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Purpose Targeted next-generation sequencing (NGS) is widely used for simultaneously detecting clinically informative genetic alterations in a single assay. Its application in clinical settings requires the validation of NGS gene panels. In this study, we aimed to validate a targeted hybridization capture-based DNA panel (ONCOaccuPanel) using the Illumina MiSeq sequencing platform. The panel allows the simultaneous detection of single-nucleotide variants (SNVs), insertions, deletions, and copy number changes of 323 genes and fusions of 17 genes in solid tumors.Materials and Methods We used 16 formalin-fixed paraffin-embedded (FFPE) tumor samples with previously known genetic mutations and one reference material (HD827) for validation. Moreover, we sequenced an additional 117 FFPE tumor samples to demonstrate the clinical utility of this panel.Results Validation revealed a 100% positive percentage agreement and positive predictive value for the detection of SNVs, insertions, deletions, copy number changes, fusion genes, and microsatellite instability–high types. We observed high levels of reproducibility and repeatability (R2 correlation coefficients=0.96-0.98). In the limit of detection assessment, we identified all clinically relevant genes with allele frequencies > 3%. Furthermore, the clinical application of ONCOaccuPanel using 117 FFPE samples demonstrated robust detection of oncogenic alterations. Oncogenic alterations and targetable genetic alterations were detected in 98.2% and 27.4% cases, respectively.Conclusion ONCOaccuPanel demonstrated high analytical sensitivity, reproducibility, and repeatability and is feasible for the detection of clinically relevant mutations in clinical settings.

Epstein-Barr virus (EBV)-associated gastric cancer is a distinct subtype of stomach adenocarcinoma. Although previous studies have investigated its clinicopathological characteristics, there is a lack of research focusing on advanced EBV-associated gastric cancer. In this study, we performed a comprehensive review of advanced EBV-associated gastric cancer cases.We retrospectively collected 18 consecutive cases of surgically resected advanced EBV-associated gastric cancer. Clinicopathological parameters were investigated using histological review, immunohistochemistry, and a review of the electronic medical records of the hospital.The predominant histological pattern of advanced EBV-associated gastric cancer, according to the Laurén classification, was intestinal-type adenocarcinoma with varying degrees of differentiation. However, focal areas showing conventional gastric carcinoma with a lymphoid stromal pattern were found in all cases except one. In addition to the previously described histological patterns of EBV-associated gastric cancer, one case displayed chronic granulomatous inflammation-like histology with barely identifiable malignant epithelial cells. Another case had a pure signet-ring cell carcinoma component showing nuclear positivity for the EBV-encoded RNA in situ hybridization assay. Remarkably, five out of 18 cases (27.8%) showed aberrant p53 expression on immunostaining, which is known to occur rarely in EBV-associated gastric cancer. All cases with aberrant p53 expression had intestinal-type adenocarcinoma-like components.Advanced EBV-associated gastric cancer had distinct histology and a higher rate of aberrant p53 immunostaining pattern than conventional EBV-associated gastric cancer. Therefore, their biological behavior should be investigated separately.

Invasive mucinous adenocarcinoma (IMA) of the lung frequently presents with diffuse pneumonic-type features or multifocal lesions, which are regarded as a pattern of intrapulmonary metastases. However, the genomics of multifocal IMAs have not been well studied. We performed whole exome sequencing on samples taken from 2 to 5 regions in seven patients with synchronous multifocal IMAs of the lung (24 regions total). Early initiating driver events, such as KRAS, NKX2-1, TP53, or ARID1A mutations, are clonal mutations and were present in all multifocal IMAs in each patient. The tumor mutational burden of multifocal IMAs was low (mean: 1.13/mega base), but further analyses suggested intra-tumor heterogeneity. The mutational signature analysis found that IMAs were predominantly associated with endogenous mutational process (signature 1), APOBEC activity (signatures 2 and 13), and defective DNA mismatch repair (signature 6), but not related to smoking signature. IMAs synchronously located in the bilateral lower lobes of two patients with background usual interstitial pneumonia had different mutation types, suggesting that they were double primaries. In conclusion, genomic evidence found in this study indicated the clonal intrapulmonary spread of diffuse pneumonic-type or multifocal IMAs, although they can occur in multicentric origins in the background of usual interstitial pneumonia. IMAs exhibited a heterogeneous genomic landscape despite the low somatic mutation burden. Further studies are warranted to determine the clinical significance of the genomic characteristics of IMAs in expanded cohorts.

Background/Aim: Targeted next-generation sequencing (NGS) is a well-established technique to detect pathogenic alterations in tumors. Indeed, it is the cornerstone of targeted therapy in precision medicine. We investigated the clinical utility of next-generation sequencing in real-world cases. Patients and Methods: We retrospectively selected six representative cancer cases, wherein targeted NGS played a pivotal role in the diagnosis and treatment of patients. Additionally, we analyzed three cases with rare, unusual pathogenic alterations. Results: Our NGS analysis revealed that four patients had TPR-ROS1, EGFR-RAD51, and NCOA4-RET fusions and MET exon 14 skipping mutation, respectively, which can be treated with targeted therapy. Furthermore, we used NGS as a diagnostic tool to confirm the origin of unknown primary malignant tumors in two cases. Interestingly, NGS also helped us identify the following cases: patients exhibiting BRCA1 and TP53 mutations that exhibited histological and immunohistochemical characteristics consistent with endometrioid carcinoma, patients with high-grade serous carcinoma not possessing a TP53 mutation, and patients with small cell lung cancer with a ERBB2 mutation and displaying no loss of RB1. Conclusion: We recommend targeted NGS for the diagnoses and targeted therapy of cancer patients.

Gastric cancer (GC) is one of the most common lethal malignant neoplasms worldwide, with limited treatment options for both locally advanced and/or metastatic conditions, resulting in a dismal prognosis. Although the widely used morphological classifications may be helpful for endoscopic or surgical treatment choices, they are still insufficient to guide precise and/or personalized therapy for individual patients. Recent advances in genomic technology and high-throughput analysis may improve the understanding of molecular pathways associated with GC pathogenesis and aid in the classification of GC at the molecular level. Advances in next-generation sequencing have enabled the identification of several genetic alterations through single experiments. Thus, understanding the driver alterations involved in gastric carcinogenesis has become increasingly important because it can aid in the discovery of potential biomarkers and therapeutic targets. In this article, we review the molecular classifications of GC, focusing on The Cancer Genome Atlas (TCGA) classification. We further describe the currently available biomarker-targeted therapies and potential biomarker-guided therapies. This review will help clinicians by providing an inclusive understanding of the molecular pathology of GC and may assist in selecting the best treatment approaches for patients with GC.

Herein, we describe the first case of renal intravascular large B cell lymphoma in Korea occurring in a 66-year-old female. She presented with mild fever and dyspnea. On physical and laboratory evaluations, hemophagocytic lymphohistiocytosis was suspected, but the bone marrow biopsy results were unremarkable. During the work-up, massive proteinuria developed, which led to a renal biopsy. The renal architecture was relatively well-preserved, but the glomeruli were hypercellular with the infiltration of atypical, large lymphoid cells with increased nucleus-cytoplasm ratio and clumped chromatin. Similar cells were also present in the peritubular capillaries. The tumor cells exhibited membranous staining for CD20 and CD79a. After the diagnosis of intravascular large B cell lymphoma, the patient received rituximab-based chemotherapy under close follow-up.

Background/Aim: Poorly differentiated thyroid carcinoma (PDTC), anaplastic thyroid carcinoma (ATC), and advanced DTC have poor outcomes. Materials and Methods: We performed next-generation sequencing in nine selected aggressive thyroid cancers. Results: Among the nine patients, the driver gene mutations BRAF V600E (3/9) and NRAS Q61K (1/9) were detected. Other oncogenic mutations included ERBB2 (1/9) and CDK4 (1/9). Telomerase reverse transcriptase (TERT) promoter mutation was found in five cases. Among tumor suppressor genes, mutations in TP53 (3/9), ARID1A (1/9), APC (1/9), MEN1 (1/9), DICER1 (1/9), and MED12 (1/9) were identified. RET fusions were found in two cases, one with PTDC and the other with ATC. The ATC with RET fusion also harbored TP53 and TERT promoter mutations. None of the PDTC cases had BRAF or RAS gene alterations. Conclusion: Since genetic alterations with therapeutic and prognostic implications were detected using next-generation sequencing, this technique is recommended to be performed for patients with aggressive thyroid cancer.

The methylation status of the OThe MGMT promoter methylation status and its association with clinicopathological parameters were retrospectively analysed in a cohort of 316 patients with GBM with wild-type IDH.MGMT methylation was significantly associated with ATRX chromatin remodeler (ATRX) loss and completion of the standard Stupp protocol. The median durations of overall and progression-free survival for the unmethylated, low-methylated (10-39%), and hypermethylated (≥40%) groups were 15, 23, and 30 months and 11, 18, and 21 months, respectively. However, the improvement in the survival of the hypermethylated group was not statistically significant.We suggest a possible association between MGMT methylation status and ATRX mutations in GBM with wild-type IDH.

Infections with multiple human papilloma virus (HPV) types have been reported, but their role in cervical carcinogenesis has not been fully elucidated. In this study, 236 cases with multiple HPV infection were examined and compared to 180 cases with single HPV infection. HPV genotyping was performed with cervico-vaginal swab specimens using multiplex (real-time) polymerase chain reaction (PCR). In multiple HPV infection, the most prevalent HPV genotype was HPV 53, followed by HPV 16, 58, 52, and 68. HPV 33, 35, 39, 51, 52, 53, 58, and 68 were high-risk-HPV (HR-HPV) genotypes that were more frequently detected in multiple HPV infection compared to that in single HPV infection. The association between multiple HPV infection and high-grade SIL (HSIL) was significantly stronger compared to that of single HPV infection and HSIL (p = 0.002). Patients with multiple HPV infection displayed persistent and longer duration of the HPV infection compared to patients with single HPV infection. Multiple HPV infections have distinct clinicopathologic characteristics. Since it is associated with persistent HPV infection, HSIL, and different HR-HPV strains in contrast to single HPV infection, the presence of multiple HPV infection should be reported, close follow up is warranted.

Background/aim Well-differentiated papillary mesothelioma (WDPM) is histologically characterized by papillary architecture with fibrovascular cores, lined by bland mesothelial cells. We recently experienced a case of WDPM associated with multiple peritoneal inclusion cysts, which prompted us to initiate a comprehensive review of previously diagnosed WDPM cases. Materials and methods The clinicopathological characteristics and immunophenotype of 12 cases of peritoneal WDPM were investigated using a review of electronic medical records, pathological examination, and immunostaining. Results The patients' ages ranged from 23 to 75 years. No patient had endometriosis or a previous history of asbestos exposure. Ten tumors were detected incidentally during surgery for other causes. Most tumors appeared as a small, single nodule on the peritoneal surface, but in three cases, WDPM presented as multiple lesions. All but one patient had no symptoms. All the patients examined are still well without postoperative recurrence. Histologically, all cases demonstrated typical papillary architecture with fibrovascular cores. The mesothelial cells lining the papillae consisted mostly of single row of cells, although areas of proliferation to multiple layers were observed in a few cases. Their nuclei appeared bland, but two cases exhibited mild nuclear atypia and prominent nucleoli. Immunostaining revealed that the mesothelial cells were positive for D2-40, cytokeratin 5/6, cytokeratin 7, and Wilms' tumor 1. Conclusion We herein demonstrated the clinicopathological characteristics of peritoneal WDPMs. WDPM has distinct pathological features. Although all cases we examined were uneventful after surgery, further surveillance is recommended since the biological behavior of WDPM is still uncertain.

IntroductionMethylation status of the O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter plays key role in glioblastoma (GBM) with respect to the patient’s responses to temozolomide chemotherapy and disease prognosis. Although the cut-off value of MGMT methylation (≥10%) is currently widely used to dichotomize the MGMT status as “methylated” or “unmethylated” in pyrosequencing (PSQ) analysis, it is still unclear whether it reflects the actual MGMT methylation status of the patients. Thus, we investigated if there is a so called “hyper-hypermethylation cut-off value” that confers additional survival benefits in IDH-wild-type GBM patients. MethodsWe retrospectively analyzed a cohort of 110 isocitrate dehydrogenase (IDH)-wild-type GBM patients who underwent gross total resection followed by the standard treatment. Predictive “hyper-hypermethylated” cutoff values that yielded maximal differences in survival were investigated. ResultsThe estimated hyper-hypermethylated cutoff value was 40%. The median OS values for unmethylated, low-methylated, and hyper-hypermethylated groups were 16, 17, and 23 months, respectively. With regard to disease progression, the median progression-free survival (PFS) values were 11, 14, and 16 months, respectively. The hyper-hypermethylated MGMT cutoff values were correlated with significantly improved overall survival (OS), compared to the low-methylated (p=0.039, HR=3.86, CI=1.1 to 13.8) or unmethylated groups (p=0.023, HR=3.92, CI=1.21 to 12.6). ConclusionThus, in addition to the standard MGMT cut-off point of 10%, we suggest that a so-called “hyper-hypermethylated” MGMT cut-off point may confer additional survival benefits in IDH-wild-type GBM patients.

Background/aim In recent years, the genomic landscape of neuroendocrine tumors (NETs) of the lung has been investigated. However, more data are necessary to elucidate the heterogeneous nature of NETs, especially in East Asian patients. Patients and methods A total of 64 patients who underwent surgical resection for lung NETs [26 typical or atypical carcinoid tumors, 21 large-cell neuroendocrine carcinomas (LCNECs), and 19 small-cell lung carcinomas (SCLCs)] were enrolled, and samples from 46 patients were subjected to targeted next-generation sequencing. Results Co-mutations of tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) were detected in 15%, 42%, and 93% of carcinoid tumors, LCNECs, and SCLCs, respectively. Oncogenic or targetable genetic alterations identified in this study included mutations of KRAS proto-oncogene (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), ALK receptor tyrosine kinase (ALK), mitogen-activated protein kinase kinase 1 (MAP2K1), and isocitrate dehydrogenase 1 (IDH1), as well as amplifications of erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 1 (FGFR1), CD274 molecule (CD274), and MYCN proto-oncogene (MYCN). These alterations were more frequently found in high-grade NETs than in carcinoid tumors (33.3% vs. 7.7%). Programmed cell death-ligand 1 expression was strongly associated with the LCNEC subtype among NETs (p=0.002). Conclusion The mutational status of TP53 and RB1 was significantly associated with NET subtypes in East Asian patients. Targeted therapy or immunotherapy may serve as a treatment option in a subset of patients with high-grade NETs.

The histologic features of renal oncocytoma (RO) are similar to those for the more aggressive chromophobe renal cell carcinoma (ChRCC). To assess immunohistochemical markers of the two, the sensitivity and specificity of cytokeratin 7 (CK7) and C-kit, as well as hepatocyte nuclear factor-1β (HNF-1β), were analyzed. Typical cases of ChRCC and RO at Severance Hospital between July 2014 and July 2018 were selected retrospectively. Among 44 cases, 17 were unanimously compatible with ChRCC, 16 were RO, and 11 cases were indeterminate. Samples from all selected cases were used for immunostaining with antibodies against CK7, C-kit, HNF-1β, and CD10. Immunostaining demonstrated complete loss of HNF-1β expression in 11 out of 17 (64.7%) ChRCC cases and a partial, but significant loss in > 50% of tumor cells in the remaining 6 cases (35.3%). In contrast, HNF-1β expression was preserved in tumor cells of RO cases. Fourteen of 17 ChRCC cases (82.4%) were diffusely positive for CK7, whereas cases of RO were focal positive or negative. C-kit staining did not show a significant difference between ChRCC and RO. Two of five ChRCC cases showing diffuse immunoreactivity for CD10 had poor prognoses of local invasion, distant metastasis, or death. Loss of HNF-1β expression is a useful marker with which to diagnose ChRCC, especially in cases with confusing histologic findings or equivocal CK7 staining. Additionally, CD10 staining in high-grade ChRCC aids in diagnosis and prediction of the clinical prognosis.

Extramammary Paget disease (EMPD) is a rare cutaneous neoplasm. Perianal Paget disease (PPD) is a subset of EMPD manifesting perianal lesions. Two cases of PPD in Severance Hospital are described in this article. A 65-year-old female and 78-year-old male patients visited our institution because of an unhealed perianal skin lesion despite treatment for a long period with topical agents. PPD was diagnosed by skin biopsies in both cases, and the patients underwent surgical treatment. Clinical manifestations, preoperative work-ups, and surgical treatments including different reconstruction methods are described in detail. As only sporadic PPD cases have been reported and no standard treatment has been established, we hope that our experience could contribute to improving the diagnosis and treatment of PPD patients.

Objective Histologic types are correlated with prognosis in lung adenocarcinoma. The acinar/papillary type is most common, although this group comprises heterogeneous tumor types. However, the prognostic factors in this group have not been well studied. Therefore, we investigated the prognostic factors of acinar/papillary lung adenocarcinomas and attempted to define the so-called “intermediate grade” group of lung adenocarcinoma. Materials and methods We classified surgically resected invasive non-mucinous adenocarcinomas of the lung and analyzed their clinicopathological features and prognostic factors, focusing on the acinar/papillary type. Results A total of 301 cases with stage I-III lung adenocarcinoma were enrolled, of which 193 were acinar/papillary type (64.1%). In survival analysis of the entire cohort, acinar/papillary types showed intermediate survival compared with lepidic and micropapillary/solid types. In the univariate survival analysis for acinar/papillary types, stage, age, lymphovascular invasion, spread through air spaces, presence of micropapillary or solid pattern, and programmed death-ligand 1 (PD-L1) positivity were associated with poor recurrence-free survival and overall survival. In multivariate analysis, spread through air spaces and PD-L1 expression were independent poor prognostic factors of recurrence-free survival and overall survival in the acinar/papillary cohort, respectively. Conclusions Evaluation of spread through air spaces and PD-L1 expression may be useful to stratify patients with acinar/papillary lung adenocarcinomas in terms of prognosis.

Malignant mesothelioma is a highly lethal cancer. V-set immunoregulatory receptor (VSIR, also known as V-domain Ig suppressor T-cell activation, VISTA), a negative immune checkpoint regulator, was reported to be expressed in malignant mesothelioma; however, its detailed expression pattern and clinicopathological significance have not been elucidated. We examined the expression of VSIR and CD274 and CD8+ tumor-infiltrating lymphocytes in a total of 124 samples from 66 patients with malignant mesothelioma and analyzed the clinicopathological characteristics and their relationship with the immunohistochemical findings. A total of 553 non-small cell lung carcinomas were also evaluated for VSIR expression. VSIR expression was higher in epithelioid type mesothelioma (p

Primary effusion lymphoma (PEL) is a B-cell non-Hodgkin's lymphoma that is usually characterized by lymphomatous effusions in the body cavity without any detectable tumor masses. According to the World Health Organization (WHO) schema for tumor classification, PEL is defined by the presence of human herpesvirus 8 (HHV8) in malignant lymphoid cells. However, a subset of effusion-based B-cell lymphoma is not HHV8-positive and exhibits different clinicopathological characteristics. The 2017 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues does not list HHV8-negative effusion-based lymphoma, which remains an underappreciated B-cell lymphoma, as an individual entity. The present study reports a case of this rare type of lymphoma with indolent clinical behavior in a 75-year-old male patient receiving only symptomatic treatment. Additionally, a review of similar cases reported in the English literature is presented.

Composite tumors of cardiac myxoma and malignant lymphoma are extremely rare, with 11 of such cases reported in the literature. A 44-year-old man presented to us with abrupt right lower leg pain. A computed tomography angiogram revealed segmental obstruction of the right common femoral artery, and embolectomy was performed. The embolectomy specimen contained several clusters of degenerated round/oval atypical cells in the fibrinous or myxoid background. Immunohistochemical examination revealed that these atypical cells were negative for cytokeratin and non-specifically expressed CD45. After one year of follow-up, the patient presented with dyspnea on exertion. Transthoracic echocardiography revealed a 5.7×1.9 cm, lobulated, echogenic, intracardiac mass, and excision was performed. Microscopically, most of the mass was composed of stellate or curved cells with eosinophilic cytoplasm in the myxoid background. However, several clusters or scattered round/oval atypical cells, similar to those observed in the embolectomy specimen, were observed. These atypical cells were positive for CD45 and CD20, with a Ki-67 labeling index of 90%. Calretinin was expressed in stellate cells but not in atypical cells. Therefore, we made a diagnosis of diffuse large B cell lymphoma (DLBCL) arising in the cardiac myxoma. Our case is an extremely rare example of a composite tumor of cardiac myxoma and DLBCL presenting with peripheral arterial emboli. Thorough histological evaluation of embolectomy and cardiac myxoma specimen is crucial for the diagnosis of composite tumor.

// Ji-Ye Kim 1, 2 , Hyang Sook Jeong 2 , Taek Chung 2 , Moonsik Kim 2 , Ji Hee Lee 2 , Woo Hee Jung 2 and Ja Seung Koo 2 1 Department of Pathology and Translational Genomics, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, Republic of Korea 2 Department of Pathology, Yonsei University College of Medicine, Severance Hospital, Seoul, Republic of Korea Correspondence to: Ja Seung Koo, email: kjs1976@yuhs.ac Keywords: PHH3, invasive breast cancer, proliferation, reproducibility, prognostic value Received: February 26, 2016 Accepted: April 25, 2017 Published: May 10, 2017 ABSTRACT Background: Established measurements of proliferation in breast cancer are Ki67 and mitotic-activity-index (MAI), with problems in reproducibility and prognostic accuracy. Phosphohistone H3 (PHH3), a relatively novel IHC marker is specific for mitosis with good reproducibility. We hypothesized that PHH3 would be more reproducible and better represent proliferation than Ki67. Results: PHH3 identified easily-missed mitosis by MAI, as demonstrated by upgrading M grade at diagnosis ( n = 29/218, evenly distributed). PHH3 accurately found hot-spots, supported by mitotic count agreement between low-power and 10HPFs (R 2 = 0.999; P = 0.001). PHH3 was more reproducible than Ki67, measured by five-rater inter-class correlation coefficient (0.904 > 0.712; P = 0.008). Finally, despite a relatively short follow-up (median 46 months; 7 recurrences) PHH3 was the only variable correlated with disease-free survival ( P = 0.043), while all other conventional clinicopathologic variables, including Ki67 ( P = 0.356), did not. Materials and Methods: We compared Ki67 and PHH3 for 218 breast cancer surgical cases diagnosed from 2012 to 2013 at Severance hospital. The most representative invasive breast cancer surgical slides were immunohistochemically stained for Ki67 and PHH3. Conclusions: Poor reproducibility and inadequate representation of proliferation of Ki67 and MAI may be improved by PHH3, allowing better accuracy in breast cancer diagnostics.

Tumor cells secrete a variety of cytokines to outgrow and evade host immune surveillance. In this context, transforming growth factor-β1 (TGF-β1) is an extremely interesting cytokine because it has biphasic effects in cancer cells and normal cells. TGF-β1 acts as a growth inhibitor in normal cells, whereas it promotes tumor growth and progression in tumor cells. Overexpression of TGF-β1 in tumor cells also provides additional oncogenic activities by circumventing the host immune surveillance. Therefore, this study ultimately aimed to test the hypothesis that suppression of TGF-β1 in tumor cells by RNA interference can have antitumorigenic effects. However, we demonstrated here that the interrelation between TGF-β isotypes should be carefully considered for the antitumor effect in addition to the selection of target sequences with highest efficacy. The target sequences were proven to be highly specific and effective for suppressing both TGF-β1 mRNA and protein expression in cells after infection with an adenovirus expressing TGF-β1 short hairpin RNA (shRNA). A single base pair change in the shRNA sequence completely abrogated the suppressive effect on TGF-β1. Surprisingly, the suppression of TGF-β1 induced TGF-β3 upregulation, and the suppression of TGF-β2 induced another unexpected downregulation of both TGF-β1 and TGF-β3. Taken together, this information may prove useful when considering the design for a novel cancer immunogene therapy.