Publications by authors named "Christopher B Umbricht"

44 Publications

Methylated markers accurately distinguish primary central nervous system lymphomas (PCNSL) from other CNS tumors.

Clin Epigenetics 2021 May 5;13(1):104. Epub 2021 May 5.

Departments of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21231, USA.

Background: Definitive diagnosis of primary central nervous system lymphoma (PCNSL) requires invasive surgical brain biopsy, causing treatment delays. In this paper, we identified and validated tumor-specific markers that can distinguish PCNSL from other CNS tumors in tissues. In a pilot study, we tested these newly identified markers in plasma.

Results: The Methylation Outlier Detector program was used to identify markers in TCGA dataset of 48 diffuse large B-cell lymphoma (DLBCL) and 656 glioblastomas and lower-grade gliomas. Eight methylated markers clearly distinguished DLBCL from gliomas. Marker performance was verified (ROC-AUC of ≥ 0.989) in samples from several GEO datasets (95 PCNSL; 2112 other primary CNS tumors of 11 types). Next, we developed a novel, efficient assay called Tailed Amplicon Multiplexed-Methylation-Specific PCR (TAM-MSP), which uses two of the methylation markers, cg0504 and SCG3 triplexed with ACTB. FFPE tissue sections (25 cases each) of PCNSL and eight types of other primary CNS tumors were analyzed using TAM-MSP. TAM-MSP distinguished PCNSL from the other primary CNS tumors with 100% accuracy (AUC = 1.00, 95% CI 0.95-1.00, P < 0.001). The TAM-MSP assay also detected as few as 5 copies of fully methylated plasma DNA spiked into 0.5 ml of healthy plasma. In a pilot study of plasma from 15 PCNSL, 5 other CNS tumors and 6 healthy individuals, methylation in cg0504 and SCG3 was detectable in 3/15 PCNSL samples (20%).

Conclusion: The Methylation Outlier Detector program identified methylated markers that distinguish PCNSL from other CNS tumors with accuracy. The high level of accuracy achieved by these markers was validated in tissues by a novel method, TAM-MSP. These studies lay a strong foundation for a liquid biopsy-based test to detect PCNSL-specific circulating tumor DNA.
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http://dx.doi.org/10.1186/s13148-021-01091-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097855PMC
May 2021

Characterization of TERT and BRAF copy number variation in papillary thyroid carcinoma: An analysis of the cancer genome atlas study.

Genes Chromosomes Cancer 2021 Jun 18;60(6):403-409. Epub 2020 Dec 18.

Department of Oncology, Johns Hopkins University, Baltimore, Maryland, USA.

Alterations in the genome, including mutations and copy number variation (CNV), can drive cancer progression. The Cancer Genome Atlas (TCGA) project studying papillary thyroid cancer (PTC) identified a number of recurrent arm-level copy number amplifications, some spanning genes that are also commonly mutated in thyroid cancer. Herein, we focus on the role of TERT and BRAF CNV in PTC, including its relation to mutation status, gene expression, and clinicopathological characteristics. Utilizing TCGA CNV data, we identified focal amplifications and deletions involving the TERT and BRAF loci. TERT amplifications are more frequent in later stage thyroid tumors; in contrast, BRAF amplifications are not associated with stage. Furthermore, TERT amplifications are more frequently found in tumors also harboring TERT mutations, the combination further increasing TERT expression. Conversely, BRAF amplifications are more frequently found in BRAF wildtype tumors, and are more common in the follicular subtype of PTC as well as classic PTCs associated with a high follicular component and a RAS-like expression profile (assessed by the BRAF/RAS score). This is the first study to examine the TCGA thyroid dataset for gene-level CNV of TERT and BRAF, and their relationship with mutation status, tumor type and tumor stage. Assessing the differences in patterns of TERT and BRAF amplifications in the context of the mutation status of these genes may provide insight into the differing roles CNV can play depending on tumor type, and may lead to a better understanding of cancer drivers in thyroid cancer.
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http://dx.doi.org/10.1002/gcc.22928DOI Listing
June 2021

Integrated Multiparametric Radiomics and Informatics System for Characterizing Breast Tumor Characteristics with the OncotypeDX Gene Assay.

Cancers (Basel) 2020 Sep 27;12(10). Epub 2020 Sep 27.

Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

Optimal use of multiparametric magnetic resonance imaging (mpMRI) can identify key MRI parameters and provide unique tissue signatures defining phenotypes of breast cancer. We have developed and implemented a new machine-learning informatic system, termed Informatics Radiomics Integration System (IRIS) that integrates clinical variables, derived from imaging and electronic medical health records (EHR) with multiparametric radiomics (mpRad) for identifying potential risk of local or systemic recurrence in breast cancer patients. We tested the model in patients ( = 80) who had Estrogen Receptor positive disease and underwent OncotypeDX gene testing, radiomic analysis, and breast mpMRI. The IRIS method was trained using the mpMRI, clinical, pathologic, and radiomic descriptors for prediction of the OncotypeDX risk score. The trained mpRad IRIS model had a 95% and specificity was 83% with an Area Under the Curve (AUC) of 0.89 for classifying low risk patients from the intermediate and high-risk groups. The lesion size was larger for the high-risk group (2.9 ± 1.7 mm) and lower for both low risk (1.9 ± 1.3 mm) and intermediate risk (1.7 ± 1.4 mm) groups. The lesion apparent diffusion coefficient (ADC) map values for high- and intermediate-risk groups were significantly ( < 0.05) lower than the low-risk group (1.14 vs. 1.49 × 10 mm/s). These initial studies provide deeper insight into the clinical, pathological, quantitative imaging, and radiomic features, and provide the foundation to relate these features to the assessment of treatment response for improved personalized medicine.
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http://dx.doi.org/10.3390/cancers12102772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601838PMC
September 2020

Exploring the epigenetic regulation of telomerase reverse transcriptase (TERT) in human cancer cell lines.

Mol Oncol 2020 10 19;14(10):2355-2357. Epub 2020 Sep 19.

Department of Surgery, Johns Hopkins University, Baltimore, MD, USA.

Telomerase regulation, including TERT promoter methylation, has been of long-standing interest to cancer biologists. Rowland et al. have now vastly expanded their ongoing characterization of TERT promoter methylation in cancer cells, analyzing the methylation patterns of 833 cell lines from 23 human cancers. They document a highly conserved pattern of hypomethylation around the proximal promoter, as well as a more heterogeneous region of hypermethylation further upstream, both associated with active TERT expression in cancer cells. They further describe the interplay between activating TERT promoter mutations and allelic methylation and transcription patterns. This valuable dataset represents the most extensive characterization of TERT promoter methylation in cancer cells to date and will help guide the future study of transcriptional regulation of telomerase. Comment on: https://doi.org/10.1002/1878-0261.12786.
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http://dx.doi.org/10.1002/1878-0261.12798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530778PMC
October 2020

Telomerase Reverse Transcriptase (TERT) Regulation in Thyroid Cancer: A Review.

Front Endocrinol (Lausanne) 2020 31;11:485. Epub 2020 Jul 31.

Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.

is the catalytic subunit of the enzyme telomerase and is essential for telomerase activity. Upregulation of expression and resulting telomerase activity occurs in the large majority of malignancies, including thyroid cancer. This upregulation results in continued cellular proliferation and avoidance of cellular senescence and cell death. In this review we will briefly introduce and telomerase activity as it pertains to thyroid cancer and, highlight the effects of on cancer cells. We will also explore in detail the different regulatory strategies and how is reactivated in thyroid cancer cells, specifically. These regulatory mechanisms include both activating single base pair promoter mutations and epigenetic changes at the promoter, including changes in CpG methylation and histone modifications that affect chromatin structure. Further, regulation includes the allele-specific regulation of the promoter in thyroid cancer cells harboring the promoter mutation. These entail allele-specific transcriptional activator binding, DNA methylation, histone modifications, and mono-allelic expression of . Lastly, copy number alterations and alternative splicing are also implicated. Both amplifications of the locus and increased full-length transcripts and decreased inactive and dominant negative isoforms result in active telomerase. Finally, the clinical significance of in thyroid cancer is also reviewed.
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http://dx.doi.org/10.3389/fendo.2020.00485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412884PMC
June 2021

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

Characterization of Allele-Specific Regulation of Telomerase Reverse Transcriptase in Promoter Mutant Thyroid Cancer Cell Lines.

Thyroid 2020 10 4;30(10):1470-1481. Epub 2020 May 4.

Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Telomerase reverse transcriptase () promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in regulation. Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors (GA binding protein transcription factor subunit alpha) and , as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). We found the mutant promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous mutant cell lines. We demonstrated that the transcriptional activators and bind only to the mutant allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, exhibits from the mutant allele only. In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous mutations.
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http://dx.doi.org/10.1089/thy.2020.0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583328PMC
October 2020

DNA methylation markers predict recurrence-free interval in triple-negative breast cancer.

NPJ Breast Cancer 2020 31;6. Epub 2020 Jan 31.

1Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.

We lack tools to risk-stratify triple-negative breast cancer (TNBC). Our goal was to develop molecular tools to predict disease recurrence. Methylation array analysis was performed on 110 samples treated by locoregional therapy obtained from institutional cohorts. Discovered marker sets were then tested by Kaplan-Meier analyses in a prospectively collected TNBC cohort of 49 samples from the no-chemotherapy arms of IBCSG trials VIII and IX, and by logistic regression in a chemotherapy-treated cohort of 121 TNBCs from combined IBCSG trials and institutional repositories. High methylation was associated with shorter recurrence-free interval in the no-chemotherapy arm of the IBCSG studies, as well as in the chemotherapy-treated patients within the combined institutional and IBCSG chemotherapy cohorts (100 marker panel,  = 0.002; 30 marker panel,  = 0.05). Chromosome 19 sites were enriched among these loci. In conclusion, our hypermethylation signatures identify increased recurrence risk independent of whether patients receive chemotherapy.
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http://dx.doi.org/10.1038/s41523-020-0145-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994477PMC
January 2020

Breast Cancer Risk in Postmenopausal Women with Medical History of Thyroid Disorder in the Women's Health Initiative.

Thyroid 2020 04 3;30(4):519-530. Epub 2020 Feb 3.

Department of Family Medicine, Brown University Warren Alpert Medical School, Providence, Rhode Island.

The association between thyroid disorders and breast cancer remains controversial, in part, due to small cohort sizes and inconsistent findings. We investigated this association in postmenopausal women to determine whether hyper- or hypothyroidism is associated with the risk of developing breast cancer and to determine whether menopausal hormone therapy (MHT) further modifies the risk. We conducted a prospective cohort study of multiethnic U.S. postmenopausal women aged 50 to 79 years enrolled in both clinical trial and observational study arms between 1993 and 1998 and followed up through February 28, 2017. Development of invasive breast cancer after enrollment was recorded and a history of hyper- or hypothyroidism before the diagnosis of breast cancer was identified. The effect modification by MHT in both study arms was analyzed. All statistical tests were two sided. Among a total of 134,122 women who were included in our study, 8137 participants developed invasive breast cancer during the follow-up period. There was a significant inverse association of invasive breast cancer among women with a history of hypothyroidism (hazard ratio [HR] 0.91, confidence interval [95% CI] 0.86-0.97) and among women who had taken levothyroxine [HR 0.89, 95% CI 0.82-0.96]. Evaluating effect modification by MHT use, the inverse association between hypothyroidism treated with thyroid replacement medications and breast cancer risk was strongest in non-MHT users [HR 0.80, 95% CI 0.69-0.93]. The results did not significantly differ by race/ethnicity. Although a history of hyperthyroidism was associated with an increased risk of invasive breast cancer [HR 1.11, 95% CI 0.91-1.35], this finding did not reach statistical significance. We did not see significant differences in the breast cancer Surveillance, Epidemiology, and End Results stages, histologic types, morphologic grades, or receptor status (estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2) according to thyroid disorder status. Compared with women with no history of thyroid disorder, hypothyroidism was associated with a lower risk of breast cancer. This was mainly seen among those who received thyroid replacement therapy and had never used MHT. Among the treatment options for hypothyroidism, levothyroxine had the strongest inverse association with breast cancer risk.
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http://dx.doi.org/10.1089/thy.2019.0426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187984PMC
April 2020

DNA Methylation Markers for Breast Cancer Detection in the Developing World.

Clin Cancer Res 2019 11 12;25(21):6357-6367. Epub 2019 Jul 12.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Purpose: An unmet need in low-resource countries is an automated breast cancer detection assay to prioritize women who should undergo core breast biopsy and pathologic review. Therefore, we sought to identify and validate a panel of methylated DNA markers to discriminate between cancer and benign breast lesions using cells obtained by fine-needle aspiration (FNA). Two case-control studies were conducted comparing cancer and benign breast tissue identified from clinical repositories in the United States, China, and South Africa for marker selection/training ( = 226) and testing ( = 246). Twenty-five methylated markers were assayed by Quantitative Multiplex-Methylation-Specific PCR (QM-MSP) to select and test a cancer-specific panel. Next, a pilot study was conducted on archival FNAs (49 benign, 24 invasive) from women with mammographically suspicious lesions using a newly developed, 5-hour, quantitative, automated cartridge system. We calculated sensitivity, specificity, and area under the receiver-operating characteristic curve (AUC) compared with histopathology for the marker panel.

Results: In the discovery cohort, 10 of 25 markers were selected that were highly methylated in breast cancer compared with benign tissues by QM-MSP. In the independent test cohort, this panel yielded an AUC of 0.937 (95% CI = 0.900-0.970). In the FNA pilot, we achieved an AUC of 0.960 (95% CI = 0.883-1.0) using the automated cartridge system.

Conclusions: We developed and piloted a fast and accurate methylation marker-based automated cartridge system to detect breast cancer in FNA samples. This quick ancillary test has the potential to prioritize cancer over benign tissues for expedited pathologic evaluation in poorly resourced countries.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825533PMC
November 2019

Measuring DNA Copy Number Variation Using High-Density Methylation Microarrays.

J Comput Biol 2019 04 21;26(4):295-304. Epub 2019 Feb 21.

5 Department of Oncology Bioinformatics, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

Genetic and epigenetic changes drive carcinogenesis, and their integrated analysis provides insights into mechanisms of cancer development. Computational methods have been developed to measure copy number variation (CNV) from methylation array data, including ChAMP-CNV, CN450K, and, introduced here, Epicopy. Using paired single nucleotide polymorphism (SNP) and methylation array data from the public The Cancer Genome Atlas repository, we optimized CNV calling and benchmarked the performance of these methods. We optimized the thresholds of all three methods and showed comparable performance across methods. Using Epicopy as a representative analysis of Illumina450K array, we show that Illumina450K-derived CNV methods achieve a sensitivity of 0.7 and a positive predictive value of 0.75 in identifying CNVs, which is similar to results achieved when comparing competing SNP microarray platforms with each other.
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http://dx.doi.org/10.1089/cmb.2018.0143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479247PMC
April 2019

Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines.

Genes Chromosomes Cancer 2019 08 10;58(8):530-540. Epub 2019 Feb 10.

Department of Surgery, The University of Virginia School of Medicine, Charlottesville, Virginia.

Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.
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http://dx.doi.org/10.1002/gcc.22735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621557PMC
August 2019

Young age at diagnosis is associated with worse prognosis in the Luminal A breast cancer subtype: a retrospective institutional cohort study.

Breast Cancer Res Treat 2018 Dec 17;172(3):689-702. Epub 2018 Sep 17.

Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.

Purpose: Although age is a recognized independent prognostic risk factor, its relative importance among molecular subtypes of Breast cancer (BCA) is not well documented. The aim of this study was to evaluate the prognostic role of age at diagnosis among different immunohistochemical subtypes of BCA.

Methods: We conducted a retrospective study of women with invasive BCA undergoing surgery at the Johns Hopkins Hospital, excluding patients presenting with stage IV breast cancer. Patients were stratified into three age groups: ≤ 40, 41-60, and > 60 years, and multivariable analysis was performed using Cox regression. We also identified differentially expressed genes (DEG) between age groups among BCA subtypes in the public TCGA dataset. Finally, we identified key driver genes within the DEGs using a weighted gene co-expression network analysis.

Results: Luminal A breast cancer patients had significantly lower 5 year disease-free survival (DFS) and distant metastasis-free survival (DMFS) in the ≤ 40 year age group compared to the 41-60 year age group, while the other molecular subtypes showed no significant association of DFS or DMFS with age. Age was a stronger outcome predictor than tumor grade or proliferative index in Luminal A BCA patients, but not other subtypes. BCA TCGA gene expression data were divided into two groups (≤ 40 years, > 40 years). We identified 374 DEGs in the Luminal A BCA subset, which were enriched in seven pathways and two modules of co-expressed genes. No age group-specific DEGs were identified in non-Luminal A subtypes.

Conclusions: Age at diagnosis may be an important prognostic factor in Luminal A BCA.
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http://dx.doi.org/10.1007/s10549-018-4950-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786966PMC
December 2018

Preoperative Molecular Markers in Thyroid Nodules.

Front Endocrinol (Lausanne) 2018 18;9:179. Epub 2018 Apr 18.

Department of Surgery, University of Virginia, Charlottesville, VA, United States.

The need for distinguishing benign from malignant thyroid nodules has led to the pursuit of differentiating molecular markers. The most common molecular tests in clinical use are Afirma Gene Expression Classifier (GEC) and Thyroseq V2. Despite the rapidly developing field of molecular markers, several limitations exist. These challenges include the recent introduction of the histopathological diagnosis "Non-Invasive Follicular Thyroid neoplasm with Papillary-like nuclear features", the correlation of genetic mutations within both benign and malignant pathologic diagnoses, the lack of follow-up of molecular marker negative nodules, and the cost-effectiveness of molecular markers. In this manuscript, we review the current published literature surrounding the diagnostic value of Afirma GEC and Thyroseq V2. Among Afirma GEC studies, sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) ranged from 75 to 100%, 5 to 53%, 13 to 100%, and 20 to 100%, respectively. Among Thyroseq V2 studies, Se, Sp, PPV, and NPV ranged from 40 to 100%, 56 to 93%, 13 to 90%, and 48 to 97%, respectively. We also discuss current challenges to Afirma GEC and Thyroseq V2 utility and clinical application, and preview the future directions of these rapidly developing technologies.
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http://dx.doi.org/10.3389/fendo.2018.00179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915469PMC
April 2018

Thyroid Nodule Diagnostic Markers in the Face of the New NIFTP Category: Time for a Reset?

Thyroid 2017 11 28;27(11):1393-1399. Epub 2017 Sep 28.

1 Endocrine Surgery, Department of Surgery, The Johns Hopkins University School of Medicine , Baltimore, Maryland.

Background: Current thyroid molecular tests are specifically designed for the differential diagnosis of nodules with indeterminate or suspicious fine-needle aspiration (FNA) cytology.

Summary: However, their clinical validity faces challenges from both variation among institutions in cancer prevalence and, most recently, the new category of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). The latter diagnosis was previously classified as malignant. Relevant to this, all molecular panels on the market today were originally tested and validated within the context of these entities being considered malignant.

Conclusion: This review examines possible effects of the NIFTP reclassification as a precancerous lesion on the original validation studies and, investigates the effect of the significant reported variability in thyroid cancer prevalence on the performance of these tests.
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http://dx.doi.org/10.1089/thy.2017.0238DOI Listing
November 2017

Positive Ultrasound-guided Lymph Node Needle Biopsy in Breast Cancer may not Mandate Axillary Lymph Node Dissection.

Ann Surg Oncol 2017 Oct 1;24(10):3004-3010. Epub 2017 Aug 1.

Rebecca Fortney Breast Center, Anne Arundel Medical Center, Annapolis, USA.

Background: The ACOSOG Z0011 (Z11) trial demonstrated that in patients with nonpalpable axillary lymph nodes (LN) and one to two positive sentinel LN (SLN), axillary LN dissection (ALND) is unnecessary.JAMA 305:569-575, [2011], Ann Surg 264:413-42, [2016] The Z11 trial did not require preoperative axillary ultrasound (axUS). In many centers, preoperative axUS is part of the standard workup of a newly diagnosed breast cancer patient, but in light of the Z11 results, its role is now questioned.

Methods: We retrospectively analyzed newly diagnosed breast cancer patients at two institutions. Inclusion criteria were patients with (1) no palpable lymphadenopathy, (2) abnormal axUS, (3) axillary LN metastasis confirmed preoperatively by axUS-lymph node needle biopsy, (4) no neoadjuvant therapy, and (5) ALND. LN disease burden was dichotomized as N1 versus N2-3. We examined relationships between clinicopathologic factors, including axUS characteristics, and LN disease burden.

Results: Of 129 included cases, 67 had N1 disease (51.9%) and 62 had N2-3 disease (48.1%). Factors significantly associated with N1 disease were tumor size ≤2 cm (p = 0.012), nonlobular histology (p = 0.013), and one suspicious LN on axUS (p = 0.008). For patients with both tumor size on imaging ≤2 cm and one abnormal LN on axUS, only 27% had N2-3 disease (p = 0.007).

Conclusions: More than half of patients without palpable adenopathy but with preoperative US-guided biopsy proven axillary LN metastases had N1 disease. For patients with both tumor size ≤2 cm and only 1 abnormal LN on axUS, 73% had N1 disease. This suggests that such patients, if they are otherwise analogous to Z11 patients, may undergo attempt at SLNB.
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http://dx.doi.org/10.1245/s10434-017-5935-yDOI Listing
October 2017

Identification of novel biomarker and therapeutic target candidates for diagnosis and treatment of follicular carcinoma.

J Proteomics 2017 08 12;166:59-67. Epub 2017 Jul 12.

Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address:

Distinguishing follicular carcinoma from follicular adenoma, based on cytomorphological features, has always been challenging to cytopathologists. Identification of biomarkers for improving diagnostic accuracy is important for clinical management. Meanwhile, it is critical to identify therapeutic target candidates for treatment of follicular carcinoma. Currently, no reliable diagnostic protein biomarkers and therapeutic targets are available. To explore novel protein biomarker and therapeutic target candidates, a liquid chromatography-tandem mass spectrometry approach was applied to analyze control, follicular adenoma, and follicular carcinoma using formalin-fixed, paraffin-embedded tissue samples. The proteomics analysis revealed 80 protein biomarker candidates for diagnosis of thyroid follicular carcinoma. The candidates were prioritized into three categories and ranked within each category. Using the proteomics data and bioinformatics results, the top seven biomarker candidates were coiled-coil-helix-coiled-coil-helix domain-containing protein 2, mitochondrial (CHCHD2), succinyl-CoA ligase [GDP-forming] subunit beta, mitochondrial (SUCLG2), stomatin-like protein 2, mitochondrial (STOML2), ES1 protein homolog, mitochondrial (C21orf33), fumarate hydratase, mitochondrial (FH), 3-hydroxyacyl-CoA dehydrogenase type-2 (HSD17B10), and electron transfer flavoprotein subunit beta (ETFB); and the top seven therapeutic target candidates were insulin receptor (INSR), Myc proto-oncogene protein (MYC), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), gastrin (GAST), N-myc proto-oncogene protein (MYCN), transforming growth factor beta-1 (TGFB1), and interleukin-4 (IL4). Immunohistochemical staining of SUCLG2 and ETFB is highly consistent with the discovery of proteomics, revealing that SUCLG2 has a sensitivity of 75% and a specificity of 80% to distinguish follicular carcinoma from follicular adenoma based on a specific cut-off score calculated from the IHC staining percentage and intensity.

Biological Significance: Distinguishing follicular carcinoma from follicular adenoma, based on cytomorphological features, has always been challenging to cytopathologists. Fourteen biomarker candidates were identified. Two of them were validated with Immunohistochemical staining. The Identification of biomarkers for improving diagnostic accuracy is important for clinical management.
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http://dx.doi.org/10.1016/j.jprot.2017.07.003DOI Listing
August 2017

Optimizing the Use of Gene Expression Profiling in Early-Stage Breast Cancer.

J Clin Oncol 2016 12 31;34(36):4390-4397. Epub 2016 Oct 31.

Hyun-seok Kim, Christopher B. Umbricht, Peter B. Illei, Ashley Cimino-Mathews, Soonweng Cho, Nivedita Chowdhury, Maria Cristina Figueroa-Magalhaes, Catherine Pesce, Stacie C. Jeter, Deborah K. Armstrong, Roisin M. Connolly, John H. Fetting, Ben Ho Park, Vered Stearns, Antonio C. Wolff, and Leslie Cope, The Johns Hopkins University School of Medicine; Kala Visvanathan, The Johns Hopkins University Bloomberg School of Public Health, Baltimore; Charles Mylander, Martin Rosman, and Lorraine Tafra, Anne Arundel Medical Center, Annapolis, MD; Bradley M. Turner, David G. Hicks, University of Rochester Medical Center, Rochester, NY; Robert S. Miller, American Society of Clinical Oncology, Alexandria, VA; and Tyler A. Jensen and Dylan V. Miller, Intermountain Healthcare, Salt Lake City, UT.

Purpose Gene expression profiling assays are frequently used to guide adjuvant chemotherapy decisions in hormone receptor-positive, lymph node-negative breast cancer. We hypothesized that the clinical value of these new tools would be more fully realized when appropriately integrated with high-quality clinicopathologic data. Hence, we developed a model that uses routine pathologic parameters to estimate Oncotype DX recurrence score (ODX RS) and independently tested its ability to predict ODX RS in clinical samples. Patients and Methods We retrospectively reviewed ordered ODX RS and pathology reports from five institutions (n = 1,113) between 2006 and 2013. We used locally performed histopathologic markers (estrogen receptor, progesterone receptor, Ki-67, human epidermal growth factor receptor 2, and Elston grade) to develop models that predict RS-based risk categories. Ordering patterns at one site were evaluated under an integrated decision-making model incorporating clinical treatment guidelines, immunohistochemistry markers, and ODX. Final locked models were independently tested (n = 472). Results Distribution of RS was similar across sites and to reported clinical practice experience and stable over time. Histopathologic markers alone determined risk category with > 95% confidence in > 55% (616 of 1,113) of cases. Application of the integrated decision model to one site indicated that the frequency of testing would not have changed overall, although ordering patterns would have changed substantially with less testing of estimated clinical risk-high or clinical risk-low cases and more testing of clinical risk-intermediate cases. In the validation set, the model correctly predicted risk category in 52.5% (248 of 472). Conclusion The proposed model accurately predicts high- and low-risk RS categories (> 25 or ≤ 25) in a majority of cases. Integrating histopathologic and molecular information into the decision-making process allows refocusing the use of new molecular tools to cases with uncertain risk.
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http://dx.doi.org/10.1200/JCO.2016.67.7195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455310PMC
December 2016

Monitoring of Serum DNA Methylation as an Early Independent Marker of Response and Survival in Metastatic Breast Cancer: TBCRC 005 Prospective Biomarker Study.

J Clin Oncol 2017 Mar 21;35(7):751-758. Epub 2016 Nov 21.

Kala Visvanathan, Johns Hopkins University School of Medicine and Bloomberg School of Public Health; MaryJo S. Fackler, Zhe Zhang, Zoila A. Lopez-Bujanda, Stacie C. Jeter, Lori J. Sokoll, Leslie M. Cope, Christopher B. Umbricht, David M. Euhus, Saraswati Sukumar, and Antonio C. Wolff, Johns Hopkins University School of Medicine, Baltimore, MD; Elizabeth Garrett-Mayer, Medical University of South Carolina, Charleston, SC; Andres Forero, University of Alabama at Birmingham, Birmingham, AL; Anna M. Storniolo, Indiana University, Bloomington, IN; Rita Nanda, University of Chicago, Chicago, IL; Nancy U. Lin, Dana-Farber Cancer Institute, Boston, MA; Lisa A. Carey, University of North Carolina, Chapel Hill, NC; and James N. Ingle, Mayo Clinic, Rochester, MN.

Purpose Epigenetic alterations measured in blood may help guide breast cancer treatment. The multisite prospective study TBCRC 005 was conducted to examine the ability of a novel panel of cell-free DNA methylation markers to predict survival outcomes in metastatic breast cancer (MBC) using a new quantitative multiplex assay (cMethDNA). Patients and Methods Ten genes were tested in duplicate serum samples from 141 women at baseline, at week 4, and at first restaging. A cumulative methylation index (CMI) was generated on the basis of six of the 10 genes tested. Methylation cut points were selected to maximize the log-rank statistic, and cross-validation was used to obtain unbiased point estimates. Logistic regression or Cox proportional hazard models were used to test associations between the CMI and progression-free survival (PFS), overall survival (OS), and disease status at first restaging. The added value of the CMI in predicting survival outcomes was evaluated and compared with circulating tumor cells (CellSearch). Results Median PFS and OS were significantly shorter in women with a high CMI (PFS, 2.1 months; OS, 12.3 months) versus a low CMI (PFS, 5.8 months; OS, 21.7 months). In multivariable models, among women with MBC, a high versus low CMI at week 4 was independently associated with worse PFS (hazard ratio, 1.79; 95% CI, 1.23 to 2.60; P = .002) and OS (hazard ratio, 1.75; 95% CI, 1.21 to 2.54; P = .003). An increase in the CMI from baseline to week 4 was associated with worse PFS ( P < .001) and progressive disease at first restaging ( P < .001). Week 4 CMI was a strong predictor of PFS, even in the presence of circulating tumor cells ( P = .004). Conclusion Methylation of this gene panel is a strong predictor of survival outcomes in MBC and may have clinical usefulness in risk stratification and disease monitoring.
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http://dx.doi.org/10.1200/JCO.2015.66.2080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455421PMC
March 2017

Human telomerase reverse transcriptase regulation by DNA methylation, transcription factor binding and alternative splicing (Review).

Int J Oncol 2016 Dec 20;49(6):2199-2205. Epub 2016 Oct 20.

Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), plays an essential role in telomere maintenance to oppose cellular senescence and, is highly regulated in normal and cancerous cells. Regulation of hTERT occurs through multiple avenues, including a unique pattern of CpG promoter methylation and alternative splicing. Promoter methylation affects the binding of transcription factors, resulting in changes in expression of the gene. In addition to expression level changes, changes in promoter binding can affect alternative splicing in a cotranscriptional manner. The alternative splicing of hTERT results in either the full length transcript which can form the active telomerase complex with hTR, or numerous inactive isoforms. Both regulation strategies are exploited in cancer to activate telomerase, however, the exact mechanism is unknown. Therefore, unraveling the link between promoter methylation status and alternative splicing for hTERT could expose yet another level of hTERT regulation. In an attempt to provide insight into the cellular control of active telomerase in cancer, this review will discuss our current perspective on CpG methylation of the hTERT promoter region, summarize the different forms of alternatively spliced variants, and examine examples of transcription factor binding that affects splicing.
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http://dx.doi.org/10.3892/ijo.2016.3743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903903PMC
December 2016

Association of BRAF V600E Mutation and MicroRNA Expression with Central Lymph Node Metastases in Papillary Thyroid Cancer: A Prospective Study from Four Endocrine Surgery Centers.

Thyroid 2016 Apr 7;26(4):532-42. Epub 2016 Mar 7.

1 Endocrine Surgery, Department of Surgery, The Johns Hopkins University School of Medicine , Baltimore, Maryland.

Background: Studies have demonstrated an association of the BRAF(V600E) mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAF(V600E) mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM).

Methods: Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAF(V600E) mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC.

Results: Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAF(V600E) almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAF(V600E) mutation to predict CLNM independently. While BRAF(V600E) was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAF(V600E) was not significantly associated with CLNM.

Conclusion: In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAF(V600E) mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers.
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http://dx.doi.org/10.1089/thy.2015.0378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827320PMC
April 2016

MicroRNA Expression and Association with Clinicopathologic Features in Papillary Thyroid Cancer: A Systematic Review.

Thyroid 2015 Dec 8;25(12):1322-9. Epub 2015 Oct 8.

1 Endocrine Surgery Section, Department of Surgery, The Johns Hopkins University School of Medicine , Baltimore, Maryland.

Background: Studies have suggested that microRNAs (miR) may be useful prognostic markers and are associated with aggressive clinicopathologic features in papillary thyroid cancer (PTC). This systematic review examined associations between miRs and aggressive clinicopathologic features in PTC.

Methods: A literature search was performed within the PubMed, Embase, Cochrane, Web of Science, and Scopus databases for papers published prior to November 24, 2014. The search was performed by combining the concepts "thyroid tumor" with "microRNA" and by using "and" as the Boolean operator. Upon retrieval of candidate studies, full-text publications were reviewed in their entirety and selected if they examined the prognostic significance between miR expression and established aggressive clinicopathologic features of PTC.

Results: Fifteen studies from 13 unique groups that included 807 patients were reviewed. Most of the studies were retrospective, and none included patients who had undergone routine central lymph node dissection. Expression levels of miRs-21, -34b, -130b, -135b, -146b, -151, -181b, -199b-5p, -221, -222, -451, -623, -1271, -2861, and let-7e showed significant association with at least one aggressive feature, such as large tumor size, extrathyroidal extension, multifocality, lymphovascular invasion, lymph node metastases, distant metastasis, advanced American Joint Cancer Committee stage, and presence of the BRAF(V600E) mutation. Herein we summarize the literature with regard to these associations.

Conclusion: Further studies are needed to investigate whether miRs are independent predictors of aggressive clinicopathologic features before it can be recommended that miR expression levels should be incorporated into the management algorithm for patients with PTC. A well-designed prospective study is needed to assess these potential associations.
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http://dx.doi.org/10.1089/thy.2015.0193DOI Listing
December 2015

Do breast cancer cell lines provide a relevant model of the patient tumor methylome?

PLoS One 2014 26;9(8):e105545. Epub 2014 Aug 26.

Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

It is well documented that tumor cells undergo dramatic genetic and epigenetic changes during initial establishment as cell lines and in subsequent serial passaging, and that the resultant cell lines may have evolved significantly from the primary tumors from which they were derived. This has potential implications due to their widespread use in drug response experiments and studies of genomic function. One approach to optimizing the design of such cell line studies is to identify and use the cell lines that faithfully recapitulate critical features of primary tumors. To evaluate the epigenetic fidelity of breast cancer cell lines in the context of primary tumors, we performed methylation profiling of 55 well-characterized breast cancer cell lines on the Illumina HumanMethylation27 BeadChip platform, and compared them to publicly available methylation profiles of primary breast tumors. We found that the DNA methylation profiles of breast cancer cell lines largely retain the features that characterize primary tumors, although there are crucial differences as well. We describe these similarities and differences between primary tumors and breast cancer cell lines in detail, and develop a quantitative measure of similarity that is used to score each cell line with respect to how faithfully its methylation profile mirrors that of primary tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105545PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144876PMC
May 2015

Lower vitamin D levels in surgical hyperparathyroidism versus thyroid patients.

Am Surg 2014 May;80(5):505-10

Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

Low vitamin D levels have been shown to be associated with primary hyperparathyroidism, but it is unclear whether vitamin D deficiency may be an etiologic factor in the development of primary hyperparathyroidism. To investigate this, we compared preoperative vitamin D levels of patients undergoing surgery for primary hyperparathyroidism with those of patients undergoing surgery for benign thyroid disease. With Institutional Review Board approval, data were collected prospectively on patients undergoing parathyroidectomy or thyroidectomy by one surgeon between March 2006 and July 2011. Patients were excluded if they underwent simultaneous thyroid and parathyroid surgery, had secondary or tertiary hyperparathyroidism, if no preoperative vitamin D level was measured, or if they took vitamin D supplements. Inclusion criteria were met by 219 patients who underwent parathyroidectomy and 186 patients who underwent thyroid surgery. Patient age, sex, race, and preoperative vitamin D levels (vitamin D 25-OH; normal, 32 to 100 pg/mL) were collected. Statistical analysis was performed using linear regression. Vitamin D levels were significantly lower in the parathyroid group compared with the thyroid group (23.8 vs 28.5 pg/mL; P < 0.001). This difference was also observed after adjustment for age, sex, and race with a mean difference of 4.87 pg/mL (P < 0.001). Statistically significant associations between lower vitamin D levels and patients younger than 50 years (P = 0.048), male sex (P = 0.03), and nonwhite race were identified (P < 0.001). Patients with primary hyperparathyroidism are more likely to have lower vitamin D levels than a control surgical population. Further study is needed to determine whether low vitamin D levels may be an etiologic factor associated with the development of hyperparathyroidism.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362715PMC
May 2014

Does BRAF V600E mutation predict aggressive features in papillary thyroid cancer? Results from four endocrine surgery centers.

J Clin Endocrinol Metab 2013 Sep 22;98(9):3702-12. Epub 2013 Aug 22.

Endocrine Surgery, Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.

Background: Existing evidence is controversial regarding the association between BRAF mutation status and aggressive features of papillary thyroid cancer (PTC). Specifically, no study has incorporated multiple surgical practices performing routine central lymph node dissection (CLND) and thus has patients who are truly evaluable for the presence or absence of central lymph node metastases (CLNMs).

Methods: Consecutive patients who underwent total thyroidectomy and routine CLND at 4 tertiary endocrine surgery centers were retrospectively reviewed. Descriptive and bivariable analyses examined demographic, patient, and tumor-related factors. Multivariable analyses examined the odds of CLNM associated with positive BRAF status.

Results: In patients with classical variant PTC, bivariate analysis found no significant associations between BRAF mutation and aggressive clinicopathologic features; multivariate analysis demonstrated that BRAF status was not an independent predictor of CLNM. When all patients with PTC were analyzed, including those with aggressive or follicular subtypes, bivariate analysis showed BRAF mutation to be associated with LNM, advanced American Joint Committee on Cancer (AJCC) stage, and histologic subtype. Multivariable analyses showed BRAF, age, size, and extrathyroidal extension to be associated with CLNM.

Conclusion: Although BRAF mutation was found to be an independent predictor of central LNM in the overall cohort of patients with PTC, this relationship lost significance when only classical variant PTC was included in the analysis. The usefulness of BRAF in predicting the presence of LNM remains questionable. Prospective studies are needed before BRAF mutation can be considered a reliable factor to guide the treatment of patients with PTC, specifically whether to perform prophylactic CLND.
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http://dx.doi.org/10.1210/jc.2013-1584DOI Listing
September 2013

A tertiary center's experience with second review of 3885 thyroid cytopathology specimens.

J Clin Endocrinol Metab 2013 Apr 22;98(4):1450-7. Epub 2013 Feb 22.

Departments of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

Background: Although the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) has standardized the diagnostic terminology for thyroid fine-needle aspiration (FNA), morphological interpretation remains subjective, and interobserver discrepancies are expected. This study quantifies the frequency and magnitude of these discrepancies in a single tertiary center's experience and elucidates key factors that are associated with changes in diagnosis.

Methods: Institutional consultation for 3885 thyroid cytological samples over 45 months were reviewed. BSRTC classification made by the sending institution was compared with that of our institution. An ANOVA was performed to determine factors that may be associated with interinstitutional diagnostic differences. Histopathology diagnoses were available for 1049 (27%) nodules; the malignancy rates for inside and outside BSRTC classifications were calculated.

Results: There were 937 1-step changes and 301 ≥2-step diagnostic discrepancies comprising 24% and 8% of all cases, respectively. Second review decreased the indeterminate rate 38% to 28% (P < .000001). Indeterminate diagnostic category before second review, low specimen cellularity, Hashimoto's thyroiditis, and low volume of consults from the sending institution were associated with discordance. Of the 1049 thyroid nodules operated for which unequivocal histopathology was available, the malignancy rates for the BSRTC categories before and after second review were compared. Categorical upgrades were associated with a malignancy rate of 84%, whereas downgrades were associated with a malignancy rate of 38% (P < .000001).

Conclusion: This is the largest series to date of thyroid cytology second review. The BSRTC classification changed 32% of the time, potentially resulting in significant changes in clinical and surgical management. Because certain specimen characteristics (indeterminate diagnostic category before second review, low specimen cellularity, Hashimoto's thyroiditis, and low volume of consults from the sending institution) were particularly associated with a diagnosis change, morphological second review may be of potential benefit in these settings.
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http://dx.doi.org/10.1210/jc.2012-3898DOI Listing
April 2013

Is BRAF mutation associated with lymph node metastasis in patients with papillary thyroid cancer?

Surgery 2012 Dec 11;152(6):977-83. Epub 2012 Oct 11.

Endocrine Surgery Section, Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

Background: Some have proposed using V600E BRAF mutation status to dictate the surgical management of patients with papillary thyroid cancer (PTC). However, well-designed studies examining BRAF association with aggressive clinicopathologic features of PTC, including the presence of lymph node metastases (LNM), in patients who have undergone routine central lymph node dissection (CLND), are lacking.

Methods: Under institutional review board approval, 63 patients diagnosed with PTC on fine-needle aspiration who underwent total thyroidectomy and CLND were included. BRAF mutation status was determined in fresh frozen or intraoperative fine-needle aspiration samples with a colorimetric assay. Associations between BRAF mutation status and clinicopathologic features of PTC were examined using Chi-square and multivariate logistic regression analyses.

Results: BRAF mutation was found to be significantly associated with race only on Chi-square analysis. BRAF mutation was not found to be significantly associated with the presence of LNM (P = .167). On multivariate analysis, only size and venous/lymphatic invasion were significantly associated with LNM.

Conclusion: This small series underscores the prematurity in utilizing BRAF mutation status to determine the surgical management of patients with PTC, specifically whether or not to perform a CLND.
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http://dx.doi.org/10.1016/j.surg.2012.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715093PMC
December 2012

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer.

Epigenetics 2012 Jul 1;7(7):710-9. Epub 2012 Jul 1.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based "mutector assay" was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.
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http://dx.doi.org/10.4161/epi.20524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414391PMC
July 2012

Three-gene molecular diagnostic model for thyroid cancer.

Thyroid 2012 Mar 26;22(3):275-84. Epub 2012 Jan 26.

Endocrine Surgery Section, Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.

Background: The preoperative diagnosis of thyroid nodules primarily depends upon fine needle aspiration (FNA) cytology. However, up to 25% of FNA samples have associated "suspicious or indeterminate", but not diagnostic cytologic reports, resulting in difficulty deciding appropriate clinical management for these patients. We hypothesize that the use of molecular markers as an adjunct to FNA cytology can improve the distinction of benign from malignant nodules that have associated suspicious or indeterminate cytology.

Methods: Using microarray analysis, we previously identified and reported on 75 genes useful in the distinction of benign versus malignant thyroid nodules. In the present study, we have further validated the expression of 14 of these markers in a large number of thyroid samples by immunohistochemistry (IHC) analysis of 154 thyroid tumors and quantitative real-time RT-PCR (QRT-PCR) analysis of 95 FNA samples. Of the 154 tumors analyzed by IHC, 44 samples (29%) had associated suspicious or indeterminate FNA cytology.

Results: Receiver operating characteristic using three-gene model, (HMGA2, MRC2, and SFN) analysis for the detection of malignant nodules resulted in areas under the curve (AUCs) of≥0.95 (80% sensitivity; 100% specificity) and≥0.84 (71% sensitivity; 84% specificity) for the IHC data in tumors, and QRT-PCR data in FNA samples, respectively.

Conclusions: Our results suggest that a three-gene model for the cytological diagnosis of indeterminate thyroid nodules is both feasible and promising. Implementation of this as an adjunct to thyroid cytology may significantly impact the clinical management of patients with suspicious or indeterminate thyroid FNA nodules.
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http://dx.doi.org/10.1089/thy.2011.0169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286810PMC
March 2012

Genome-wide methylation analysis identifies genes specific to breast cancer hormone receptor status and risk of recurrence.

Cancer Res 2011 Oct 8;71(19):6195-207. Epub 2011 Aug 8.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

To better understand the biology of hormone receptor-positive and-negative breast cancer and to identify methylated gene markers of disease progression, we carried out a genome-wide methylation array analysis on 103 primary invasive breast cancers and 21 normal breast samples, using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Estrogen and/or progesterone receptor-positive tumors displayed more hypermethylated loci than estrogen receptor (ER)-negative tumors. However, the hypermethylated loci in ER-negative tumors were clustered closer to the transcriptional start site compared with ER-positive tumors. An ER-classifier set of CpG loci was identified, which independently partitioned primary tumors into ER subtypes. A total of 40 (32 novel and 8 previously known) CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER subtype-specific loci was validated in silico, using an independent, publicly available methylome dataset from the Cancer Genome Atlas. In addition, we identified 100 methylated CpG loci that were significantly associated with disease progression; the majority of these loci were informative particularly in ER-negative breast cancer. Overall, the set was highly enriched in homeobox containing genes. This pilot study shows the robustness of the breast cancer methylome and illustrates its potential to stratify and reveal biological differences between ER subtypes of breast cancer. Furthermore, it defines candidate ER-specific markers and identifies potential markers predictive of outcome within ER subgroups.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-1630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308629PMC
October 2011