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Background: Lymph node (LN)-related risk factors have been updated to predict long-term outcomes in patients with papillary thyroid carcinoma (PTC). However, those factors’ analytic appropriateness and general applicability must be validated. This study aimed to assess LN-related risk factors, and suggest new LN-related risk categories. Methods: This multicenter observational cohort study included 1232 patients with PTC with N1 disease treated with a total thyroidectomy and neck dissection followed by radioactive iodine remnant ablation. Results: The median follow-up duration was 117 months. In the follow-up period, structural recurrence occurred in 225 patients (18.3%). Among LN-related variables, the presence of extranodal extension (p < 0.001), the maximal diameter of metastatic LN foci (p = 0.029), the number of retrieved LNs (p = 0.003), the number of metastatic LNs (p = 0.003), and the metastatic LN ratio (p < 0.001) were independent risk factors for structural recurrence. Since these factors showed a nonlinear association with the hazard ratio of recurrence-free survival (RFS) rates, we calculated their optimal cutoff values using the K-means clustering algorithm, selecting 0.2 cm and 1.1 cm for the maximal diameter of metastatic LN foci, 4 and 13 for the number of metastatic LN, and 0.28 and 0.58 for the metastatic LN ratio. The RFS curves of each subgroup classified by these newly determined cutoff values showed significant differences (p < 0.001). Each LN risk group also showed significantly different RFS rates from the others (p < 0.001). Conclusions: In PTC patients with an N1 classification, our novel LN-related risk estimates may help predict long-term outcomes and design postoperative management and follow-up strategies. After further validation studies based on independent datasets, these risk categories might be considered when redefining risk stratification or staging systems.

Objective: Primary tumour size (PTS) is known to be a significant prognostic factor in patients with papillary thyroid carcinoma (PTC) undergoing thyroidectomy. Although cutoff values of 2 and 4 cm are widely used in surgical extent decision and long-term outcomes predictions, the effectiveness of arbitrary cutoff values in stratifying patients for target outcomes is questionable. This study aimed to determine new optimal cutoffs of PTS., Design, Patient and Measurements: Patients (n = 529) with PTC who underwent total thyroidectomy were retrospectively reviewed. After risk factor analysis for structural recurrence, the optimal cutoffs of PTS were automatically calculated using restricted cubic spline analysis and X-tile software. Subgroups were classified based on the newly determined cutoff values. Both the recurrence-free survival (RFS) and the proportion of patients in each response-to-therapy category, using the dynamic risk stratification system (DRS), were compared between the subgroups., Results: PTS was confirmed as an independent risk factor for structural recurrence. The optimal cutoff values were calculated as 1.4 and 3.0 cm. The subgroups stratified using these newly determined cutoffs showed significantly different RFS and DRS based on the response to initial therapy, but the subgroups classified using the cutoffs of 2 and 4 cm did not., Conclusions: The newly determined cutoff values of PTS may be useful in better stratifying patients with PTC undergoing thyroidectomy for target outcomes and can be considered as a new definition in staging and risk stratification systems., (© 2021 John Wiley & Sons Ltd.)

Purpose: To evaluate and compare the diagnostic outcomes of ultrasonography (US)-guided fine needle aspiration (FNA) and core needle biopsy (CNB) performed on the same thyroid nodule using a surgical specimen for direct comparison., Materials and Methods: We included 89 thyroid nodules from 88 patients from February 2015 to January 2016. The inclusion criterion was thyroid nodules measuring ≥ 20 mm (mean size: 40.0 ± 15.3 mm). Immediately after surgical resection, FNA and subsequent CNB were performed on the surgical specimen under US guidance. FNA and CNB cytopathologic results on the specimen were compared with the surgical diagnosis., Results: Among the 89 nodules, 30 were malignant and 59 were benign. Significantly higher inconclusive rates were seen in FNA for malignant than benign nodules (80.0% vs. 39.0%, p < 0.001). For CNB, conclusive and inconclusive rates did not differ between benign and malignant nodules ( p = 0.796). Higher inconclusive rates were seen for FNA among cancers regardless of US features, and in the subgroup of size ≥ 40 mm (62.5% vs. 22.9%, p = 0.028). Eleven cancers were diagnosed with CNB (36.7%, 11/30), while none was diagnosed using FNA., Conclusion: In this experimental study using surgical specimens, CNB showed a potential to provide improved diagnostic sensitivity for thyroid cancer, especially when a conclusive diagnosis is limited with FNA., Competing Interests: Conflicts of Interest: The authors have no potential conflicts of interest to disclose., (Copyrights © 2022 The Korean Society of Radiology.)

Background: Long-term management and follow-up strategies in patients with intermediate-risk papillary thyroid carcinoma (PTC) according to the American Thyroid Association (ATA) are still controversial due to the paucity of data on unique risk factors or a risk stratification system predictive of long-term outcomes., Patients and Methods: This study included 649 patients with PTC who underwent an initial surgical treatment. Retrospectively enrolled patients were categorized according to the ATA risk stratification system. Intermediate-risk patients were further categorized into subgroups by the number of ATA intermediate risk factors. The recurrence-free survival (RFS) rates of these subgroups were compared with those of low- and high-risk patient groups. Additionally, the patients were classified according to their response to the initial therapy using the dynamic risk stratification (DRS) system, and the percentages of patients in each category were compared among the subgroups., Results: The median follow-up period was 102 months. Structural recurrence occurred in 9.2% of all enrolled patients (60/649) and in 13.0% of intermediate-risk patients (40/308). Patients with two or more current intermediate risk factors had a poorer RFS than patients with only one risk factor (p < 0.001) and showed a comparable RFS to high-risk patients (p > 0.050). The percentages of patients with an excellent response category for DRS significantly decreased with an increase in the number of intermediate risk factors., Conclusion: Subclassification according to the number of intermediate risk factors may be useful to better predict the RFS and the response to initial therapy in patients with intermediate-risk PTC., (© 2022. Society of Surgical Oncology.)

Objective: The characteristics of metastatic lymph nodes (MLNs) have been investigated as important predictors of recurrence and progression in papillary thyroid cancer (PTC). However, clinically applicable risk stratification systems are limited to the assessment of size and number of MLNs. This study investigated the predictive value of detailed characteristics of MLNs in combination with currently used risk stratification systems., Design and Methods: We retrospectively characterized 2811 MLNs from 9014 harvested LNs of 286 patients with N1 PTC according to the maximum diameter of MLN (MDLN), maximum diameter of metastatic focus (MDMF), ratio of both diameters (MDMFR), lymph node ratio (LNR, number of MLNs/number of total harvested LNs), presence of extranodal extension (ENE), desmoplastic reaction (DR), cystic component, and psammoma body., Results: Factors related to the size and number of MLNs were associated with increased risk of recurrence and progression. Extensive presence of ENE (>40%) and DR (≥50%) increased the risk of recurrence/progression. The combination of MDLN, LNR, ENE, and DR had the highest predictive value among MLN characteristics. Combination of these parameters with ATA risk stratification or 1-year response to therapy improved the predictive power for recurrence/progression from a Harrell's C-index of 0.781 to 0.936 and 0.867 to 0.960, respectively., Conclusions: The combination of currently used risk stratification systems with detailed characterization of MLNs may improve the predictive accuracy for recurrence/progression in N1 PTC patients.

Transforming growth factor-β (TGF-β) plays crucial roles in the development of focal segmental glomerulosclerosis, but key molecular pathways remain unknown. Here, we identified the regulation of mammalian target of rapamycin complex1 (mTORC1) by TGF-β via ERK1/2 in the Adriamycin-induced murine model of focal segmental glomerulosclerosis. Adriamycin administration elicited early activation of TGF-β-ERK1/2-mTORC1 in podocytes, which persisted at later stages of albuminuria and glomerulosclerosis. Phosphorylation of the TGF-β receptor-I (TGF-βRI), Smad3, ERK1/2 and ribosomal protein S6 were evident in the glomeruli of adriamycin-treated mice. Targeting TGFβ-RI and mTORC1 with pharmacological inhibitors suppressed TGF-β signaling in glomeruli and significantly reduced albuminuria, glomerulosclerosis, protein levels of collagen 4α3, plasminogen activator inhibitor-1, and vimentin and restored mRNA levels of podocyte markers. Low dose US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor trametinib/GSK1120212 blunted TGF-β1-induced mTORC1 activation in podocytes, ameliorated up-regulation of TGF-β, plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, fibronectin and α-smooth muscle actin and prevented albuminuria and glomerulosclerosis with improved serum albumin. In cultured podocytes, this pathway was found to be associated with translation of fibrogenic collagen 4α3 and plasminogen activator inhibitor-1, without influencing their transcription. Notably, rapamycin suppressed upstream p-TGF-βRI, p-Smad3 and p-ERK1/2, and trametinib down-regulated upstream p-Smad3 in ex vivo and in vivo studies, indicating that harmful paracrine signaling among glomerular cells amplified the TGF-β-ERK1/2-mTORC1 axis by forming a positive feedback loop. Thus, an accentuated TGF-β-ERK1/2-mTORC1 pathway is suggested as a central upstream mediator to develop proteinuria and glomerulosclerosis. Hence, preventing activation of this vicious loop by trametinib may offer a new therapeutic strategy for glomerular disease treatment., (Copyright © 2019 International Society of Nephrology. All rights reserved.)

Background: Oxytocin (OXT) has been reported to act as a growth regulator in various tumor cells. However, there is a paucity of data on the influence of OXT on cell proliferation of corticotroph adenomas. This study aimed to examine whether OXT affects cell growth in pituitary tumor cell lines (AtT20 and GH3 cells) with a focus on corticotroph adenoma cells., Methods: Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were conducted with AtT20 cells to confirm the effects of OXT on hormonal activity; flow cytometry was used to assess changes in the cell cycle after OXT treatment. Moreover, the impact of OXT on proliferating cell nuclear antigen (PCNA), nuclear factor κB, and mitogen-activated protein kinase signaling pathway was analyzed by Western blot., Results: OXT treatment of 50 nM changed the gene expression of OXT receptor and pro-opiomelanocortin within a short time. In addition, OXT significantly reduced adrenocorticotropic hormone secretion within 1 hour. S and G2/M populations of AtT20 cells treated with OXT for 24 hours were significantly decreased compared to the control. Furthermore, OXT treatment decreased the protein levels of PCNA and phosphorylated extracellular-signal-regulated kinase (P-ERK) in AtT20 cells., Conclusion: Although the cytotoxic effect of OXT in AtT20 cells was not definite, OXT may blunt cell proliferation of corticotroph adenomas by altering the cell cycle or reducing PCNA and P-ERK levels. Further research is required to investigate the role of OXT as a potential therapeutic target in corticotroph adenomas., Competing Interests: No potential conflict of interest relevant to this article was reported., (Copyright © 2019 Korean Endocrine Society.)

Objective: Tumor location in papillary thyroid microcarcinoma (PTMC) might determine tumor outgrowth from the thyroid gland. However, the clinical implications of tumor location and minimal extrathyroid extension (mETE) have not been well elucidated. We aimed to investigate the relationship between tumor location and mETE to predict the aggressiveness of PTMC., Methods: A total of 858 patients with PTMC were grouped according to tumor location on ultrasonography: central (cPTMC) and peripheral PTMC (pPTMC). PTMC without mETE (PTMC-mETE(-)) was divided further according to margin shape: encapsulated (E-) or infiltrative (I-). To understand the molecular biologic characteristics of PTMC presenting with an I-margin and mETE, transcriptome data from TCGA-THCA were analyzed using Gene Set Enrichment Analysis (GSEA)., Results: pPTMC (n = 807, 94.1%) accounted for the majority of cases; mETE was identified only in pPTMC (403/807; 49.9%). pPTMC-mETE(+) showed aggressive clinical characteristics that increased the odds ratio (OR) for lymph node metastasis (LNM). Interestingly, subgroup analysis of PTMC-mETE(-) revealed that the I-margin also increased the OR for LNM, independent of other clinical factors. GSEA of TCGA-THCA data suggested coordinated upregulation of genes related to epithelial-mesenchymal transition (EMT) in PTC with mETE. Immunohistochemical staining for laminin subunit gamma 2 (LAMC2), CD59, E-cadherin and vimentin showed that these markers of EMT were associated with progressive changes in E-margin PTMC-mETE(-), I-margin PTMC-mETE(-) and pPTMC-mETE(+)., Conclusion: mETE related to peripheral location of PTMC is an important predictor of tumor invasiveness, as is the I-margin, which presents with EMT features similar to mETE. I-margin PTMC-mETE(-) and pPTMC-mETE(+) might reflect the pattern of invasive PTMC.

Background: Hürthle cell-rich nodules (HCNs) encompass non-neoplastic to malignant lesions. There is paucity of literature on the frequency distribution of HCNs among Bethesda categories, histologic follow-up, risk of malignancy (ROM), and risk of neoplasia (RON). The objective of this retrospective, multi-institutional study was to determine the prevalence of the cytologic diagnostic category and surgical outcomes of patients with HCN., Methods: Nine tertiary health centers representing 6 Asian countries participated. Cases were retrieved from respective databases. The Bethesda System for Reporting Thyroid Cytopathology was used. Cytology results were correlated with surgical diagnoses., Results: Of 42,190 thyroid aspirates retrieved, 760 (1.8%) had a Hürthle cell predominance. Most (61%) were categorized as atypia of undetermined significance/follicular lesion of undetermined significance, Hürthle cell type" (AUS-H); 35% were categorized as follicular neoplasm, Hürthle cell type (FN-H); and 4% were categorized as suspicious for malignancy (SFM). Histologic follow-up was available for 288 aspirates (38%). Most were benign on resection (66%), and the most common histologic diagnosis was Hürthle cell adenoma (28.5%). The ROM for AUS-H, FN-H, and SFM, as calculated on resected nodules, was 32%, 31%, and 71%, respectively; and the RON was 47%, 81%, and 77%, respectively. The 5 institutions that had an AUS-H:HCN ratio below 0.5 diagnosed HCN less frequently as AUS-H than as FN-H., Conclusions: This is the largest, contemporary, multi-institutional series of HCNs with surgical follow-up. Although there was wide interinstitutional variation in prevalence and surgical outcomes, there was no significant difference in the ROM among institutions. The categories AUS-H and FN-H had a similar ROM for resected nodules., (© 2019 American Cancer Society.)

Introduction: papillary thyroid microcarcinoma is a well-known malignant neoplasm with good prognosis. The known prognostic factors are patient's age, multifocality, and extrathyroidal extension. CD9 and CD82, members of the tetraspanin family, are expressed in numerous cancer cells and play many roles associated with the cellular process., Material and Methods: we investigated the immunohistochemical expression of CD9 and CD82 in papillary thyroid microcarcinoma and analysed the clinicopathological and prognostic significance. For the retrospective analysis, we collected the cases of 553 PTMC patients who had undergone thyroidectomy., Results: The group with lymph node metastasis showed higher immunostaining intensity for CD9 than the group without metastasis (p = 0.002). In multivariate analysis, high CD9 intensity (OR = 1.58 in 3+, p = 0.0025) correlated with lymph node metastasis., Conclusion: We suggest CD9 as a predictive prognostic factor for lymph node metastasis in PTMC.

We reviewed the current status of thyroid fine-needle aspiration cytology (FNAC) in Korea. Thyroid aspiration biopsy was first introduced in Korea in 1977. Currently, radiologists aspirate the thyroid nodule under the guidance of ultrasonography, and cytologic interpretation is only legally approved when a cytopathologist makes the diagnosis. In 2008, eight thyroid-related societies came together to form the Korean Thyroid Association. The Korean Society for Cytopathology and the endocrine pathology study group of the Korean Society for Pathologists have been updating the cytologic diagnostic guidelines. The Bethesda System for Reporting Thyroid Cytopathology was first introduced in 2009, and has been used by up to 94% of institutions by 2016. The average diagnosis rates are as follows for each category: I (12.4%), II (57.9%), III (10.4%), IV (2.9%), V (3.7%), and VI (12.7%). The malignancy rates in surgical cases are as follows for each category: I (28.7%), II (27.8%), III (50.6%), IV (52.3%), V (90.7%), and VI (100.0%). Liquid-based cytology has been used since 2010, and it was utilized by 68% of institutions in 2016. The categorization of thyroid lesions into "atypia of undetermined significance" or "follicular lesion of undetermined significance" is necessary to draw consensus in our society. Immunocytochemistry for galectin-3 and BRAF is used. Additionally, a molecular test for BRAF in thyroid FNACs is actively used. Core biopsies were performed in only 44% of institutions. Even the institutions that perform core biopsies only perform them for less than 3% of all FNACs. However, only 5% of institutions performed core biopsies up to three times more than FNAC.

Background: The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) has standardized the reporting of thyroid cytology specimens. The objective of the current study was to evaluate the nationwide usage of TBSRTC and assess the malignancy rates in each category of TBSRTC in Korea., Methods: Questionnaire surveys were used for data collection on the fine needle aspiration (FNA) of thyroid nodules at 74 institutes in 2012. The incidences and follow-up malignancy rates of each category diagnosed from January to December, 2011, in each institute were also collected and analyzed., Results: Sixty out of 74 institutes answering the surveys reported the results of thyroid FNA in accordance with TBSRTC. The average malignancy rates for resected cases in 15 institutes were as follows: nondiagnostic, 45.6%; benign, 16.5%; atypical of undetermined significance, 68.8%; suspicious for follicular neoplasm (SFN), 30.2%; suspicious for malignancy, 97.5%; malignancy, 99.7%., Conclusions: More than 80% of Korean institutes were using TBSRTC as of 2012. All malignancy rates other than the SFN and malignancy categories were higher than those reported by other countries. Therefore, the guidelines for treating patients with thyroid nodules in Korea should be revisited based on the malignancy rates reported in this study.

Background: For patients with papillary thyroid carcinoma (PTC), lymph node metastasis is associated with an increased recurrence rate. The purpose of this study was to investigate whether the vascular endothelial growth factor (VEGF), microvessel density (MVD), and vascular index (VI) can predict lymph node metastasis in patients with PTC., Methods: From January 2011 to October 2011, 202 patients with PTCs underwent preoperative staging ultrasound evaluation. To evaluate vascularity, we measured the VI, VEGF expression, and MVD., Results: The VI was significantly correlated with MVD (p = .009). On multivariate analysis, young age showed a significant correlation with lymph node metastasis (p < .001; p < .001; p < .001). However, the other clinicopathologic features, VEGF, MVD, and VI failed to show any significant correlations with lymph node metastasis., Conclusion: Although the VI showed significant correlation with MVD, it was not significantly correlated to lymph node metastasis. © 2016 Wiley Periodicals, Inc. Head Neck 39: 334-340, 2017., (© 2016 Wiley Periodicals, Inc.)

BRAF (V600E) mutation is the most commonly detected genetic alteration in thyroid cancer. Unlike its high treatment response to selective BRAF inhibitor (PLX4032) in metastatic melanoma, the treatment response in thyroid cancer is reported to be low. The purpose of this study is to investigate the resistance mechanism responsible for this low treatment response to BRAF inhibitor in order to maximize the effect of targeted therapy. We examined the expression of feedback regulation mechanisms and alterations in the upper signal transduction pathway in thyroid cancer cell lines harboring BRAF mutation. Also, we investigated the effect of dual inhibition from combinatorial therapy. Two thyroid cancer cell lines, 8505C (anaplastic thyroid cancer) and BCPAP (papillary thyroid cancer) were selected and treated with PLX4032 and its drug sensitivity were examined and compared. Further investigation on the changes in signals responsible for the different treatment response to PLX4032 was carried out and the same experiment was performed on orthotopic xenograft mouse models. Unlike BCPAP cells, 8505C cells presented drug resistance to PLX4032 treatment and this was mainly due to increased expression of c-Met. Effective inhibitions of c-Met, p-AKT, and p-ERK were achieved after dual treatment with BRAF inhibitor (PLX4032) and c-Met inhibitor (PHA665752). Similar results were confirmed by in vivo study with orthotopic xenograft mouse model. c-Met-mediated reactivation of the PI3K/AKT pathway and MAPK pathway contributes to the relative insensitivity of BRAF (V600E) mutant anaplastic thyroid cancer cells to PLX4032. Dual inhibition of BRAF and c-Met leads to sustained treatment response. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Objective: To evaluate the clinical significance and compare the imaging features according to the Thyroid Image Reporting and Data System (TIRADS) between atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) subcategories., Design & Patients: A total of 192 thyroid nodules in 188 patients (mean age: 50·2 ± 11·8 years) that had been initially diagnosed as AUS/FLUS on US-guided fine needle aspiration (US-FNA) were included. One cytopathologist retrospectively reviewed the cytology slides, subcategorizing cytology results into AUS and FLUS. A TIRADS category was assigned to each thyroid nodule according to the number of suspicious US features. Clinical, US features and malignancy rates were compared between the two subcategories., Results: Of the 192 AUS/FLUS lesions, 149 (77·6%) were subcategorized as AUS and 43 (22·4%) as FLUS. Of the 192 AUS/FLUS nodules, 82 (42·7%) were malignant. The malignancy rates between AUS and FLUS subcategories were not significantly different, 45·6% to 32·6%, respectively (P = 0·127). When applying TIRADS, significant differences were seen in TIRADS category between benign and malignant nodules in the AUS subcategory (P < 0·001), but not in the FLUS subcategory (P = 0·414). The malignancy rates in TIRADS categories 3, 4a, 4b, 4c and 5 were 15·4%, 22·2%, 33·3%, 57·1% and 80·0% (P < 0·001) in AUS nodules and 40·0%, 50·0%, 23·5%, 22·2% and 0·0% (P = 0·414) in FLUS nodules, respectively., Conclusion: Suspicious US features are useful in predicting malignancy among AUS subcategories but not in FLUS subcategories. Subcategorization into AUS and FLUS cytology may be helpful in deciding upon treatment or management of thyroid nodules., (© 2015 John Wiley & Sons Ltd.)

The objective of the study was to evaluate whether texture analysis using histogram and gray level co-occurrence matrix (GLCM) parameters can help clinicians diagnose lymphocytic thyroiditis (LT) and differentiate LT according to pathologic grade. The background thyroid pathology of 441 patients was classified into no evidence of LT, chronic LT (CLT), and Hashimoto's thyroiditis (HT). Histogram and GLCM parameters were extracted from the regions of interest on ultrasound. The diagnostic performances of the parameters for diagnosing and differentiating LT were calculated. Of the histogram and GLCM parameters, the mean on histogram had the highest Az (0.63) and VUS (0.303). As the degrees of LT increased, the mean decreased and the standard deviation and entropy increased. The mean on histogram from gray-scale ultrasound showed the best diagnostic performance as a single parameter in differentiating LT according to pathologic grade as well as in diagnosing LT., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The purpose of the study was to compare the diagnostic utility of the combined conventional smear (CS) and liquid-based preparation (LBP) compared to CS with respect to the non-diagnostic rate, the atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS) rate, and the diagnostic performances for malignancy. This retrospective study was approved by the Institutional Review Board, and the need to obtain informed consent was waived. In our institution, thyroid FNAs were processed with CS before October 2012, and LBP has been used in combination with CS after October 2012. On-site evaluation for the adequacy of CS was not performed. This study included nodules 5 mm or larger with FNAs performed by faculties: 811 nodules in 773 patients for CS, and 926 nodules in 894 patients for combined CS and LBP. Nodules with surgery or either benign or malignancy cytology on initial or repeat FNA were regarded to have the reference standards needed to calculate diagnostic performances. The proportion of Bethesda categories and diagnostic performances were compared between the two groups with z test. The non-diagnostic rate, the AUS or FLUS rate, and the diagnostic performances for malignancy were similar (All P > 0.05). The follicular neoplasm (FN) or suspicious for FN rate decreased from 1.2 to 0.3 % (P = 0.034). The benign rate increased from 51.4 to 57.0 % (P = 0.019). Combined CS and LBP decreased FN or suspicious for FN diagnoses, and increased benign diagnoses compared to CS with comparable non-diagnostic rates and AUS or FLUS rates, and comparable diagnostic performances.

Purpose: To evaluate the clinical value of additional von Kossa staining on cytology in thyroid nodules with echogenic spots on ultrasound (US)., Materials and Methods: 342 thyroid nodules were analyzed for cytology and von Kossa staining was performed to detect microcalcifications. We compared diagnostic performances and accuracies of FNA and FNA with von Kossa staining to detect malignancy. We evaluated associations of aggressive pathologic features and von Kossa positivity in the surgically confirmed papillary thyroid carcinoma (PTC) group., Results: Two hundred and thirty two (67.8%) nodules were malignant and 110 (32.2%) were benign on cytopathology. Compared to cytology alone, additional von Kossa staining slightly improved sensitivity from 89.7% to 90.9% (P=0.081). In 207 cases of surgically confirmed PTC, von Kossa positivity was an independent predictor of central lymph node metastasis in PTCs by multivariate analysis (odds ratio, 2.218; P=0.021)., Conclusions: Adding von Kossa staining to cytology improved the sensitivity of thyroid nodules with echogenic spots on US. Positive von Kossa stains in PTCs may also be useful in predicting central lymph node metastasis., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Objective: The objective of this study was to investigate differences in dynamic contrast-enhanced MRI (DCE-MRI) parameters on the basis of the status of human papillomavirus (HPV) and epidermal growth factor receptor (EGFR) biomarkers in patients with squamous cell carcinoma (SCC) of the oral cavity and oropharynx by use of histogram analysis., Materials and Methods: A total of 22 consecutive patients with oral cavity and oropharyngeal SCC underwent DCE-MRI before receiving treatment. DCE parameter maps of the volume transfer constant (K(trans)), the flux rate constant (kep), and the extravascular extracellular volume fraction (ve) were obtained. The histogram parameters were calculated using the entire enhancing tumor volume and were compared between the patient subgroups on the basis of HPV and EGFR biomarker statuses., Results: The cumulative histogram parameters of K(trans) and kep showed lower values in the HPV-negative and EFGR-overexpression group than in the HPV-positive EGFR-negative group. These differences were statistically significant for the mean (p = 0.009), 25th, 50th, and 75th percentile values of K(trans) and for the 25th percentile value of kep when correlated with HPV status in addition to the mean K(trans) value (p = 0.047) and kep value (p = 0.004) when correlated with EGFR status. No statistically significant difference in ve was found on the basis of HPV and EGFR status., Conclusion: DCE-MRI is useful for the assessment of the tumor microenvironment associated with HPV and EGFR biomarkers before treatment of patients with oral cavity and oropharyngeal SCC.

Dacomitinib, an irreversible pan-HER inhibitor, had shown modest clinical activity in squamous cell carcinoma of head and neck (SCCHN) patients. Therefore, validated predictive biomarkers are required to identify patients most likely to benefit from this therapeutic option. To characterize the genetic landscape of cisplatin-treated SCCHN genomes and identify potential predictive biomarkers for dacomitinib sensitivity, we performed whole exome sequencing on 18 cisplatin-resistant metastatic SCCHN tumors and their matched germline DNA. Platinum-based chemotherapy elevated the mutation rates of SCCHN compared to chemotherapy-naïve SCCHNs. Cisplatin-treated SCCHN genomes uniquely exhibited a novel mutational signature characterized by C:G to A:T transversions at CCR sequence contexts that may have arisen due to error-prone translesional synthesis. Somatic mutations in REV3L, the gene encoding the catalytic subunit of DNA polymerase ζ involved in translesional synthesis, are significantly enriched in a subset of patients who derived extended clinical benefit to dacomitinib (P = 0.04). Functional assays showed that loss-of-function of REV3L dramatically enhanced the sensitivity of SCCHN cells to dacomitinib by the loss of both translesion synthesis and homologous recombination pathways. Our data suggest that the 'platinum' mutational signature and inactivation of REV3L may inform treatment options in patients of recurrent SCCHN.

Purpose: The goal of this study was to validate the ultrasonography (US) and cytopathological features that are used in the diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) and to characterize the role of BRAF (V600E) mutation analysis in the diagnosis of FVPTC., Methods: From May 2012 to February 2014, 40 thyroid nodules from 40 patients (mean age, 56.2 years; range, 26 to 81 years) diagnosed with FVPTC were included in this study. The US features of the nodules were analyzed and the nodules were classified as probably benign or suspicious for malignancy. Twenty-three thyroid nodules (57.5%) underwent BRAF (V600E) mutation analysis. Clinical information and histopathologic results were obtained by reviewing the medical records of the patients., Results: Thirty nodules (75.0%) were classified as suspicious for malignancy, while 10 (25.0%) were classified as probably benign. Seven of the eight nodules (87.5%) with atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) cytology showed suspicious US features, while one of the two nodules (50.0%) with follicular neoplasm cytology presented suspicious US features. Five of the 23 nodules (21.7%) that underwent BRAF (V600E) mutation analysis had positive results, all of which were diagnosed as suspicious for malignancy or malignant based on cytology. None of the nodules with benign, AUS/FLUS, or follicular neoplasm cytology were positive for the BRAF (V600E) mutation., Conclusion: US features allow nodules to be classified as suspicious for malignancy, and the presence of suspicious US features in nodules with ambiguous cytology may aid in the diagnosis of FVPTC. BRAF (V600E) mutation analysis is of limited value in the diagnosis of FVPTC.

Purpose: Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1α (HIF-1α) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1α-induced invasive characteristics., Materials and Methods: Cells were cultured under controlled hypoxic environments (1% O₂) or normoxic conditions. The effect of hypoxia on HIF-1α, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1α and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1α, tissue analysis was done., Results: Hypoxia induces HIF-1α expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1α via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1α with RNA interference suppressed hypoxia-induced HIF-1α and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1α. These were confirmed in the orthotopic FTC model., Conclusion: Hypoxia induced HIF-1α, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.

Objectives: The purpose of this study was to investigate the level of conclusive results obtained with ultrasound (US)-guided core needle biopsy (CNB) and how this method should be applied for diagnosis of thyroid nodules., Methods: From January 2013 to November 2014, US-guided CNB was performed in 84 thyroid lesions of 83 patients. Based on CNB pathologic reports, thyroid nodules were divided into 2 categories: conclusive (nodules reaching a definite pathologic diagnosis suggesting benignity or malignancy) and inconclusive (nodules that were not able to reach a definite diagnosis because of ambiguous results). Medical records and US examinations were reviewed and compared., Results: The mean age of the 83 patients ± SD was 49.7 ± 14.1 years. Of the 84 thyroid nodules, 73 (86.9%) were diagnosed as benign or malignant and 11 (13.1%) as inconclusive by CNB pathologic analysis. Among the 11 nodules with inconclusive results, the possibility of a follicular neoplasm was suggested in 8 nodules (72.7%). No significant difference was seen in tumor size and US features when comparing the nodules with conclusive and inconclusive results (all P >.05)., Conclusions: Ultrasound-guided CNB may have supplemental roles in addition to US-guided fine-needle aspiration for diagnosis of selected cases. A considerable proportion of inconclusive results are seen on US-guided CNB, especially for diagnosis of follicular lesions, which must be considered when using this method for diagnosis of thyroid nodules., (© 2015 by the American Institute of Ultrasound in Medicine.)

Some morphologic parameters have been studied to help predict the BRAF(V600E) mutation using cytopathologic specimens, which can indicate which nodules should undergo further testing. The aim of this study was to investigate the value of cytomorphologic parameters to predict the BRAF(V600E) mutation in nodules read as "suspicious for malignancy" on cytology. This study included 142 resected nodules which were diagnosed as "suspicious for malignancy" on cytology in 142 patients. At our institution, BRAF(V600E) mutation analysis was performed at the request of the referring clinicians based on the clinical features of the patients, or the judgment of the radiologists performing US-FNA because suspicious US features were observed on the targeted nodule during this study period. Cytology smears were re-reviewed to assess the presence and amount of polygonal eosinophilic (plump) cells and microfollicles, and the presence of intranuclear pseudoinclusions, irregular nuclear membranes, nuclear grooves, sickles cells, psammoma bodies, and cystic changes. We evaluated the diagnostic performances of the cytomorphologic features to predict the BRAF(V600E) mutation. Polygonal eosinophilic (plump) cells, microfollicles, intranuclear pseudoinclusions, sickle cells, and cystic changes were significantly associated with the BRAF(V600E) mutation. The mutation was not present in all 6 thyroid nodules with microfollicles larger than 20% on cytology. Additionally, polygonal eosinophilic (plump) cells larger than 20%, cystic changes, and sickle cells on cytology had a high specificity of 95%, 96.7%, and 81.7%, respectively. Excluding 6 nodules with microfollicles larger than 20% on cytology, there were 82 (60.3%) nodules with the BRAF(V600E) mutation among the 136 nodules. Among the 136 nodules, there were 95 nodules with polygonal eosinophilic (plump) cells larger than 20%, cystic changes, or sickle cells on cytology. Of the 95 nodules, 69 (72.6%) had the mutation. Cytomorphologic features can help select nodules for the BRAF(V600E) mutation test among nodules read as "suspicious for malignancy" on cytology., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

We investigated the clinicopathological implications of leptin-signaling proteins and Epstein-Barr virus (EBV)-infection status in gastric carcinomas. Immunohistochemistry for leptin signalling-related proteins (leptin, leptin-receptor, pSTAT3, ERK, pAkt, mTOR and HIF-1 alpha), and in situ hybridization for EBV-encoded small RNAs was performed in 343 cases of gastric carcinomas. The siRNA against leptin-receptor was transfected into three stomach cancer cell lines, and western blot for caspase 3 was performed. The TNM stage was a prognostic factor in all 343 patients, and was negatively correlated with expression of leptin, pSTAT3, ERK, pAkt, mTOR and HIF-1 alpha (P < 0.05). Leptin-receptor expression was correlated with poor survival in 207 patients of the advanced gastric cancer (AGC) subgroup, 139 of the Lauren diffuse group, and in 160 patients with lymph node metastasis (P < 0.05, respectively). Additionally, in stomach cancer cells, cleaved caspase 3 level increased by leptin-receptor inhibition, that is, apoptosis increased. Interestingly, EBV-positive AGC (n = 29) tended to show better survival of patients than EBV-negative AGC (n = 178) (P = 0.06). pAkt expression was related with a good survival of 32 patients (9%) in the EBV-positive subgroup, but was not an independent prognostic factor. Among, leptin signaling-related proteins, expressions of leptin-receptor and mTOR were different between EBV-positive subgroup and EBV-negative subgroup (P < 0.05, respectively). In conclusion, leptin-signaling proteins and EBV status show different significance on patient survival, according to subsets of gastric carcinomas. The leptin-receptor may predict poor patient prognosis in the AGC, Lauren diffuse and lymph node metastasis subgroups, while EBV-positive status can show a good prognosis in the AGC. Each leptin signaling-related protein may be differently involved in carcinogenesis of EBV-negative and EBV-positive subsets.

The object of this study is to evaluate the additional role of RAS mutation in detecting thyroid malignancy among BRAF mutation-negative nodules diagnosed as atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) on cytology.From December 2009 to December 2011, 202 BRAF mutation-negative thyroid nodules diagnosed as AUS/FLUS cytology in 201 patients were included in this study. RAS mutation analysis was performed using residual material from ultrasonography-guided fine needle aspiration (US-FNA) cytology testing for K-RAS, N-RAS, and H-RAS codons 12/13 and 61 point mutations. The authors evaluated the association between RAS mutation status and cytopathologic characteristics.Of the 202 BRAF mutation-negative thyroid nodules with AUS/FLUS cytology, 4 were considered insufficient for mutation analysis. Of the 198 thyroid nodules, 148 (74.7%) were confirmed as benign and 50 (25.3%) as malignant. Thirty-one (15.7%) of the 198 thyroid nodules were positive for any RAS mutation, 4 positive for K-RAS 12/13, 26 for N-RAS 61, and 1 positive for H-RAS 61. Seven (22.6%) of the RAS mutation positive nodules were malignant, 1 with K-RAS 12/13, 6 with N-RAS 61. Twenty-four (77.4%) of the 31 nodules positive for K-RAS 12/13 (N = 3), N-RAS 61 (N = 20), or H-RAS 61 (N = 1) mutations were proven benign. None of the 198 thyroid nodules were positive for K-RAS 61, N-RAS 12/13, or H-RAS 12/13 mutations.N-RAS 61 mutation is the most common mutation detected among BRAF mutation-negative nodules with AUS/FLUS cytology. RAS mutation has limited value in predicting malignancy among BRAF mutation-negative thyroid nodules with AUS/FLUS cytology and further, investigation is anticipated to evaluate the true role of RAS mutation in thyroid malignancy.

Kinase suppressor of RAS1 (KSR1) is a scaffold protein implicated in RAS-mediated RAF activation. However, the molecular function of KSR in papillary thyroid cancer (PTC) is unknown. Thus, this study aimed to characterize the role of KSR1 in patients with PTC. qRT-PCR and immunohistochemistry (IHC) revealed inter-tumor heterogeneities in the expression of KSR1 in PTC tissues. Interestingly, BRAFV600E-positive PTC showed higher KSR1 mRNA expression than BRAFV600E-negative PTC (P<0.001). Gene Set Enrichment Analysis (GSEA) using public repositories showed that high KSR1 expression coordinately upregulated Notch signaling (nominal P=0.019, false discovery rate (FDR) q-value=0.165); this finding was supported by GeneNetwork analysis, indicating that KSR1 expression is positively correlated with NOTCH1 expression (ρ=0.677, P=6.15×10(-9)). siRNA against KSR1 (siKSR1) significantly decreased ERK phosphorylation induced by BRAFV600E, resulting in reduced expression of NOTCH1 and HES1, targets of Notch signaling. GSEA revealed that high KSR1 expression was also associated with downregulation of genes related to oxidative phosphorylation (OxPhos). Consistent with this, electron microscopy showed that PTCs with high KSR1 expression exhibited structural defects of the mitochondrial cristae. Furthermore, siKSR1-transfected BCPAP and 8505C cells generated fewer colonies in colony-forming assays. In addition, GSEA showed that high expression of KSR2 and connector enhancer of KSR1 (CNKSR1) also coordinately upregulated Notch signaling (KSR2: nominal P=0.0097, FDR q-value=0.154 and CNKSR1: nominal P<0.0001, FDR q-value=0.00554), and high CNKSR2 was associated with downregulation of the OxPhos gene set (nominal P<0.0001, FDR q-value <0.0001). In conclusion, KSR1 is coordinately regulated with Notch signaling and OxPhos in PTC, because its scaffold function might be required to sustain the proliferative signaling and metabolic remodeling associated with this type of cancer., (© 2015 Society for Endocrinology.)

Purpose: The goals of this study were to investigate the clinical activity, safety, and biomarkers of dacomitinib, an irreversible tyrosine kinase inhibitor of EGFR, HER2, and HER4, in recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M-SCCHN)., Experimental Design: Patients were eligible if the diseases were not amenable to curative treatment and had progressed on platinum-based chemotherapy, and were treated with dacomitinib 45 mg/day. The primary endpoint was objective response rate by RECISTv1.1. Exploratory analysis included the characterization of somatic mutation, gene copy number, gene expression, p16(INK4A) expression by IHC, and investigation of their relationship with clinical outcomes., Results: Forty-eight patients were evaluable for efficacy and toxicity. Ten patients (20.8%) had partial responses and 31 patients (65%) had stable diseases. The median progression-free survival (PFS) and overall survival (OS) were 3.9 months [95% confidence interval (CI), 2.9-5.0] and 6.6 months (95% CI, 5.4-10.3). Adverse events were mostly grade 1-2. Mutations in the PI3K pathway (PIK3CA, PTEN) and high expression of inflammatory cytokines (IL6, IL8, IL1A, IL1B, IL4, and TNF) were significantly associated with shorter PFS (2.9 vs. 4.9 months without mutations, P = 0.013; 2.8 vs. 9.9 months with low expression, P = 0.004). Those harboring PI3K pathway mutations or high inflammatory cytokine expression had shorter median OS (6.1 vs. 12.5 months lacking PI3K pathway mutations and with low inflammatory cytokine expression, P = 0.005)., Conclusions: Dacomitinib demonstrated clinical efficacy with manageable toxicity in platinum-failed R/M-SCCHN patients. Screening of PI3K pathway mutation and inflammatory cytokine expression may help identify which R/M-SCCHN patients are likely to gain benefit from dacomitinib., (©2014 American Association for Cancer Research.)

A decision to perform fine needle aspiration (FNA) on thyroid nodules mainly depends on sonographic features. We investigated if lymph node metastasis (LNM) risk differed by tumor size of thyroid cancers without suspicious sonographic features. Three hundred sixty patients with thyroid cancers with benign looking sonographic features were grouped by nodule size on ultrasonography (US) (≤ or >1 cm). The clinicopathologic parameters were compared between the groups. A multivariate analysis was performed to discover the independent factors predicting the presence of LNM. The nodules greater than 10 mm on US (n = 157) demonstrated a larger tumor size on histology (17.9 ± 14.5 vs. 5.6 ± 2.4 mm, P < 0.001), a lower frequency of classical papillary thyroid carcinoma (PTC) (58.6 vs. 87.2 %, P < 0.001), and a higher frequency of follicular variant PTC and follicular thyroid carcinoma (19.7 and 17.8 % vs. 9.4 and 1.5 %, respectively, P < 0.01). In subgroup analysis of 269 patients with classical PTC, the larger nodule size on US was associated with a higher prevalence of LNM (28.3 vs. 14.7 %, P = 0.007). A multivariate analysis revealed that classical PTC, extrathyroidal extension, and the US nodule size >10 mm were independent predictive factors of LNM after adjusting for age, sex, TSH level, and multifocality. Thyroid cancers larger than 10 mm with benign US features are more likely to be nonclassical PTC than those with smaller diameters. The larger ones also have an increased risk of LNM in classical PTC. These cases require a more aggressive approach to FNA.

Background: The aim of this study was to evaluate the diagnostic utility of PAI at detecting thyroid microcalcifications at 700 nm laser wavelengths., Methods: This study included 36 resected samples in 18 patients. To evaluate the PA manifestation of microcalcifications in PAI, gray level histogram and co-occurrence matrix (COM) texture parameters were extracted from the 3 fixed ROI US and PA images, respectively, per sample. We compared the textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without microcalcifications., Results: On specimen US, the mean value (2748.4 ± 862.5) of samples with microcalcifications on specimen radiography was higher than that (1961.9 ± 780.2) of those without microcalcifications (P = 0.007). However, there were no significant differences in textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without when applying both the mean value of the three slices of thyroid specimens and the value of the thyroid specimen slice which had the highest value of the mean values in specimen US., Conclusion: PAI did not show significant PA contrast on thyroid microcalcifications indicating that the experimental setup and protocols should be enhanced, e.g., method of complete blood rejection from ex vivo specimens, the multi-wavelength spectroscopic PA imaging method which can solely extract the PA signal from microcalcifications even with high spectral interferences, or PA imaging with narrower slice thickness using 2-dimensional array transducer, etc.

Although a hyperplastic polyp (HP) shares morphological and molecular features with a sessile serrated adenoma/polyp (SSA/P), HPs and SSA/Ps are considered nonneoplastic and neoplastic epithelial polyps, respectively. Because HPs and SSA/Ps cover the morphological spectrum, we hypothesized that an intermediate serrated polyp (ISP) might exist between an HP and an SSA/P in terms of both morphological and molecular aspects. An ISP was defined as a serrated lesion that carries distorted crypts (columnar crypt dilation, irregularly branching crypts, or horizontally arranged basal crypts) in less than 3 consecutive crypts. We analyzed HPs (microvesicular, n = 16, and goblet cell-rich, n = 28), ISPs (n = 44), and SSA/Ps (n = 26) for their methylation status of 8 CpG island methylator phenotype panel markers and mutation status of KRAS/BRAF. The number of methylated markers and BRAF mutation frequency increased in the order of HP, ISP, and SSA/P. Microvesicular HPs and goblet cell-rich HPs are distinct from each other, based on the high frequency of BRAF and KRAS mutation, respectively, but are not different in the number of methylated panel markers. Proximally located microvesicular HPs and ISPs were higher in the number of methylated markers but lower in the frequency of BRAF mutation than distally located ones. However, SSA/Ps did not show any difference in the number of methylated markers and the frequency of BRAF mutation between proximally and distally located lesions. Our findings that serrated polyps, intermediate between HPs and SSA/Ps in terms of morphological features, display molecular alterations intermediate between those of HPs and SSA/Ps suggest the presence of ISPs between HPs and SSA/Ps., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study reports a case of anaplastic transformation from a well-differentiated thyroid carcinoma in a young patient. The first recurrent tissue contained poorly differentiated foci that revealed lower thyroglobulin, thyroid transcription factor 1 (TTF-1), and galectin-3 expression than the well-differentiated area. However there was no increased p53 or Ki-67 expression in the poorly differentiated foci, nor in the well-differentiated area. The tissue subsequently relapsed and revealed only anaplastic features, complete loss of thyroglobulin, TTF-1, and galectin-3 expression and revealed an increase in p53 and Ki-67 expression. The BRAF V600E and BRAF V600V mutation were found in the initially diagnosed papillary thyroid carcinoma and the poorly differentiated foci of the recurring papillary thyroid carcinoma; however, only the BRAF V600V mutation was found in the anaplastic carcinoma. These results suggest that overexpression of p53 and Ki-67 contributed to the anaplastic transformation. We also found that the BRAF type changed during the tumor relapse.

CD9 and CD82, members of the tetraspanin family, act as metastasis suppressors in many human malignant tumors, but the role of these molecules is not well known in clear cell renal cell carcinoma (CCRCC). This study was designed to evaluate the immunohistochemical expression of CD9 and CD82 in 644 cases of CCRCC and to determine the clinicopathologic and prognostic significance of their expression. The percentage of positive tumor cells was evaluated, and the expression was classified into 2 categories: low expression (less than 10% positive cells) or high expression (more than 10% positive cells) for CD9 expression and negative (no positive cells) or positive for CD82 expression. CD9 high expression was found in 303 (47.0%) patients, and CD82 positivity was found in 98 (15.2%) patients. High expression of CD9 was statistically associated with older patients (p=0.003). The cases showing positive immunoreactivity for CD82 exhibited a high stage (p<0.001) and high nuclear grade (p<0.001). The overall, cancer-specific and progression-free survival rates were significantly higher in patients with a CD82-negative profile compared to patients with a CD82-positive profile (p<0.001). Although the biological function of CD82 in CCRCC remains unclear, the CCRCC patients with CD82 positive expression show poor prognosis., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Focal CpG island hypermethylation and diffuse genomic hypomethylation signify the changes in the DNA methylation status in cancer cells. ALU and LINE-1 repetitive DNA elements comprise ~28% of the human genome. PCR-based measurements of these repetitive DNA elements can be used as a surrogate marker of the genomewide methylation content. Our study aimed to identify the timing of ALU and LINE-1 hypomethylations during multistep gastric carcinogenesis and their prognostic implications in gastric cancer (GC). In our study, we analyzed the methylation statuses of ALU and LINE-1 in 249 cases of gastric biopsy samples and another independent set of 198 cases of advanced GC by pyrosequencing. Regardless of the Helicobacter pylori infection status, a significant decrease in the ALU methylation levels was noted during the transitions from chronic gastritis to intestinal metaplasia and from gastric adenoma to GC. LINE-1 methylation decreased during the transition from intestinal metaplasia to gastric adenoma and no further decrease occurred during the transition from gastric adenoma to GC. A low LINE-1 methylation status was strongly associated with poor prognosis in GC. A multivariate analysis revealed that LINE-1 methylation status was an independent prognostic factor. Our findings suggest that ALU and LINE-1 hypomethylations are early events during multistep gastric carcinogenesis. Furthermore, the LINE-1 methylation status can be used as a molecular biomarker to define a subset of GC patients with poor prognosis., (Copyright © 2011 UICC.)

Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate the promoter CpG island methylation of 15 genes in relation to breast cancer subtype, and to investigate whether the patterns of CpG island methylation in each subtype are associated with their cancer stem cell phenotype represented by CD44+/CD24- and ALDH1 expression. We performed MethyLight analysis of the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochemistry in 36 luminal A, 33 luminal B, 30 luminal-HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer. The number of CpG island loci methylated differed significantly between subtypes, and was highest in the luminal-HER2 subtype and lowest in the basal-like subtype. Methylation frequencies and levels in 12 of the 15 genes differed significantly between subtypes, and the basal-like subtype had significantly lower methylation frequencies and levels in nine of the genes than the other subtypes. CD44+/CD24- and ALDH1+ putative stem cell populations were most enriched in the basal-like subtype. Methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors than in CD44+/CD24-cell (-) tumors, even within the basal-like subtype. ALDH1 (+) tumors were also less methylated than ALDH1 (-) tumors. Our findings showed that promoter CpG island methylation was different in relation to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have distinct patterns of CpG island methylation according to molecular subtypes and these are associated with different stem cell phenotypes of the tumor.

This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions [flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS)] to invasive ductal carcinoma (IDC). We performed MethyLight analysis for the methylation status of 57 promoter CpG island loci in 20 IDCs and their paired normal breast tissues. After selecting 15 CpG island loci showing breast cancer-specific DNA methylation, another set of normal breast tissue (n = 10), ADH/FEA (n = 30), DCIS (n = 35), and IDC (n = 30) of the breast were analyzed for these loci. We found six new methylation markers of breast cancer, namely DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, and TMEFF2, in addition to APC, GSTP1, HOXA10, IGF2, RARB, RASSF1A, RUNX3, SCGB3A1 (HIN-1), and SFRP1. The number of methylated genes increased stepwise from normal breast to ADH/FEA and DCIS, while IDC did not differ from DCIS. Methylation levels and frequencies of APC, DLEC1, HOXA1, and RASSF1A promoter CpG islands were significantly higher in ADH/FEA than in normal breast tissue. GRIN2B, GSTP1, HOXA1, RARB, RUNX3, SFRP1, and TMEFF2 showed higher methylation levels and frequencies in DCIS than in ADH/FEA. DICS and IDC did not differ in the methylation levels or frequencies for most CpG island loci except SFRP1 and HOXA10. Our findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and was similar in IDC and DCIS, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression.

Ex-adenoma carcinoma (EAC) is a carcinoma with contiguous adenoma element in its vicinity which provides a morphological evidence for adenoma-carcinoma sequence. During multistep colorectal carcinogenesis, promoter CpG island hypermethylation has been known to increase in a stepwise manner whereas diffuse genomic hypomethylation has been known to be an early event and not progress. However, some controversies exist. EAC is a good model to study the timing of hypermethylation and hypomethylation changes during multistep carcinogenesis, which this study aimed to elucidate. We analyzed 39 cases of EAC for their methylation status in eight DNA methylation markers of CpG island methylator phenotype (CIMP) panel, ten CIMP-nonrelated, cancer-specific markers, and three repetitive DNA elements (ALU, LINE-1, and SAT2) using MethyLight assay or combined bisulfite restriction analysis. Twenty-two cases of cancers had contiguous tubulovillous adenomas and 17 cases had contiguous tubular adenomas. Regardless of CIMP markers or nonrelated markers, a significant increase in the number of methylated genes was found from normal mucosa to adenoma, whereas no increase was found from adenoma to carcinoma. Both ALU and LINE-1 showed a significant decrease of methylation levels from normal mucosa to adenoma (p < 0.05), but there is no difference between adenoma and cancer. However, SAT2 methylation level exhibited a stepwise decrease from normal mucosa to adenoma to cancer. Our findings suggest that morphological progression from traditional adenoma to carcinoma does not appear to be accompanied by increases in promoter CpG island hypermethylation or repetitive DNA hypomethylation, except for SAT2 hypomethylation which showed continuous progression during multistep carcinogenesis.

Gastric carcinoma (GC) is one of the human cancers in which promoter CpG island hypermethylation is frequently found. CpG island methylator phenotype (CIMP) refers to a subset of GCs which harbor concordant methylation of multiple promoter CpG island loci. However, little is known regarding clinicopathological features of CIMP-positive (CIMP-high) GC. Our study aimed to characterize clinicopathological features of CIMP-high GC. We analyzed 196 cases of GCs for their methylation status in 16 cancer-specific CpG island loci using MethyLight assay and arbitrarily defined CIMP-high GC as those with methylation at 13 or more CpG island loci. With exclusion of microsatellite instability-positive GC and EBV-positive GC from the analysis, CIMP-high GC (n = 10, 6.7%) demonstrated tendency toward higher cancer stage, infiltrative growth type, poor differentiation, and diffuse or mixed type of Lauren classification. CIMP-high GC showed significantly shortened survival compared with that of CIMP-negative GC. When CIMP-negative GC (methylation at 12 or less) was divided into CIMP-intermediate and CIMP-low (methylation at one or none), CIMP-low exhibited better clinical outcome than CIMP-intermediate. Hypermethylation at 14 CpG island loci or more was closely associated with poor clinical outcome and found to be an independent prognostic factor. Our findings that CIMP-high GCs were featured with characteristic clinicopathological parameters, including poor prognosis are distinct from previous studies. More extensive, large-scaled study is necessary to validate the findings of the present study.

CpG island methylator phenotype (CIMP) refers to a subset of colorectal cancers (CRCs) that are characterized by concordant hypermethylation of multiple CpG island loci. CIMP+ CRCs have peculiar clinicopathological features. However, controversy exists over prognostic implications of CIMP in CRCs. We analyzed 320 cases of CRCs for their CIMP status using the MethyLight assay and determined clinicopathological features and prognostic implications of CIMP alone or in combination with microsatellite instability (MSI). With methylation of five or more markers among eight markers examined, CIMP+ tumors were significantly associated with female gender, proximal tumor location, poor differentiation, nodal metastasis, more advanced cancer, BRAF mutations, MSI, and poor prognosis (all P values <0.05). Ogino's combined eight-marker panel outperformed the Ogino and the Laird five-marker panels in detecting these features. Of the four molecular subtypes generated by the combination of CIMP and MSI status, the CIMP+/MSI- subtype showed the worst clinical outcome (P = 0.0003). However, poor prognosis of CIMP+/MSI- subtype was found to be attributed to BRAF mutation. In conclusion, the CIMP+/MSI- subtype tends to present with distinct clinicopathological and molecular features and shows the worst clinical outcome among the four molecular subtypes of CRCs.

Biliary intraepithelial neoplasia (BilIN) is the premalignant lesion of extrahepatic cholangiocarcinoma (EHC), and there are no published data regarding epigenetic changes throughout disease progression from normal biliary epithelia to BilIN to EHC. The objective of this study was to identify the occurrence of CpG island hypermethylation and repetitive DNA hypomethylation in BilIN. A total of 50 EHCs, 31 BilINs, and 31 normal cystic duct samples were analyzed for their methylation status in seven genes and two repetitive DNA elements. The number of methylated genes increased with disease progression (normal bile duct, 0.6; BilIN, 2.0; EHC, 3.6; P < 0.001). The methylation level of examined genes was significantly higher in BilIN than in normal samples (TMEFF2, HOXA1, NEUROG1, and RUNX3, P < 0.05) and in EHC than in BilIN samples (TMEFF2, HOXA1, NEUROG1, RUNX3, RASSF1A, and APC, P < 0.05). Long interspersed nucleotide element-1 (LINE-1) and juxtacentromeric satellite 2 (SAT2) methylation levels were markedly lower in EHC than in normal duct and BilIN samples, and BilIN samples showed a decrease of SAT2 methylation levels but no decrease of LINE-1 methylation levels compared to normal samples. These findings suggest that most of cancer-specific CpG island hypermethylation occur in the stage of BilIN and that CpG island hypermethylation seems to occur earlier than repetitive DNA element hypomethylation.