting a meta-analysis.

Methods: To identify all studies that are qualified for meta-analysis, we conducted a computerized literature search of MEDLINE database (before November, 2010). Two investigators independently extracted the data and reached a consensus on all items.

Results: According to our search limits, twenty-two case-control studies, including 8,575 lung cancer cases and 9,484 controls, were selected for meta-analysis. Significant heterogeneity was found among those twenty-two case-control studies, when excluding two studies that were not accorded with Hard-Weinberg Equilibrium, the remains performed well homogeneity. Compared with Ser326 genotype, Cys326 genotype can significantly increase risk of lung cancer (OR=1.24, 95%CI: 1.10-1.39, P < 0.001), especially in Asian ethnic population and hospital population (OR=1.28, 95%CI: 1.11-1.49; OR=1.26, 95%CI: 1.09-1.46).

Conclusions: The present study indicates that the hOGG1 Ser326Cys polymorphism is associated with risk of lung cancer, Cys326 genotype can significantly increase risk of lung cancer.
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http://dx.doi.org/10.3779/j.issn.1009-3419.2011.03.05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999670PMC
March 2011

[Mutational analysis of hOGG1 gene promoter in patients with non-small cell lung cancer].

Zhongguo Fei Ai Za Zhi 2011 Mar;14(3):199-204

Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou, China.

Background And Objective: 8-hydroxygumine DNA glycosylase 1 (OGG1) is a DNA repair enzyme, which can repair damaged DNA by excising 8-dihydro-8-oxoguanine (8-OH-G). Polymorphisms in human OGG1 gene (hOGG1) may alter glycosylase activity, thereby affects its repair to the damaged DNA, resulting in contribution to carcinogenesis. However, an association of genetic variants of hOGG1 promoter with non-small cell lung cancer (NSCLC) remains unclear. The present study aims to explore whether there are mutations in the promoter region of hOGG1 and the association of the potential genetic variants with NSCLC.

Methods: Forty lung cancer patients were enrolled from January, 2003 to December, 2005 in the first affiliated hospital of Soochow University. PCR-SSCP followed by direct sequencing were performed to detect mutations within the promoter region of the hOGG1 gene in NSCLC and corresponding paracancerous lung tissues.

Results: No abnormal mutation was found in the promoter region of the hOGG1 gene in 40 patients with NSCLC. However, a SNP rs159153 in hOGG1 was significantly associated with higher TNM stage (P=0.008). Moreover, lower frequency of lymph node metastasis was observed in smoker patients with NSCLC (P=0.034).

Conclusions: The SNP rs159153 in the promoter region of the hOGG1 gene and smoking history may effectively forebode the aggressiveness and metastatic potential of NSCLC.
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http://dx.doi.org/10.3779/j.issn.1009-3419.2011.03.04DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999654PMC
March 2011

The role of interleukin-15 polymorphisms in adult acute lymphoblastic leukemia.

PLoS One 2010 Oct 25;5(10):e13626. Epub 2010 Oct 25.

Laboratory of Cellular and Molecular Tumor Immunology, Cyrus Tang Hematology Center, Department of Hematology, Jiangsu Institute of Hematology, The First Affiliated Hospital, Soochow University, Suzhou, China.

Background: Interleukin-15 (IL-15) plays important roles in the immune system and in the development of hematopoietic cells. Previous studies revealed that five SNPs in IL-15, rs10519612, rs10519613, rs35964658, rs17007695 and rs17015014, were significantly associated with childhood Acute Lymphoblastic Leukemia (ALL) treatment response. In adult ALL, the expression of IL-15 was also correlated with the immunophenotypes of ALL. Therefore, we hypothesize that SNPs of IL-15 might also be associated with adult ALL.

Methods And Findings: We genotyped the above five SNPs of IL-15 gene by PCR-RFLP assays in adult ALL case-control studies. The current study included 121 adult ALL patients and 263 healthy controls. IL-15 genotypes and haplotypes were determined and the associations with the risk of ALL were analyzed by logistic regression. SNPs rs10519612 and rs17007695 were significantly associated with ALL (P = 0.013 and P = 0.001). We observed a 2-fold and 2.4-fold excess risk of developing ALL for the rs10519612 CC and rs17007695 TC genotype carriers compared with non-carriers, respectively. Haplotype analysis revealed that haplotypes ACAC, CAGT and CCAT were significantly associated with adult B-ALL, while haplotype CCAT conferred susceptibility to T-ALL.

Conclusion: These findings suggest that IL-15 gene polymorphisms are significantly associated with ALL in adult Chinese population.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013626PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963612PMC
October 2010

[Defective expression of TGFBR3 gene and its molecular mechanisms in non-small cell lung cancer cell lines].

Zhongguo Fei Ai Za Zhi 2010 May;13(5):451-7

Soochow University Laboratory of Cancer Molecular Genetics, Suzhou 215123, China.

Background And Objective: It has been reported that defective expression of TGFBR3 was found in non-small cell lung cancer (NSCLC). However, its molecular mechanisms remain unclear. The aim of this study is to investigate expression of TGFBR3 in NSCLC cell lines and normal human bronchial epithelial cell (HBEpiC), and to explore potential molecular mechanisms underlying inactivation of TGFBR3 gene.

Methods: Western blot was performed to determine the expression of TGFBR3 in HBEpiC and NSCLC cell lines. Automatic image analysis was carried out to estimate relative expression of TGFBR3 protein. We screened for mutation of the promoter region of TGFBR3 gene using DNA direct sequencing. Bisulfite-sodium modification sequencing was used to detect the methylation status of TGFBR3 promoter.

Results: TGFBR3 protein level was abnormally reduced in NSCLC cell lines as compared with HBEpiC. There was significant difference in TGFBR3 expression between the highly metastatic cell line 95D and non-metastatic cell lines, including LTEP-alpha-2, A549 and NCI-H460. No mutation and methylation was found in upstream sites -165 to -75 of the proximal promoter of TGFBR3 in HBEpiC and NSCLC cell lines. Hypermethylation was shown in upstream sites -314 to -199 of the distal promoter of TGFBR3 in HBEpiC and NSCLC cell lines.

Conclusion: Reduced expression of TGFBR3 was observed in NSCLC cell lines, especially in 95D, suggesting that TGFBR3 might play an important role in development and progression of NSCLC and correlate with NSCLC invasion and migration. The methylation event occurring at TGFBR3 promoter is not a major cause for reduction of TGFBR3 expression.
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http://dx.doi.org/10.3779/j.issn.1009-3419.2010.05.14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6000709PMC
May 2010

CpG island methylator phenotype involving chromosome 3p confers an increased risk of non-small cell lung cancer.

J Thorac Oncol 2010 Jun;5(6):790-7

Soochow University Laboratory of Cancer Molecular Genetics, School of Basic Medicine and Biological Sciences, Medical College of Soochow University, Suzhou, People's Republic of China.

Purpose: This study aims to explore the association of CpG island methylator phenotype (CIMP) involving tumor suppressor genes on short arm of chromosome 3 (3p) with increased risk of non-small cell lung cancer (NSCLC).

Methods And Materials: In this study, four NSCLC cell lines were cultured, and peripheral blood mononuclear cell (PBMC) specimens from 80 patients with NSCLC and 80 matched controls were collected for 3p-involved CIMP (3pCIMP) analysis. 3pCIMP was referred to as having at least three synchronously methylated genes of 3p per sample. Methylation-specific polymerase chain reaction was performed to examine the methylation status of each gene. DNA demethylation of NSCLC cell lines was achieved through the treatment with 5-aza-deoxycytidine. Logistic regression was used to assess odds ratios and 95% confidence intervals, which were adjusted for gender, age, and smoking status.

Results: Demethylation experiment showed that 3pCIMP status could play an important role in NSCLC cell proliferation. A total of 97.5% of PBMC specimens from NSCLC patients presented promoter methylation of any one of six genes (hOGG1, RAR-B, SEMA3B, RASSF1A, BLU, or FHIT) on 3p. Interestingly, 3pCIMP+ was found in 43.8% of NSCLC PBMC specimens and only in 6.3% of normal PBMC samples. The data suggest that 3pCIMP status is significantly associated with NSCLC and normal PBMC samples (p 0.001). More importantly, the results show that 3pCIMP positive carriers have a 12.8-fold increased risk of NSCLC (adjusted odds ratio, 12.8; 95% confidence interval, 4.38 -37.4, p 0.001) in Chinese population.

Conclusions: This is the first evidence of an association between PBMC 3pCIMP and risk for NSCLC.
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http://dx.doi.org/10.1097/jto.0b013e3181d862f5DOI Listing
June 2010

[Meta Analysis of Association between Polymorphisms in Promoter Region of MMPs gene and Risk of Lung Cancer.].

Zhongguo Fei Ai Za Zhi 2009 May;12(5):381-6

Laboratory of Medical Genetics, School of Basic Medicine & Biological Sciences, Medical College of Soochow University, Suzhou 215123, China.

Background: Lung cancer is one of the most common cancers worldwide, especially in China. It has been proved that matrix metalloproteases (MMPs) play a key role in malignant cell metastasis. There have been a considerable number of studies investigating MMP polymorphisms in relation to various cancers. However, the associations of MMP polymorphisms with risk of lung cancer are lack of consistency. The aim of this study is to assess the association of MMP polymorphisms with risk of lung cancer by conducting a Meta -analysis from all eligible casecontrol studies published to date.

Methods: To identify all studies that examined the association of polymorphisms in the promoter of MMP1, MMP2 and MMP9 with lung cancer, we conducted a computerized literature search of PubMed and MEDLINE database (before March, 2009). Two investigators independently extracted the data and reached a consensus on all items.

Results: Eight case-control studies, including 4 467 lung cancer cases and 4 051 controls, were selected for Meta -analysis to better assess the purported associations of common MMPs polymorphisms with risk of lung cancer. Our results suggest that MMP2 -735C/T polymorphism is significantly modified risk of lung cancer. Comparing with the wild -735C allele, the variant T allele decreased risk of developing lung cancer (OR=0.72, 95%CI: 0.61-0.85, P =0.0001). Other polymorphisms, including MMP1 -1607 1G/2G, MMP2 -1306C/T and MMP9 -1562C/T, are not associated with risk of lung cancer.

Conclusions: The present study indicates that the MMP2 -735C/T polymorphism is associated with the risk of lung cancer. Lager studies should be required to warrant the association of MMP9 -1562C/T polymorphism with risk of lung cancer.
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http://dx.doi.org/10.3779/j.issn.1009-3419.2009.05.017DOI Listing
May 2009