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Objective 4-1BB is a costimulatory immune-activating molecule. Increased amounts of this protein have previously been found in the plasma of patients with oropharyngeal and oral cancer. Here, we focused on this molecule that functions as part of the immune system. We investigated 4-1BB in the peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) of patients with head and neck squamous cell cancer (HNSCC). Materials and Methods The expression level of 4-1BB in the PBMCs was determined using real-time polymerase chain reaction (PCR). The TIMER (Tumor Immune Estimation Resource) web server was utilized to approximate the 4-1BB level in HNSCC TILs. Moreover, 4-1BB immunohistochemistry (IHC) was used to validate TILs in four organs of HNSCC, including oral cancer (OC), oropharyngeal cancer (OPC), sinonasal cancer (SNC), and laryngeal cancer (LC), in both the tumor area and adjacent normal epithelium. The difference in 4-1BB expression levels in various groups was assessed using a Kruskal-Wallis test and an independent sample t-test. Results The level of 4-1BB expression in PBMCs was highest in OPC, followed by OC and healthy controls (HC). Significant differences were discovered between HC and OPC and between OC and OPC. Bioinformatics revealed a substantial correlation between 4-1BB expression level and lymphocyte infiltration in HNSCC, including B cells, CD8+ T cells, and CD4+ T cells. IHC validation in HNSCC tissue revealed that the average number of 4-1BB positive TILs in all four HNSCC subtypes was considerably greater than the number of lymphocytes seen in adjacent normal tissue. Interestingly, the number of lymphocytes that were 4-1BB positive increased in relation to the TIL level. Conclusion A higher number of 4-1BB expression levels were found in the PBMCs and TILs of HNSCC patients, implying that 4-1BB may be a promising approach for HNSCC patients to improve their immune function. It is important to study and create a treatment that uses 4-1BB medicine as well as existing drugs.

Objective: The Azzopardi phenomenon, known as the deoxyribonucleic acid deposition on various structures due to cellular necrosis, has never been reported in non-neoplastic eyes. Methods: We report a case of a 48-year-old man who had congenital nystagmus with poor vision in both eyes, presented with decreased vision and photophobia in his left eye. An exudative retinal detachment was found, which did not respond to systemic steroid treatment. Glaucoma due to occlusio pupillae was later developed. Laser iridotomy and anti-glaucoma medications decreased intraocular pressure to an acceptable level. Vision in the left eye gradually deteriorated during the 10-year clinical course. Evisceration was finally performed due to persistent dull aching ocular pain along with signs of ocular hypotony Results: Histopathological examination showed phthisis bulbi and focal nodular retinal gliosis. The Azzopardi phenomenon was found at the retinal vessel walls, within the retinal layers and along the internal limiting membrane. There was neither evidence of intraocular tumors nor foreign bodies. Conclusion: This case demonstrated that the Azzopardi phenomenon could be present in a non-neoplastic eye with a longstanding disease that proceeds to phthisis bulbi.

Background Assessing nuclear features is diagnostically challenging in the aspect of thyroid pathology. The aim of this study was to determine whether pathologists could distinguish BRAF-like and RAS-like nuclear features morphologically and identify morphological features to differentiate thyroid tumors with RAS-like mutations from encapsulated papillary thyroid carcinoma (PTC) with predominant follicular growth and BRAFV600E mutation. Methods Representative whole slide images of 16 encapsulated thyroid tumors with predominant follicular growth were reviewed by 12 thyroid pathologists using a web browser-based image viewer. Total nuclear score was calculated from semi-quantitatively scored eight nuclear features. The molecular profile of RAS and BRAF genes was determined by Sanger sequencing. Results Total nuclear score ranging 0 to 24 could differentiate BRAF-like tumors from RAS-like tumors with a cut-off value of score 14. The interobserver agreement was the highest for the assessment of nuclear pseudoinclusions (NPIs) but the lowest for nuclear elongation and sickle-shaped nuclei. NPIs were found in tumors with BRAFV600E mutation, but not in tumors with RAS-like mutations. Total nuclear scores were significantly higher for tumors with BRAFV600E than for those with RAS-like mutations (Pl0.001). Conclusion Our results suggest that NPIs and high nuclear scores have diagnostic utility as rule-in markers for differentiating PTC with BRAFV600E mutation from benign or borderline follicular tumors with RAS-like mutations. Relaxation of rigid criteria for nuclear features resulted in an overdiagnosis of PTC. Immunostaining or molecular testing for BRAFV600E mutation is a useful adjunct for cases with high nuclear scores to identify true PTC.

Object Papillary thyroid cancer (PTC) has an excellent prognosis. However, patients with such, if refract to radioiodine treatment, increase recurrent and mortality rates. Tumor aggressiveness in primary tumor of PTC expresses CXCR4 chemokine receptor. Thus, CXCR4 expression of the tumor may predict response to radioiodine treatment. Materials and methods Retrospective review of seventy-four PTC patients, treated with total/near-total thyroidectomy and radioiodine treatment at King Chulalongkorn Memorial Hospital from January 2007 to 2013, were classified as non-radioiodine-refractory (non-RAIR) or RAIR treatment response. All histopathologic diagnoses were reviewed and paraffin blocks were retrieved for CXCR4 immunostaining, determined by automated digital imaging analysis for intensity and extension. The scores were compared between primary tumour and adjacent normal thyroid tissue as well as between the tissue of non-RAIR and that of RAIR. Factors determining type of RAI response were analyzed. Results CXCR4 immunostaining scores of PTC is significantly higher than normal thyroid [2.03 (0.52) and 1.48 (0.75)] [mean (SD)] (P = 0.0001). CXCR4 immunostaining scores in RAIR are potentially higher than non-RAIR [1.95 (0.54) and 2.13 (0.47) (P = 0.149)]. Odds ratio of CXCR4 immunostaining score for predicting RAIR treatment is 1.99 (P = 0.150). CXCR4 immunostaining scores positively associate with tumor size (R = 0.298, P = 0.01); whereas no significant association with other clinicopathologic factors. Conclusion Our data support the notion that CXCR4 are significantly expressed in PTC tumor over normal thyroid tissues. However, there is no clinical association with radioiodine treatment response.

Thyroid gland involvement of Langerhans cell histiocytosis (LCH) is extremely rare in both systemic and isolated disease. The role of viral infection in LCH development is not yet fully understood. Although several viruses are proposed as etiologic factors, such as Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6), they seem to play a bystander role in LCH. A 29-year old female patient with a prior history of multisystemic LCH (pituitary gland and skull bone), presented with a thyroid nodule. The patient underwent a total thyroidectomy and the histological examination revealed nodular lesions composed of sheets and clusters of histiocytes in the inflammatory background. The histiocytes stained positive for S-100 and CD1a and were negative for HHV-8, cytomegalovirus, and VE1 (anti-BRAF(V600E)) on immunohistochemistry. The EBER in situ hybridization for EBV showed frequent positive-stained cells. The conventional PCR analysis for EBV was positive and qPCR analysis confirmed a significant DNA copy number difference (p = 0.02) between the tumor and adjacent non-neoplastic thyroid tissue. PCR analysis for HHV-6, HPV, HSV was negative in both tumor and benign samples. In conclusion, the presented case showed a rare thyroid involvement by LCH associated with EBV infection, which has not been reported before. Further studies are required to investigate a possible etiologic link between EBV infection and LCH.

Background Papillary thyroid carcinoma (PTC) accounts for the majority of diagnoses of thyroid carcinoma. BRAFV600E mutation is the most common genetic alteration in PTC, which has diagnostic and prognostic significance. The rate of BRAFV600E mutation in PTC from Thailand has not been reported. Our purpose was to estimate the prevalence of BRAF mutation in a large institutional series using an affordable approach, which combined mutation-specific immunohistochemistry (IHC) with VE1 antibody and tissue microarray (TMA). Methods A total of 476 PTC cases plotted on TMA were employed for determining the mutation status in this study. The cancer tissue of initial 100 cases (pilot study) were analyzed for BRAFV600E mutation by using both direct sequencing and VE1 immunostaining. For the subsequent PTC cases, VE1 IHC was used as an alternative to direct sequencing for the detection of mutation. Univariate and multivariate analyses were done to determine the association of clinicopathological variables with BRAFV600E mutation. Results In the pilot study, VE1 IHC showed excellent analytical performance (κ=0.884) for detecting BRAFV600E mutation in PTC TMA as compared to direct sequencing. The prevalence of BRAFV600E in the whole cohort was 60.9% by using VE1 IHC. The mutation was commonly seen in tall cell (92.9%) and classic (70.2%) variants of PTC. Multivariate analysis (P

Background: Various molecular biomarkers, including epidermal growth factor receptor (EGFR), have gained importance as predictors of head and neck cancer disease progression.Aims: This study aimed ...

We present the conceptual study investigated the capacity of minichromosome maintenance-2 (MCM-2), Ki-67, and epidermal growth factor receptor (EGFR) to assess the severity and progression of laryngeal squamous cell carcinoma (LSCC) disease and to study the correlations among these markers. A total of 30 patients with LSCC with immunohistochemistry (IHC) staining for MCM-2, Ki-67 and EGFR were examined. Mean expression levels of the three markers were evaluated for comparing between early and advanced stages of LSCC. The mean MCM-2, Ki-67, and EGFR expression levels were significantly decreased in advanced-stage compared with early-stage LSCC. Pearson correlation analysis showed a statistically significant correlation between the MCM-2 and Ki-67. Regarding subgroup analyses, MCM-2, Ki-67, and EGFR showed significant differences between early- and advanced-stage LSCC with non-recurrence, while for the recurrent subgroup LSCC, only MCM-2 revealed a significant difference between early- and advanced-stage LSCC. Altogether, these results support the role for downregulation of MCM-2, Ki-67 and EGFR in advanced-stage LSCC and correlation of MCM-2 and Ki-67 expressions that would be a promising strategy to predict prognosis of LSCC including severity and progression. We contextualize our findings and advocate the position of the biological markers, especially MCM-2, as an emerging evaluation tool for LSCC disease.

Background There has been a sharp rise in the incidence of human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) in many countries. Patients with HPV-positive OPSCC have a more favorable prognosis compared with HPV-negative OPSCC, leading to investigation and adoption of de-escalation treatment protocols. The baseline rate of HPV prevalence in certain populations is of epidemiologic significance. We aimed to evaluate the rate of high-risk HPV in a large cohort of Thai patients, including OPSCC, oral SCC (OSCC) and laryngeal SCC (LSCC). Methods In total, 504 patients with HN cancer (110 OPSCC, 260 OSCC and 134 LSCC) who had been treated in Chulalongkorn University between 2010 and 2016 formed the sample set. All histological slides were reviewed to validate the diagnosis and render the histological type as keratinizing (K), non-keratinizing (NK) or non-keratinizing with maturation (NK-M). Immunohistochemistry with p16 was performed in all cases and scored semiquantatively. Positive and equivocal cases were tested by the high-risk HPV DNA in situ hybridization (ISH). Validation with quantitative polymerase-chain reaction (qPCR) was performed in p16-positive OPSCC. Results The OPSCC were represented by NK (7.3%), NK-M (16.4%) and K (76.4%) types, with an HPV incidence of 100, 22.2 and 4.7%, respectively. The average HPV prevalence in OPSCC was 14.5%. The concordance with p16/ISH was 51.6%, while concordance of the NK morphology with positive HPV ISH was 100%. ISH-qPCR concordance in p16-positive OPSCC was 72.7%. Patients with HPV-positive OPSCC had significantly more tumors with a NK histologic type, tonsillar location, earlier clinical stage, less association with smoking, and, finally, better outcome and longer survival time. In non-OPSCC, p16-positive HPV-associated cancers were found in only 1.5% of OSCC (4/260) and LSCC (2/134). Conclusion A low rate of HPV-related OPSCC was found in Thai patients. The NK morphology was an excellent predictor of high-risk HPV infection in OPSCC. For OPSCC patients, HPV-positive ones had a significantly longer survival time than HPV-negative ones. There was a lack of p16-positive HPV-related OSCC and LSCC. Morphology and p16 status had a poor predictive value for detecting HPV in OSCC and LSCC.

Immunophenotype of thyroglossal duct (TGD) cysts, including lining epithelium and thyroid remnants, is scarcely addressed in the literature. There is indirect evidence that C cells may be derived from progenitor cells of the midline thyroid primordium. This is supported by the recent concept of the endodermal origin of lateral thyroid anlagen and several case reports. We aimed to search for neuroendocrine cells in TGD cysts and to characterize immunophenotype of the thyroid follicles and epithelial lining of TGD. Out of 98 TGD cysts, 70% contained both cyst-lining epithelium and thyroid follicles, whereas 30% possessed only cyst-lining epithelium. Specimens eligible for immunohistochemistry (n = 61) were stained for thyroid-specific and neuroendocrine markers. Thyroid remnants were positive for thyroid transcription factor 1 (TTF-1) and other thyroid tissue-specific markers and negative for calcitonin. TGD epithelium showed strong p63 positivity. We found that respiratory epithelium in 9.8% of TGDs contained neuroendocrine cells positive for calcitonin, chromogranin A, and synaptophysin but negative for carcinoembryonic antigen. In 44.2% of the cases, we detected compact buds, microscopic structures appearing as nests of epithelial cells with a biphasic population of basal (p63+) and central (TTF-1+) cells. Thyroid remnants in TGD expressed full spectrum of thyroid-specific markers and contained no C cells. Instead, calcitonin-expressing neuroendocrine cells were found among the respiratory epithelium of TGD. These cells can be a potential source of neuroendocrine tumors mimicking medullary carcinoma in median anlage derivatives. We also discovered precursor compact buds with dual immunophenotype and proposed a concept of their morphogenesis.

BACKGROUND: Papillary thyroid carcinoma (PTC) accounts for the majority of diagnoses of thyroid carcinoma. BRAF(V600E) mutation is the most common genetic alteration in PTC, which has diagnostic and prognostic significance. The rate of BRAF(V600E) mutation in PTC from Thailand has not been reported. Our purpose was to estimate the prevalence of BRAF mutation in a large institutional series using an affordable approach, which combined mutation-specific immunohistochemistry (IHC) with VE1 antibody and tissue microarray (TMA). METHODS: A total of 476 PTC cases plotted on TMA were employed for determining the mutation status in this study. The cancer tissue of initial 100 cases (pilot study) were analyzed for BRAF(V600E) mutation by using both direct sequencing and VE1 immunostaining. For the subsequent PTC cases, VE1 IHC was used as an alternative to direct sequencing for the detection of mutation. Univariate and multivariate analyses were done to determine the association of clinicopathological variables with BRAF(V600E) mutation. RESULTS: In the pilot study, VE1 IHC showed excellent analytical performance (κ=0.884) for detecting BRAF(V600E) mutation in PTC TMA as compared to direct sequencing. The prevalence of BRAF(V600E) in the whole cohort was 60.9% by using VE1 IHC. The mutation was commonly seen in tall cell (92.9%) and classic (70.2%) variants of PTC. Multivariate analysis (P

Although, in the thyroid field, there have been several internationally standard diagnostic and management guidelines for practitioners from every country to follow but there is still variation among physicians across the globe in taking care of patients and pathology specimens with thyroid nodules due to dissimilar backgrounds of each place. Thailand is one among several countries that tries to maintain standard of care to meet international requirements although, with some limitations, not all recommendations are followed. To understand how approaches of thyroid nodules are performed throughout the country and how the healthcare system of the whole kingdom is managed including what major current limitations, the authors conducted a survey among physicians consisting of surgeons, radiologists and pathologists who are involved in the management of the thyroid lesions by personal phone contact and custom online questionnaires. Performances of fine needle aspiration of different institutes are also evaluated by analyzing the data from available publications and compared their results with other series from the literature. Shortage of pathologists and un-equal resource allocation are the major problems concerning administrative aspect while a lack of awareness in new terminologies or diagnostic systems (e.g., NIFTP, UMP, Bethesda, TI-RADS) among physicians, particularly clinicians, are the problems of training and continuous medical education. Another point of concern is a rather poor performance in fine needle aspiration from the Thai series in which there are high rate (30.5%) of non-diagnostic category (Bethesda I) and false negativity as demonstrated by relatively high risk of malignancy (9.6%) in Bethesda II compared with other series from the literature. Regarding molecular studies, despite their usefulness in improving diagnostic accuracy, especially for cytology specimens, these ancillary tools are not routinely used in our practice since they are available in only few referral centers. This review provided a background information of the perspective on how thyroid nodules are managed in the Thai settings.

BackgroundInvasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis.MethodsTissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced.ResultsPolymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species.ConclusionPolymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.

Background The accurate preoperative identification of medullary thyroid carcinoma (MTC) is challenging due to the rarity of tumor and variable cytologic appearance. The Asian experience with diagnosing MTC by fine-needle aspiration (FNA) was scarcely reported. Methods Cases of MTC with available FNA slides were enrolled from 13 hospitals representing 8 Asia-Pacific countries. Clinicopathological information, including sample preparation technique, staining method, original cytologic diagnosis and review diagnosis were collected. Results Of a total of 145 MTC cases retrospectively recruited, 99 (68.3%) were initially interpreted as MTC/suspicious for MTC (S-MTC). The distribution of original FNA diagnostic categories was not associated with the staining method or sample preparation technique. The staining methods used were Papanicolaou, hematoxylin-eosin and Romanowsky stains. Liquid-based cytology (LBC) was used only in three countries. After reviewing all cases, the diagnostic rate of MTC/S-MTC increased to 91.7% (133/145). Cases with initially unrecognized MTC had either marked pleomorphism or cytology mimicking papillary carcinoma or follicular neoplasm. Although LBC provided certain benefits, there was no significant difference in diagnostic accuracy between conventional smear and LBC. Immunocytochemistry was available in 38 cases (26.2%), all of which were correctly recognized as MTC. Conclusion Our report summarizes how MTC is handled in contemporary Asian thyroid FNA practice. Although the detection rate of MTC by cytology alone is less satisfactory, integration with ancillary tests could achieve an excellent performance. The recognition of constitutive cytomorphologic features is needed for each cytopreparatory method, which may result in a lower threshold to initiate further workup for MTC.

Detection of BRAFV600E is useful for making diagnosis and risk stratification of papillary thyroid carcinoma (PTC). Molecular testing, however, is not always available for routine clinical use. To assess the clinical utility and reliability of VE1 immunohistochemistry (IHC) for detecting BRAFV600E mutation in PTC, VE1 IHC was performed on the tissue microarrays of 514 patients with PTC and was compared with Sanger sequencing results. Of 514 PTC cases, 433 (84.2%) were positive for VE1 expression. Among 6 discordant cases between VE1 IHC and Sanger sequencing, 3 initial VE1-false negative cases turned out to be true false negative on repeat testing, and 3 VE1-false positive cases showed BRAFV600E mutation using digital PCR analysis. PTCs with low variant allele fraction were positive for VE1 IHC but were not detected using sequencing. VE1 IHC showed 99.3% sensitivity, 100% specificity, 100% positive predictive value, and 96.4% negative predictive value. The BRAFV600E mutation was significantly associated with older age, multifocality, extrathyroidal extension, lymph node metastasis, and advanced tumor stage. In conclusion, VE1 IHC is a reliable method for detecting BRAFV600E mutation in PTC specimens.

Background Assessment of HER2 status is considered standard of care in the histopathologic workup of breast cancer and conveys prognostic and predictive information used to guide treatment decisions. The assessment is often carried out in a two-step approach where immunohistochemical expression of HER2 protein is first evaluated by conventional microscopy and equivocal cases are further analyzed by in-situ hybridization techniques to assess gene amplification status. In this study we compared conventional manual assessment of immunohistochemical HER2 expression with digital image analysis (DIA) and consensus manual assessment by a panel of three pathologists. Methods From our archive we retrieved sections of 109 invasive breast carcinomas stained for HER2 with corresponding HER2 score from the original pathology report. The glass slides were assessed by three pathologists to reach a consensus score. Next, the slides were scanned into whole slide images and DIA was performed using Aperio Imagescope. The scoring results were then compared with gene amplification status evaluated by dual in-situ hybridization (DISH). Results Comparing manual assessment with consensus assessment and DIA, good agreement was obtained with weighted kappa coefficients of 0.79 (manual vs. consensus) and 0.67 (manual vs. DIA). When compared with gene status assessment by DISH, agreement analysis yielded weighted kappa coefficients of 0.52 (manual vs. DISH), 0.58 (consensus vs. DISH) and 0.78 (DIA vs. DISH). There were no false negatives by any of the three methods and false positives ranging from 0.9 to 2.8%. The proportion of equivocal cases by each method was 44% (manual), 33.3% (consensus) and 14.7% (DIA). Application of DIA reduced the number of equivocal cases by 67% without increasing the proportion of false negatives. Conclusion We conclude that DIA is an accurate method to reduce the number of HER2 equivocal cases without affecting the sensitivity of the HER2 assessment.

HPV-related Multiphenotypic sinonasal carcinoma (previously known as HPV-related carcinoma with adenoid cystic carcinoma-like features) is very rare tumor of which only 55 cases are reported. All cases to date are related to high-risk types of HPV including 16, 31, 33, 35 and 56. We report of a case of a nasal mass in a 50-year-old female. Biopsy demonstrated typical features of an HPV-related multiphenotypic sinonasal carcinoma, with both basaloid and ductal type cells. Tumor cells were arranged in lobulated sheets, with focal cribriform and microcystic growth patterns. The majority of tumor cells expressed myoepithelial markers (S-100 protein, smooth muscle actin and p63) and the ductal cells were immunopositive for CD117. Polymerase chain reaction and sequencing identified HPV type 26. HPV 26 becomes the first intermediate risk type that may be associated with HPV-related multiphenotypic sinonasal carcinoma. Keywords: HPV, Type 26, Sinonasal carcinoma, Multiphenotypic

The Epstein-Barr virus (EBV) is one of the infectious agents found in stomach tissue. Recently, EBV-associated gastric carcinoma (EBVaGC) was classified as a new subtype of gastric carcinoma. To date, there is a lack of knowledge about the distribution and prevalence of EBV infection in both the normal stomach and various gastric lesions, including EBVaGC, in the Thai population. In this study, we detected EBV in the normal stomach (NS; n = 19), chronic gastritis (CG; n = 36), intestinal metaplasia (IM; n = 40), gastric dysplasia (GD; n = 15), and gastric adenocarcinoma (GC; n = 33) by polymerase chain reaction (PCR) amplification of the latent membrane protein (LMP1) gene of EBV. EBV-PCR amplification was positive in 42.1%, 36.1%, 22.5%, 13.3%, and 33.3% of NS, CG, IM, GD, and GC, respectively. For further clarification in EBVaGC, we performed EBV-encoded small RNA in situ hybridization (EBER-ISH) in PCR-positive cases of GD and GC. Four GC cases were EBER-ISH positive (12.1%), while both GD cases were EBER-ISH negative. In addition, we determined the distribution of the EBV strain (type A or B) based on EBNA3C sequence and EBV variants based on LMP1 variation (wild-type and 30-bp deletion variants; wt-LMP1 or del-LMP1). The results showed that type A and wt-LMP1 were the most prevalent in all lesions. In conclusion, EBV is common in both the NS and gastric lesions, and the frequency of EBVaGC was 12.1% in Thai patients.

Background Hypermethylation of Src homology region 2 domain-containing protein-tyrosine phosphatase 1 promoter 2 (SHP1-P2) has been proven as an epithelial-specific marker. This marker has been used for the detection of lymph node metastasis in patients with lung cancer or colon cancer. Objectives To investigate SHP1-P2 methylation in patients with squamous cell carcinoma of the head and neck (HNSCC) and determine its potential for micrometastasis detection in the lymph nodes of patients with HNSCC. Methods SHP1-P2 methylation levels were analyzed by combined methylation-specific primer TaqMan real-time PCR in 5 sample groups: normal tonsils (n = 10), microdissected squamous cell carcinoma epithelia (n = 9), nonmetastatic head and neck cancer lymph nodes (LN N0, n = 15), metastatic HNSCC histologically negative for tumor cells (LN–, n = 18), and matched cases histologically positive for tumor cells (LN+, n = 18). Results SHP1-P2 methylation of 10.27 ± 4.05% was found in normal tonsils as a lymphoid tissue baseline, whereas it was 61.31 ± 17.00% in microdissected cancer cell controls. In the 3 lymph node groups, the SHP1-P2 methylation levels were 9.99 ± 6.61% for LN N0, 14.49 ± 10.03% for LN- Nx, and 41.01 ± 24.51% for LN+ Nx. The methylation levels for LN- Nx and LN+ Nx were significantly different (P = 0.0002). Receiver operating characteristic curve analysis of SHP1-P2 methylation demonstrated an area under the curve of 0.637 in distinguishing LN N0 from LN– Nx. Conclusions SHP1-P2 methylation was high in HNSCC, and low in lymphoid tissues. This methylation difference is concordant with lymph node metastasis.

Several Western studies showed that the recent introduction of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) significantly decreased risk of malignancy for cytological diagnostic categories. We aimed to determine the impact of NIFTP on risk of malignancy within a cohort of thyroid nodules from Asian countries, and to compare distribution of diagnostic categories between NIFTP and invasive encapsulated follicular variant of papillary thyroid carcinoma (eFV-PTC). Consecutive thyroid fine-needle aspirates from six institutions were retrospectively analysed. Histopathology slides with a diagnosis of eFV-PTC were reviewed and reclassified into invasive eFV-PTC and NIFTP. The risk of malignancy was calculated with and without NIFTP. Of 11,372 thyroid nodules, 2044 had available surgical follow-up. NIFTP was diagnosed in 59 cases, which constituted 2.9% of all excised nodules, and 5.3% of malignant nodules. Preoperative cytological diagnoses for NIFTP were non-diagnostic (10.2%), benign (18.6%), atypia of undetermined significance/follicular lesion of undetermined significance (22.0%), follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) (32.2%), suspicious for malignancy (SM) (11.9%), and malignant (5.1%). The only category which showed a relative reduction in risk of malignancy after reclassification of more than 20%, was FN/SFN (24.4%). There was a significantly higher prevalence of benign cytology in NIFTP (p = 0.04) and SM/malignant in invasive eFV-PTC (p = 0.05). A majority of NIFTP cases were classified in indeterminate categories, which decreased the corresponding risk of malignancy. However, the magnitude of NIFTP impact was much lower than in the Western reports. Asian countries may not experience significant effects of NIFTP reclassification on the practice of thyroid cytopathology.

(1) Background: Accurate preoperative identification of medullary thyroid carcinoma (MTC) is challenging due to a spectrum of cytomorphologic features. However, there is a scarcity of studies describing the cytomorphologic features as seen on fine-needle aspiration (FNA) smears prepared using different staining methods. (2) Methods: We performed a retrospective study on MTC cases with available FNA slides from 13 hospitals distributed across 8 Asia-Pacific countries. The differences in the constitutive cytomorphologic features of MTC with each cytopreparatory method were recorded. A comparative analysis of cytologic characteristics was carried out with appropriate statistical tests. (3) Results: Of a total of 167 MTC samples retrospectively recruited, 148 (88.6%) were interpreted as MTC/suspicious for MTC (S-MTC). The staining methods used were Papanicolaou, hematoxylin-eosin, and Romanowsky stains. Seven out of the eleven cytologic criteria can be readily recognized by all three cytopreparatory methods: high cellularity, cellular pleomorphism, plasmacytoid cells, round cells, dyshesive cells, salt-and-pepper chromatin, and binucleation or multinucleation. An accurate diagnosis was achieved in 125 (84.5%) of the 148 samples whose FNAs exhibited five or more atypical features. Conclusions: The present work is the first study on MTC to compare the morphological differences among the cytologic staining techniques. We investigated the constitutive features and the reliability of diagnostic parameters. A feasible scoring system based upon cytomorphologic data alone is proposed to achieve a high degree of diagnostic accuracy.

Background Oral squamous cell carcinoma (OSCC) is an aggressive human malignancy. Because of late diagnosis and recurrence of OSCC, the treatment of patients with OSCC is often ineffective. Thus, finding novel biomarkers of OSCC are essential. Here we derived a methylation marker by utilizing methylation microarray data and testing its capacity in cross-sectional study designed for OSCC detection and screening. Methods According to bioinformatics analysis of total of 27,578 cg sites, cg22881914 of Nidogen 2 (NID2) methylation was selected for evaluation. Next, we confirmed the methylation status by bisulfite sequencing from the microdissected OSCC cells in comparison with the microdissected oral epithelia. Subsequently, we developed a simple technique using real-time PCR with the specific probe to examine the ability for the detection of OSCC in the oral epithelial samples, which included 103 oral rinse and 82 oral swab samples. Results Based on the comparison of microdissected tissue, cg22881914 of NID2 was proved to be methylated in most OSCC cells but unmethylated in the normal oral epithelia. Furthermore, the methylated NID2-relied quantitative PCR approach has demonstrated that this marker assists in distinguishing among patients with OSCC from normal oral epithelia, smokers, and patients with oral lichen planus using the non-invasive oral rinse and swab samples. Conclusions Specific methylation at cg22881914 of NID2 of OSCC could be used as an important potential marker for detecting OSCC. Thus, to certify the utility of this marker, further studies with a larger sample size are needed.

Introduction: Cholesteatoma of the paranasal sinuses is uncommon. Its clinical characteristics are an expanding growth of the affected paranasal sinuses consisting of keratinizing squamous epithelium with bony wall destruction. Among involved paranasal sinuses, sphenoid sinus cholesteatoma is the least common. Case presentation: An 82-year-old female diabetic patient presented with subacute onset of fever after experiencing chronic progressive headaches for more than 20 years. Nasal endoscopy found purulent discharge from left sphenoethmoidal recess. Computed tomography (CT) scan of the paranasal sinus showed soft tissue lesions that totally filled the left sphenoid sinus with posterior and inferior wall destruction. There was no evidence of connection to the left mastoid cavity. Management and outcome: Left sphenoidotomy was performed. Histopathology revealed cholesteatoma. Two months after surgery, she became worse and CT showed extensive skull base destruction. The patient underwent bilateral sphenoidectomy and craniotomy with surgical debridement of osteomyelitis of the skull base. She received long-term intravenous ertapenam and sitafloxacin for treating drug-resistant Klebsiella infection. The osteomyelitis could not be controlled, and she died. Discussion: Progressive headache can be caused by an uncommon disease such as sphenoid sinus cholesteatoma, which is a surgical condition. Complicating osteomyelitis of the skull base requires extensive debridement surgery and should be anticipated.

Background This study aims to compare histopathology of nasal polyp and ethmoid mucosa for diagnosing eosinophilic mucin rhinosinusitis (EMRS). Methodology Patients with chronic rhinosinusitis with polyps (CRSwNP) were enrolled. Using eosinophilic mucin as a reference, histopathology of polyp apex, polyp pedicle and ethmoid mucosa was compared for density of tissue eosinophil and sensitivity for diagnosing EMRS. Associations with asthma were assessed for each site. Results Thirty patients with CRSwNP were enrolled. When polyp apex, polyp pedicle and ethmoid mucosa were assessed for tissue eosinophilia, consistent results were reported in 16 patients (53%). Median tissue eosinophil was greater in polyp apex (58, IQR: 7-100) than ethmoid mucosa (10, IQR: 2-21), but not different from polyp pedicle (22, IQR: 1-96). Sensitivity for diagnosing EMRS were 100% (95%CI: 47.8 - 100) for polyp apex, 60% (95%CI: 14.7 - 94.7) for polyp pedicle, 80% (95%CI: 28.4 â€" 99.5) for ethmoid mucosa. Associations with asthma were significant for polyp pedicle, and ethmoid mucosa but not polyp apex. Conclusion Density of tissue eosinophil was greater in nasal polyp than in ethmoid mucosa. Histopathology of polyp apex had good sensitivity for diagnosing EMRS. Polyp pedicle and ethmoid mucosal eosinophilia associated with asthma.

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive undifferentiated follicular-derived thyroid neoplasm. This rare type of thyroid carcinoma (1–2% of all thyroid malignancies) usually develops in elderly patients, presenting as a rapidly growing large and deeply infiltrative thyroid mass. ATC is almost universally rapidly lethal with a median survival less than 6 months. It is typically diagnosed based on clinical symptoms and history. The major role of the pathologist is to differentiate ATC from other primary and secondary thyroid malignancies, which is necessary to direct a treatment. The tumor cells typically show complete loss of differentiation toward follicular cell lineage. Most cases display a mixture of spindle cell, giant cell, and epithelioid patterns rather than pure morphology. Irrespective of the pattern, malignant cells demonstrate a high degree of pleomorphism and brisk mitoses, accompanied by extensive invasion and tumor necrosis. More than half of adequately sampled cases of ATC contain the well-differentiated component. Cases of differentiated thyroid cancer with ATC foci (

Changes in the methylation level of genes containing LINE-1 alter host gene regulation. Aim: This study demonstrates that paracrine signaling of breast cancer influences the epigenetic regulation of stromal cells. Methods: We proved in vitro and in vivo breast cancer promoted LINE-1 methylation exists exclusively in female stromal cells. Results: Genes containing LINE-1 of breast cancer stromal cells were upregulated. Furthermore, one of the genes, MUC-1, was demonstrated to have expression in plasma cells from the lymph nodes of patients with lymph node metastasis or micrometastasis. Conclusion: Breast cancer sends a paracrine signal to stroma cells causing LINE-1 epigenetic regulation. Moreover, the regulated genes in stroma cells are potential biomarkers for detecting breast cancer micrometastasis.

Epstein-Barr virus-associated undifferentiated (lymphoepithelial) carcinoma is a malignancy that most commonly arises in the nasopharynx but can also occur in other locations including the lacrimal sac. Generally, this tumor strongly expresses cytokeratin, making the diagnosis straightforward. In the absence of confirmatory immunohistochemistry, the diagnosis can be problematic, particularly for tumors arising in unusual locations. Only 3 cases arising in the lacrimal sac in association with Epstein-Barr virus have been reported in the English literature, and all showed typical pathologic findings. The authors report a fourth case, unique in that it showed negative immunostaining for all cytokeratins tested. The clue to the nature of the tumor came from identification of Epstein-Barr virus by in-situ hybridization and demonstration of tonofilaments by electron microscopy. This case demonstrates that a multimodal approach may be needed in the diagnosis of Epstein-Barr virus-associated carcinoma, especially when occurring in uncommon locations.

The Epstein-Barr virus (EBV) is one of the infectious agents found in stomach tissue. Recently, EBV-associated gastric carcinoma (EBVaGC) was classified as a new subtype of gastric carcinoma. To date, there is a lack of knowledge about the distribution and prevalence of EBV infection in both the normal stomach and various gastric lesions, including EBVaGC, in the Thai population. In this study, we detected EBV in the normal stomach (NS; n = 19), chronic gastritis (CG; n = 36), intestinal metaplasia (IM; n = 40), gastric dysplasia (GD; n = 15), and gastric adenocarcinoma (GC; n = 33) by polymerase chain reaction (PCR) amplification of the latent membrane protein (LMP1) gene of EBV. EBV-PCR amplification was positive in 42.1%, 36.1%, 22.5%, 13.3%, and 33.3% of NS, CG, IM, GD, and GC, respectively. For further clarification in EBVaGC, we performed EBV-encoded small RNA in situ hybridization (EBER-ISH) in PCR-positive cases of GD and GC. Four GC cases were EBER-ISH positive (12.1%), while both GD cases were EBER-ISH negative. In addition, we determined the distribution of the EBV strain (type A or B) based on EBNA3C sequence and EBV variants based on LMP1 variation (wild-type and 30-bp deletion variants; wt-LMP1 or del-LMP1). The results showed that type A and wt-LMP1 were the most prevalent in all lesions. In conclusion, EBV is common in both the NS and gastric lesions, and the frequency of EBVaGC was 12.1% in Thai patients.

Objectives: To determine whether the spectrum of oral pathology in children seen at a medical institution differs from studies derived from dental facilities. Methods: Oral biopsy records from paediatric patients (

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer epithelium, making it a promising target for molecular imaging and therapy. Recently, several studies found unexpected PSMA radiotracer uptake by thyroid tumors, including radioiodine-refractory (RAIR) cancers. PSMA expression was reported in tumor-associated endothelium of various malignancies, however it has not been systematically addressed in thyroid tumors. We found that PSMA was frequently expressed in microvessels of thyroid tumors (120/267), but not in benign thyroid tissue. PSMA expression in neovasculature was highly irregular ranging from 19% in benign tumors to over 50% in thyroid cancer. Such heterogeneity was not directly attributed to endothelial cell proliferation as confirmed by immunostaining with proliferation-associated endothelial marker CD105. PSMA expression was associated with tumor size (p = 0.02) and vascular invasion in follicular carcinoma (p = 0.03), but not with other baseline histological, and clinical parameters. Significant translational implication is that RAIR tumors and high-grade cancers maintain high level of PSMA expression, and can be targeted by PSMA ligand radiopharmaceuticals. Our study predicts several pitfalls potentially associated with PSMA imaging of the thyroid, such as low expression in oncocytic tumors, absence of organ specificity, and PSMA-positivity in dendritic cells of chronic thyroiditis, which is described for the first time.

a b s t r a c t Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Lep- tospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Ham- sters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development. © 2012 Elsevier GmbH. All rights reserved.

Background: Men who have sex with men (MSM) are at high risk of developing human papillomavirus (HPV)-related anal cancer. We compared HPV genotypes in anal tissues (Bx) and anal liquid-based cytology fluid (LBC) from HIV-positive and HIV-negative MSM. Methods: Bx (32 normal, 41 low-grade squamous intraepithelial lesions (LSIL) and 22 high-grade squamous intraepithelial lesions (HSIL)), along with LBC from the same visit, were selected from 61 HIV-positive and 34 HIV-negative MSM who enrolled into a prospective cohort in Bangkok, Thailand. HPV genotyping was performed on Bx and LBC. Results: Any HPV and high-risk HPV (HR-HPV) prevalence were 63.2% and 60.0% in Bx and 71.6% and 62.1% in LBC, respectively. HIV-positive MSM had higher rates of HR-HPV genotypes detection (70.5% vs. 47.1%, p=0.03) in LBC than HIV-negative MSM. HPV16 (27%) was the most common HR-HPV found in HSIL tissue. In HIV-positive MSM, the frequency of HR-HPV detection increased with histopathologic grading in both Bx and LBC samples. HSIL was associated with the presence of any HR-HPV(OR 7.6 (95%CI 1.8â31.9); P=0.006) in LBC and in Bx((OR 5.6 (95%CI 1.4â22.7); P=0.02). Conclusions: Our data strongly support the integration of HR-HPV screening on LBC samples, along with HPV vaccination, into an anal cancer prevention program. Keywords: Human papillomavirus, Anal tissues, Men who have sex with men, HIV, Thailand

Methylation of promoter 2 of the SHP1 gene is epithelial cell specific, with reported potential as a lymph node metastatic marker.To demonstrate SHP1-P2 methylation-specific quantitative PCR effectiveness in detecting colorectal cancer (CRC) DNA in lymph nodes.SHP1-P2 methylation levels were measured in lymph nodes of CRC patients and compared with pathological findings and patient prognosis.Lymph nodes of CRC metastatic patients without microscopically detectable cancer cells had higher SHP1-P2 methylation levels than lymph nodes of controls (0.001). In addition, a higher SHP1-P2 methylation level was associated with a shorter duration until adverse disease events, metastasis, recurrence and death (r2 = 0.236 and p value = 0.048). Studying two cohorts of 74 CRC patients without microscopic lymph node metastases showed that only the cohort containing samples with high SHP1-P2 methylation levels had a significant hazard ratio of 3.8 (95%CI = 1.02 to 14.2).SHP1-P2 methylation PCR can detect CRC cancer DNA in lymph nodes even if cancer cells are not visible under a microscope, confirming applicability as a potential universal lymph node metastatic marker.