Purpose: The aim of this study was to develop a formalin-fixed paraffin-embedded (FFPE) tissue based multiplex immunochemistry (mIHC) method for high-throughput comprehensive tissue imaging and demonstrate its feasibility, validity, and usefulness. Materials and Methods: The mIHC protocol was developed and tested on tissue microarray slides made from archived gastric cancer (GC) tissue samples. On a single FFPE slide, cyclic immunochemistry for multiple markers of immune cells and cytokeratin for tumor cells was performed; hematoxylin staining was used for demarcation of nuclei. Whole slides were digitally scanned after each cycle. For interpretation of mIHC results, we performed computer-assisted image analysis using publicly available software. Results: Using mIHC, we were able to characterize the tumor microenvironment (TME) of GCs with accurate visualization of various immune cells harboring complex immunophenotypes. Spatial information regarding intratumoral and peritumoral TME could be demonstrated by digital segmentation of image guided by cytokeratin staining results. We further extended the application of mIHC by showing that subcellular localization of molecules can be achieved by image analysis of mIHC results. Conclusion We developed a robust method for high-throughput multiplex imaging of FFPE tissue slides. The feasibility and adaptability of mIHC suggest that it is an efficient method for in situ single-cell characterization and analysis.

Remarkable developments in immuno-oncology have changed the landscape of gastric cancer (GC) treatment. Because immunotherapy intervenes with tumor immune response rather than directly targeting tumor cells, it is important to develop a greater understanding of tumor immunity. This review paper summarizes the tumor immune reaction and immune escape mechanisms while focusing on the role of T cells and their co-inhibitory signals, such as the immune checkpoint molecules programmed death-1 and programmed deathligand 1 (PD-L1). This paper also describes past clinical trials of immunotherapy for patients with GC and details their clinical implications. Strong predictive markers are essential to improve response to immunotherapy. Microsatellite instability, Epstein-Barr virus, PD-L1 expression, and tumor mutational burden are now regarded as potent predictive markers for immunotherapy in patients with GC. Novel immunotherapy and combination therapy targeting new immune checkpoint molecules such as lymphocyte-activation gene 3, T cell immunoglobulin, and mucin domain containing-3, and indoleamine 2,3-dioxygenase have been suggested, and trials are ongoing to evaluate their safety and efficacy. Immunotherapy is an important treatment option for patients with GC and has great potential for improving patient outcome, and further research in immuno-oncology should be carried out.

BACKGROUND: This study was performed to analyze cytologic diagnosis of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and its impact on the risk of malignancy (ROM) in the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). METHODS: Five thousand five hundred and forty-nine cases of thyroid fine-needle aspiration cytology (FNAC) diagnosed between 2012 and 2014 were included in this study. Diagnostic categories based on TBSRTC were compared with final surgical diagnoses, and the ROM in each category was calculated both when NIFTP was included in malignant lesions and when excluded from malignant lesions. RESULTS: Of the 5,549 thyroid FNAC cases, 1,891 cases underwent surgical resection. In final diagnosis, 1,700 cases were revealed as papillary thyroid carcinoma (PTC), and 25 cases were reclassified as NIFTP. The cytologic diagnoses of NIFTP were non-diagnostic in one, benign in five, atypia of undetermined significance (AUS) in 14, follicular neoplasm in two, and suspicious for malignancy in three cases. Collectively, NIFTP/encapsulated follicular variant of PTC (EFVPTC) were more frequently classified as benign, AUS, or follicular neoplasm and less frequently categorized as malignant compared to conventional PTCs. Exclusion of NIFTP from malignant diagnoses resulted in a slight decrease in malignancy rates in non-diagnostic, benign, AUS, follicular neoplasm, and suspicious for malignancy categories without any statistical significance. CONCLUSIONS: The decrease in the ROM was not significant when NIFTP was excluded from malignant lesions. In thyroid FNACs, NIFTP/EFVPTCs were mostly classified into indeterminate categories. Therefore, it might be feasible to separate NIFTP/EFVPTC from conventional PTC on FNAC to guide clinicians to conservative management for patients with NIFTP/EFVPTC.
Biopsy, Fine-Needle ; Diagnosis ; Humans ; Thyroid Gland ; Thyroid Neoplasms

One of the complications of permanent pacemaker implantation is unintended phrenic nerve stimulation. A 15-year-old boy with a permanent pacemaker presented with chest discomfort due to synchronous chest wall contraction with pacing beats. Even after reprogramming of the pacemaker, diaphragmatic stimulation persisted. Therefore, we performed thoracoscopic phrenic nerve insulation using a Gore-Tex patch to insulate the phrenic nerve from the wire. A minimally invasive approach using a thoracoscope is a feasible option for retractable phrenic nerve stimulation after pacemaker implantation.
Adolescent ; Humans ; Male ; Minimally Invasive Surgical Procedures ; Pacemaker, Artificial ; Phrenic Nerve* ; Polytetrafluoroethylene ; Thoracic Wall ; Thoracoscopes ; Thoracoscopy ; Thorax

Rosai-Dorfman disease (RDD) is a rare non-Langerhans cell histiocytosis characterized by an accumulation of activated histiocytes within the affected tissues. It is a heterogeneous disease that includes the classical (nodal) and extra-nodal variants. The cutaneous form of the disease without the characteristic lymphadenopathy is rare and is often misdiagnosed as other dermatologic diseases. Misdiagnosis as lymphoproliferative and infectious diseases such as lymphoma and tuberculosis have been reported in the literature. Herein, we report a case of facial cutaneous RDD with successful surgical treatment. In addition, we provide dermoscopic findings and literature review as dermoscopy can be a useful adjuvant tool in the diagnosis of cutaneous RDD.

BACKGROUND: Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. METHODS: To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), beta-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for beta-actin (97 bp) were performed. RESULTS: All formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and beta-actin by PCR. hGH was successfully detected in all MFPE samples, but in only 46.7% of the FFPE samples. Prolonged formalin fixation resulted in fewer GAPDH and beta-actin PCR products, and amplification of hGH was not successful. The PCR and reverse transcription-PCR results were significantly affected by the duration of decalcification. The yield, purity, and integrity of mRNA progressively decreased with increased storage periods of paraffin blocks. CONCLUSIONS: Fixation and storage should therefore be standardized in order to improve the quality of molecular pathologic diagnosis.
Actins ; Diagnosis ; DNA ; Formaldehyde ; Gastrointestinal Neoplasms ; Glyceraldehyde 3-Phosphate ; Human Growth Hormone ; Humans* ; Oxidoreductases ; Paraffin ; Polymerase Chain Reaction ; RNA ; RNA, Messenger

PURPOSE: Molecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients’ survival with CRC. MATERIALS AND METHODS: The expression of EGFR ligands, including heparin binding epidermal growth factor-like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH. RESULTS: Unlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands–/EGFR+ group showed a significant association with the worst disease-free survival (DFS; p=0.018) and overall survival (OS; p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis. CONCLUSION: Ligand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.
Betacellulin ; Colorectal Neoplasms* ; Disease-Free Survival ; Epidermal Growth Factor* ; Heparin ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Incidence ; Ligands ; Multivariate Analysis ; Prognosis* ; Receptor, Epidermal Growth Factor* ; RNA, Messenger ; Silver ; Transforming Growth Factors

Purpose: Angiosarcoma is a highly aggressive mesenchymal tumor. Although systemic chemotherapy is often considered for the inoperable or metastatic angiosarcoma, the outcome of such treatment is unsatisfactory and poorly delineated. Materials and Methods: We reviewed electronic medical records of 75 patients with angiosarcoma who were treated with systemic chemotherapy for inoperable or metastatic disease. Patients were classified as having liver involvement if they had either primary or metastatic hepatic lesions. Results: Among the patients evaluated, 51 patients were male (68%) and 24 patients (32%) had primary cutaneous angiosarcoma. Liver involvement was present in 28 patients (37.3%). A total of 59 patients received first-line weekly paclitaxel (wPac) and showed an objective response rate (ORR) of 23.7% (n=14), a median progression free survival (mPFS) of 4.0 months (95% confidence interval [CI] 3.0–6.1), and a median overall survival (mOS) of 10.2 months (95% CI 7.0–14.6). Among patients without liver involvement, patients receiving wPac (n=35) had significantly prolonged mPFS (5.8 vs. 3.2 months, respectively, p=0.014) with a tendency for prolonged mOS (13.8 vs. 11.6 months, respectively, p=0.13) than those receiving other regimens (n=12). A total of 24 patients received second- or later-line pazopanib monotherapy and showed an ORR of 16.7% (n=4), a mPFS of 2.4 months (95% CI 1.8–4.3) and a mOS of 5.4 months (95% CI 3.5–NA). Conclusion Treatment with first-line wPac and later-line pazopanib seems to provide survival benefit, especially for patients with advanced angiosarcoma without liver involvement.

Purpose: Lymphovascular invasion is a criterion for non-curative resection in patients who have undergone endoscopic submucosal dissection (ESD) for early gastric cancer (EGC). We aimed to determine the rate of extragastric metastasis (EGM) and identify the predictors of EGM in patients with negative resection margins (R0 resection) and lymphovascular invasion in post-ESD pathology. Materials and Methods: A total of 2,983 patients underwent ESD for EGC. Among them, 110 had a pathology of R0 resection and positive lymphovascular invasion. Patients underwent additional gastrectomy (n=63) or further follow-up without gastrectomy (n=47). Results: The 110 patients were assigned to one of the 3 groups according to ESD indications based on post-ESD pathology. The first group satisfied the absolute indication for ESD (n=18), the second group satisfied the expanded indications for ESD (n=34), and the last group satisfied the beyond indication (n=58). The number of occurrences of EGM in each group was 1 (5.6%), 3 (8.8%), and 3 (5.2%), respectively. The logistic regression analysis adjusted for age, sex, tumor size, and indication for ESD, showed that larger tumor size was associated with EGM (odds ratio, 1.76; 95% confidence interval, 1.00–3.10; P=0.048). In contrast, ESD indication criteria did not affect EGM (P=0.349). Conclusions Tumor size was the only predictive indicator for EGM in patients who underwent R0 resection and lymphovascular invasion on post-ESD pathology. Even patients with pathology corresponding to the absolute indication criteria of ESD had lymphovascular invasion, which means that they require additional gastrectomy due to the risk of EGM.

Purpose: The purpose of this study was to reveal the clinicopathological characteristics and prognostic implications associated with fibroblast growth factor receptor 1 (FGFR1) amplification in colorectal cancers (CRCs). Materials and Methods: We measured the copy number of FGFR1 by droplet digital polymerase chain reaction (ddPCR), and analyzed the FGFR1 expression by immunohistochemistry, in 764 surgically resected CRCs (SNUH2007 dataset, 384 CRCs; SNUH Folfox dataset, 380 CRCs). Results: CRCs with ≥ 3.3 copies of the FGFR1 gene were classified as FGFR1 amplified. FGFR1 amplification was found in 10 of the 384 CRCs (2.6%) in the SNUH2007 dataset, and in 28 of the 380 CRCs (7.4%) in the SNUH Folfox dataset. In the SNUH2007 dataset, there was no association between the FGFR1 copy number status and sex, gross appearance, stage, or differentiation. High FGFR1 expression was associated with female sex and KRAS mutation. At the molecular level, FGFR1 amplification was mutually exclusive with BRAF mutation, microsatellite instability, and MLH1 methylation, in both SNUH2007 and SNUH Folfox datasets. Survival analysis revealed that FGFR1 amplification was associated with significantly worse clinical outcome compared with no FGFR1 amplification, in both SNUH2007 and SNUH Folfox datasets. Within the SNUH2007 dataset, CRC patients with high FGFR1 expression had an inferior progression-free survival compared with those with low FGFR1 expression. The FGFR inhibitor, PD173074, repressed the proliferation of a CRC cell line overexpressing FGFR1, but not of cells with FGFR1 amplification. Conclusion FGFR1 amplification measured by ddPCR can be a prognostic indicator of poor clinical outcome in patients with CRCs.