Kyu Sang Lee

c-MYC Copy-Number Gain Is an Independent Prognostic Factor in Patients with Colorectal Cancer

Background The aim of this study was to determine the incidence and clinicopathological significance of c-MYC gene copy-number (GCN) gain in patients with primary colorectal cancer (CRC). Methods The c-MYC GCN was investigated in 367 consecutive CRC patients (cohort 1) by using dual-color silver in situ hybridization. Additionally, to evaluate regional heterogeneity, we examined CRC tissue from 3 sites including the primary cancer, distant metastasis, and lymph-node metastasis in 152 advanced CRC patients (cohort 2). KRAS exons 2 and 3 were investigated for mutations. Results In cohort 1, c-MYC gene amplification, defined by a c-MYC:centromere of chromosome 8 ratio ≥ 2.0, was detected in 31 (8.4%) of 367 patients. A c-MYC GCN gain, defined by ≥ 4.0 c-MYC copies/nucleus, was found in 63 (17.2%) patients and was associated with poor prognosis (P = 0.015). Multivariate Cox regression analysis showed that the hazard ratio for c-MYC GCN gain was 2.35 (95% confidence interval, 1.453–3.802; P < 0.001). In a subgroup of stage II-III CRC patients, c-MYC GCN gain was significantly associated with poor prognosis by univariate (P = 0.034) and multivariate (P = 0.040) analyses. c-MYC protein overexpression was observed in 201 (54.8%) out of 367 patients and weakly correlated with c-MYC GCN gain (ρ, 0.211). In cohort 2, the c-MYC genetic status was heterogenous in advanced CRC patients. Discordance between GCN gain in the primary tumor and either distant or lymph-node metastasis was 25.7% and 30.4%, respectively. A similar frequency for c-MYC GCN gain and amplification was observed in CRC patients with both wild-type and mutated KRAS. Conclusions c-MYC GCN gain was an independent factor for poor prognosis in consecutive CRC patients and in the stage II-III subgroup. Our findings indicate that the status of c-MYC may be helpful in predicting the patients’ outcome and for managing CRC patients.

Stromal Expression of MicroRNA-21 in Advanced Colorectal Cancer Patients with Distant Metastases

Background: The aim of this study was to determine the regional heterogeneity and clinicopathological significance of microRNA-21 (miR-21) in advanced colorectal cancer (CRC) patients with distant metastasis. Methods: miR-21 expression was investigated by using locked nucleic acid– fluorescence in situ hybridization in the center and periphery of the primary cancer and in distant metastasis from 170 patients with advanced CRC. In addition, α-smooth muscle actin and desmin were evaluated to identify cancer-associated fibroblasts (CAFs) by using immunohistochemistry. Results: The miR-21 signal was observed in the cancer stroma. The expression of miR-21 (a score of 1–4) in the center and periphery of the primary cancer and in distant metastasis was observed in specimens from 133 (78.2%), 105 (61.8%), and 91 (53.5%) patients, respectively. miR-21 expression was heterogeneous in advanced CRC. Discordance between miR-21 expression in the center of the primary cancer and either the periphery of the primary cancer or distant metastasis was 31.7% or 44.7%, respectively. miR-21 stromal expression in the periphery of the primary cancer was significantly associated with a better prognosis (p=.004). miR-21 expression was significantly associated with CAFs in the center of the primary cancer (p=.001) and distant metastases (p=.041). Conclusions: miR-21 expression is observed in cancer stroma related to the CAF quantity and frequently presents regional heterogeneity in CRC. Our findings indicate that the role of miR-21 in predicting prognosis may be controversial but provide a new perspective of miR-21 level measurement in cancer specimens.

Bullous hemorrhagic dermatosis due to enoxaparin use in a bullous pemphigoid patient

Adverse reactions of subcutaneous low molecular weight heparin or unfractionated heparin could be complications by bleeding, heparin-induced thrombocytopenia, drug-induced liver injury, osteoporosis, and cutaneous reactions. Heparin-induced skin lesions vary from allergic reactions like erythema, urticaria, eczema to intradermal microvascular thrombosis associated with heparin-induced thrombocytopenia. There is a rare cutaneous complication, called bullous hemorrhagic dermatosis. We experienced this rare case of the cutaneous complication caused by enoxaparin. Several tense bullous hemorrhagic lesions occurred after 3 days of enoxaparin in a known bullous pemphigoid patient who had aortic valve replacement surgery with a mechanical prosthesis. The bullous hemorrhagic lesions were regressed after the discontinuation of enoxaparin but recurred after re-administration. The lesions were controlled by the administration of systemic corticosteroid and alternative anticoagulant. To date, less than 20 cases have been reported worldwide. This is the first case of bullous hemorrhagic dermatosis induced by enoxaparin, a low-molecular-weight heparin in Korea. This is also the first case of bullous hemorrhagic dermatosis in a known bullous pemphigoid patient.

Potential Prognostic Value of Histone Deacetylase 6 and Acetylated Heat-Shock Protein 90 in Early-Stage Breast Cancer

Purpose Histone deacetylase 6 (HDAC6) is an enzyme that deacetylates heat-shock protein 90 (HSP90). Many studies have investigated the role of HDAC6 and HSP90 in tumorigenesis and in the prognosis of cancer patients. This study aimed to evaluate the prognostic value of HDAC6 and acetylated HSP90 (acetyl-HSP90) in a cohort of breast cancer patients. Methods Immunohistochemical analysis of 314 surgical specimens obtained from patients with invasive breast cancer was carried out to assess standard pathologic factors and the expression of HDAC6 and acetyl-HSP90. Statistical analyses were performed to determine the association between HDAC6, acetyl-HSP90, and conventional clinicopathological factors, and the prognostic values of these factors were evaluated. Results HDAC6 expression did not show any correlation with other clinicopathological factors, but acetyl-HSP90 was significantly correlated with histologic grade (p=0.001) and the Ki-67 index (p=0.015). HDAC6 and acetyl-HSP90 expression were significantly associated with each other (p=0.047). Although HDAC6 was not prognostic for disease-free survival (DFS), some patients with high expression of HDAC6 experienced recurrence 5 years after diagnosis, while there was no recurrent disease after 5 years in those with low expression. Acetyl-HSP90 was significantly associated with the DFS of all patients (p=0.016) and with high HDAC6 expression (p=0.017), but not with low expression. Conclusion Expression of HDAC6 and acetyl-HSP90 are correlated. HDAC6 is proposed to be a possible predictive marker of late recurrence, and acetyl-HSP90 has prognostic value in predicting the DFS of breast cancer patients.

Prognostic relevance of programmed cell death ligand 1 expression in glioblastoma

The aim of this study was to determine the clinicopathological significance of programmed cell death ligand 1 (PD-L1) expression in glioblastoma (GBM). In a retrospective cohort of 115 consecutive patients with GBM, PD-L1 expression was determined using immunohistochemistry (IHC). Membranous and fibrillary PD-L1 staining of any intensity in > 5% neoplastic cells and tumour infiltrating immune cells (TIIs) was considered positive staining. In addition, isocitrate dehydrogenase-1 (IDH-1) (R132H) expression and cluster of differentiation 3 (CD3)-positive T-cell infiltration were investigated using IHC. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation assay and fluorescence in situ hybridization (FISH) for the assessment of 1p/19q deletion were performed. Expression of PD-L1 in tumour cells and TIIs was found in 37 (32.2%) and 6 (5.2%) patients, respectively. Kaplan–Meier analysis indicated that PD-L1 expression in tumour cells was significantly associated with poor overall survival (OS) (P = 0.017), though multivariate Cox analysis did not confirm this association (hazard ratio 1.204; P = 0.615). PD-L1 expression in TIIs did not correlate with the patient prognosis (P = 0.545). In addition, MGMT methylation and IDH-1 (R132H) expression were associated with a better prognosis (P < 0.001 and P = 0.024, respectively). The expression of PD-L1 was associated with CD3-positive T-cell infiltration (P < 0.001), and IDH-1 wild type status (P = 0.008). A deeper insight into PD-L1 expression could help to ensure the success of future immunotherapy in GBM. Our study suggested that PD-L1 target therapy might be beneficial for PD-L1-expressing GBM patients with a poor prognosis.

Programmed cell death ligand-1 protein expression and CD274/PD-L1 gene amplification in colorectal cancer: Implications for prognosis

Programmed cell death ligand‐1 (PD‐L1) detection assays have not been standardized for patients with colorectal cancer, and the prognostic value of PD‐L1 expression is unclear. We compared the PD‐L1 expression patterns in colorectal cancer samples using various immunohistochemical assays using 3 primary PD‐L1 antibodies (assay 1, MIH1; assay 2, E1L3; and assay 3, 22C3) and investigated the prognostic implication of PD‐L1 expression using each. Additionally, PD‐L1 gene amplification was evaluated using FISH. The percentage scorings and positivity rates of the 3 assays differed; the degrees of correlation and concordance between assays 2 and 3 were relatively high, whereas assay 1 was an outlier. Multivariate analyses indicated that PD‐L1 positivity in tumor cells and its negativity in tumor‐infiltrating lymphocytes were independent predictors of poorer overall and disease‐free survival in patients with colorectal cancer. PD‐L1 gene amplification was found in 2 patients (PD‐L1/CEP ratio, 5.60 and 5.84, respectively); both had strong PD‐L1 expression according to immunohistochemistry. Overall, our study showed that PD‐L1 expression status in tumor and immune cells is an independent prognostic factor in patients with colorectal cancer. Standardizations of both PD‐L1 detection using immunohistochemistry and the cut‐off for positivity are necessary. Finally, PD‐L1 gene amplification was found in a small fraction of samples, suggesting the possibility of an ancillary test for PD‐L1 evaluation.

Comparative analysis of the EGFR, HER2, c-MYC, and MET variations in colorectal cancer determined by three different measures: gene copy number gain, amplification status and the 2013 ASCO/CAP guideline criterion for HER2 testing of breast cancer

BackgroundThe purpose of this study was to explore gene copy number (GCN) variation of EGFR, HER2, c-MYC, and MET in patients with primary colorectal cancer (CRC).MethodsDual-colour silver-enhanced in situ hybridization was performed in tissue samples of 334 primary CRC patients. The amplification status (GCN ratio ≥2) and GCN gain (average GCN ≥4) data for the EGFR, HER2, c-MYC and MET genes were obtained. GCN variation was also assessed by the criterion of the 2013 ASCO/CAP guidelines for HER2 testing.ResultsAmplification of EGFR, HER2, c-MYC and MET was detected in 8 (2.4%), 20 (6.0%), 29 (8.7%), and 14 (4.2%) patients, respectively. Of 66 patients with at least one amplified gene, five exhibited co-amplification of genes studied (HER2-MET co-amplification: two patients; HER2-c-MYC co-amplification: two patients; EGFR-c-MYC co-amplification: one patient). There were 109 patients with GCN gains of one or more genes (EGFR: 11/334, HER2: 29/334, c-MYC; 60/334, MET: 48/334) and 32.1% (35/109) had multiple GCN gains. When each GCN was assessed by the criterion of the ASCO/CAP 2013 guideline for HER2 testing, 116 people showed positive or equivocal results for one or more genes. The cumulative amplification status had no association with patients’ outcome. However, the cumulative results of the GCN gain and GCN status determined according to the ASCO/CAP guideline had a significant prognostic correlation in the univariate analysis (P values of 0.006 and 0.022, respectively). In the multivariate analysis, GCN gain and GCN status were independent prognostic factors (P values of 0.010 and 0.017, respectively).ConclusionsIn this study, we evaluated GCN variation of four genes in a large sample of Korean CRC patients. The amplification status was not related to patient outcome. However, the GCN gain and GCN status according to the ASCO/CAP 2013 guideline were independent prognostic factors.

Selection of super-invasive cell population from the glioblastoma cell line and analysis of invasion-associated biomarkers

S S137 cells with vacuolated cytoplasm were noted. Immunohistochemistry performed on tumour showed positive staining with calretinin, WT1, CAM5.2 and negative staining with Berep4, CD34, and S100. Conclusion:Adenomatoid tumours mimicking testicular tumour can present a challenge for urologist to decide on the most appropriate surgery. Clinical awareness of the unusual location of adenomatoid tumour could elect patient for testis sparing surgery. 60. EPITHELIAL SPARC EXPRESSION IS CORRELATED WITH POOR SURVIVAL AS WELL AS MMP-2 EXPRESSION OF INVASIVE BREAST CARCINOMA Ji Shin Lee, Ga-Eon Kim, Jae Hyuk Lee, Jong Hee Nam, Chan Choi Department of Pathology, Chonnam National University Medical School, Gwangju, Republic of Korea Background: Invasion and metastasis are the direct causes of mortality in breast cancer patients. Secreted protein acidic and rich in cysteine (SPARC) plays a crucial role in the process of tumor invasion and metastasis in some tumors. Matrix metalloproteinases (MMPs) degrade the extracellular matrix and participate in tumor invasion and metastasis. Aims: The aims were to assess the role of SPARC alteration in the breast carcinogenesis and its association with outcome variables and MMP expression in invasive breast carcinoma (IBC). Methods: Immunohistochemical staining with anti-SPARC antibody was performed in a total of 300 patients with 26 normal breasts, 76 ductal carcinoma in situ (DCIS), and 198 IBC using tissue microarray. In addition, we also performed immunohistochemical staining for MMP-2 and MMP-9 in IBC. Results: Epithelial SPARC expression appeared to increase progressively along the continuum of neoplastic changes from normal breast epithelium to IBC (P < 0.001). The patients with high epithelial SPARC expression had a significantly poorer prognosis for disease-free and overall survival than those with low epithelial expression (p=0.002 and p=0.048, respectively). Epithelial SPARC expression was independently associated with an increased risk for poor disease-free survival. Epithelial SPARC expression was statistically correlated with MMP-2 expression (p<0.05). Conclusions: Tumor progression in breast epithelium is accompanied by increased epithelial SPARC expression. High epithelial SPARC expression may serve as a new parameter for the prognostic prediction in patients with IBC. SPARC-mediated degradation of the extracellular matrix, and its possible association with MMP-2, may contribute to the progression of IBC. 61. HEPATIC REACTIVE LYMPHOID HYPERPLASIA ASSOCIATED WITH MALIGNANT TUMOR Sik Lee, Jun Sang Bae, Woo Sung Moon Department of Pathology, Internal Medicine, Chonbuk National University, Medical School, and Research Institute for Endocrine Sciences, Jeonju, Korea Reactive lymphoid hyperplasia (RLH) is a rare benign condition that forms a mass-like lesion characterized by proliferation of non-neoplastic lymphocytes forming follicles and germinal centers. RLH is thought to represent a reactive immunological response, and some cases may arise in association with malignancy. Herein, we present four cases of hepatic RLH associated with malignant tumor clinically mimic metastatic tumor. All 4 patients were women ranging from 45 to 74 years (mean, 59 years). The hepatic lesions ranged from 0.5 to 1.5 cm. Radiologically, these lesions mimic a primary or metastatic malignancy. All 4 lesions were associated with internal malignancies; renal cell carcinoma, ovarian mucinous carcinoma, colon adenocarcinoma and bile duct carcinoma, respectively. Microscopically, the lesions are characterized by formation of follicles with polymorphic and polyclonal cell population and active germinal centers. There was no evidence of lymphoepithelial lesion or monoclonal B-cell proliferation. It can be suggested that the pathogenesis of RLH of the liver in patients with malignant tumor may be related to an immunologic abnormality that is caused by the malignant tumor itself or previous surgery for the tumors. 62. SELECTION OF SUPER-INVASIVE CELL POPULATION FROM THE GLIOBLASTOMA CELL LINE AND ANALYSIS OF INVASION-ASSOCIATED BIOMARKERS Kyu Sang Lee, Hyun Sook Jung, Seyoung Moon, Gheeyoung Choe Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Korea Background: Glioblastoma is characterized by aggressive local invasiveness, making complete surgical resection nearly impossible. To identify mechanisms of glioblastoma invasion, we isolated a subpopulation of more invasive cells and evaluated the expression of invasion related factors including integrin subunits, matrix metalloproteinase-2 (MMP2), fascin, actin and Erk1/2. Methods: We selected the first 10% of invading cells (U87-Inv) from U87MG using laminin-2 (merosin) coated Transwell filters. To characterize the more invasive cells, we used wound assay, gelatin zymography, proliferation assay, RT-PCR for integrinreceptors for merosin, western blot and immunofluorescence for fascin and actin. Results: Migration rate of U87-Inv cells exhibited about 20% increase compared with relatively lower invasive cells (U87Non). U87-Inv cells demonstrated faster wound healing, whereas lower proliferative activity. The expression levels of integrin a1, a7, and b1 increased a little higher (about 1.2-fold for b1 and 1.5-fold for a1 and a7) in U87-Inv compared with U87-Non, while the expression of integrin a6 and b4 reduced in U87-Inv (about 0.4-fold for b4 and 0.6-fold for a6). U87-Inv cells revealed increase of fascin, actin and MMP2, whereas they showed decrease of phosphorylated Erk1/2. By immunofluorescence study, U87-Inv cells revealed extensive lamellipodia expressing both fascin and actin. S138 PATHOLOGY 2016 ABSTRACT SUPPLEMENT Pathology (2016), 48(S1) Conclusions: We isolated a subpopulation of more invasive glioblastoma cells selected in merosin substrate. Our findings suggest that merosin substrate is related to the brain invasion, and that increased expression of a1, a7 and b1 integrin subunits, MMP2 activation, and fascin-actin interaction lead to increased motility and invasiveness in U87MG cell line. 63. THE SIGNIFICANCES OF NUCLEAR AND CYTOPLASMIC OVEREXPRESSION OF METASTATIC TUMOR 1 IN HEPATOCELLULAR CARCINOMA Sang Hwa Lee, Hyun Hee Chu, Eunsil Yu Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Background: Metastatic tumor antigen 1 (MTA1) protein is usually localized in the nuclei and its overexpression is suggested to be associated with vascular invasion, frequent postoperative recurrence, and poor survival in patients with hepatocellular carcinoma (HCC). Recently cytoplasmic MTA1 localization is recognized, however, its potential relationship with clinicopathological characteristics has not been examined. Aims: We investigated nuclear and cytoplasmic expressions of MTA1 to evaluate their relationship with clinicopathological characteristics in patients with HCC by immunohistochemistry. Methods: A total 263 patients with HCC who underwent resection from January 2013 to February 2015 were subjected. The patients previously treated by transarterial chemoembolization, radiofrequency ablation, and resection for recurrent HCC were excluded. Nuclear and cytoplasmic overexpressions of MTA1 were defined as positive in nucleus and cytoplasm more than 5% of tumor cells, respectively by immunohistochemistry. Results: Nuclear overexpression was found in 154 patients. It was significantly associated with female gender (p=0.039), viral etiology (p=0.02), high Edmondson-Steiner grade (p=0.022 in worst grade and p=0.008 in most grade), portal vein invasion (p=0.017), Glisson capsule invasion (p=0.026), high T stage (p=0.016), and recurrence (p=0.017). Interestingly, cytoplasmic overexpression was associated with beneficial factors for clinicopathological characteristics. Cytoplasmic overexpression was found in 183 patients. Negative for cytoplasmic overexpression was significantly associated with high histologic grade (p=0.003 in most grade), portal vein invasion (p=0.009), Glisson capsule invasion (p=0.028), presence of satellite nodule (p<0.001), high T stage (p=0.003), recurrence (p=0.02), and distant metastasis (p=0.005). A total 109 cases with negative for nuclear overexpression, negative for cytoplasmic overexpression was significantly associated with high histologic grade (p=0.01 in most grade), Glisson capsule invasion (p=0.033), presence of satellite nodule (p=0.012), high T stage (p=0.033), and distant metastasis (p=0.003). Conclusion: Nuclear overexpression of MTA1 is associated with adverse clinicopathological factors, while cytoplasmic overexpression of MTA1 is associated with beneficial clinicopathological factors in HCC. 64. CARBONIC ANHYDRASE 9 EXPRESSION IN PANCREATIC NEUROENDOCRINE TUMORS IS ASSOCIATED WITH AGGRESSIVE BEHAVIOR Sang Hwa Lee, Joo Young Kim, Soyeon An, Ki-Byung Song, Dae Wook Hwang, Song Cheol Kim, Eunsil Yu, Seung Mo Hong 1Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 2Departmen of Pathology, Korea University, Anam Hospital, Seoul, and 3Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine,