Supplementary Material for: Loss of Heterozygosity of PTEN (Encoding Phosphate and Tensin Homolog) Associated with Elevated HER2 Expression Is an Adverse Prognostic Indicator in Gastric Cancer

<b><i>Objective:</i></b> PTEN (the encoding phosphate and tensin homolog) is a well-known cancer suppressor gene and its mutation and loss of heterozygosity (LOH) occurs in various types of carcinomas. This study aimed to examine the association between LOH of PTEN and prognosis in HER2-expressing and nonexpressing gastric cancer patients. <b><i>Methods:</i></b> Fresh-frozen tumor samples of 221 gastric cancer patients with a primary diagnosis of gastric carcinoma were examined for LOH of PTEN. The results were compared with pathological parameters and the HER2 status. To elucidate the role of LOH of PTEN, the activation of the PI3K/AKT pathway was examined immunohistochemically using a phosphorylation-specific antibody. <b><i>Results:</i></b> LOH of PTEN was observed in 20% of the patients (39 of 195 cases). LOH of PTEN was associated with vascular involvement (25 of 39 cases; p = 0.0083), equivocal to positive staining for HER2 (p = 0.0080), and phospho-Akt expression (p = 0.0067). Patients with HER2-expressing gastric cancer with LOH of PTEN had a significantly worse prognosis (p = 0.0050). <b><i>Conclusions:</i></b> Although HER2 expression itself was not a prognostic factor, the combination of HER2 expression and LOH of PTEN exacerbates the malignant potential of gastric cancer through its proliferative function. © 2014 S. Karger AG, Base

Supplementary Material for: The Expression of CCAT2, a Novel Long Noncoding RNA Transcript, and rs6983267 Single-Nucleotide Polymorphism Genotypes in Colorectal Cancers

<p>Colon cancer-associated transcription 2 (<i>CCAT2</i>) was recently identified as a novel long noncoding RNA transcript encompassing the single-nucleotide polymorphism rs6983267. <i>CCAT2</i> is overexpressed in colorectal cancer (CRC) where it promotes tumor growth, metastasis, and chromosomal instability, although the clinical relevance of this enhanced expression is unknown. In this retrospective study, <i>CCAT2</i> expression was evaluated using real-time polymerase chain reaction in 149 CRC patients, and its associations with clinicopathological characteristics, outcome, rs6983267 genotypes, microsatellite status, DNA ploidy, and <i>BubR1</i> expression were analyzed. <i>CCAT2</i> expression in cancer tissue was significantly higher than in noncancer tissue (<i>p </i>< 0.001), particularly in cases of metastatic cancer (<i>p </i>< 0.001). However, relative <i>CCAT2</i> expression levels and rs6983267 genotypes were not correlated with clinicopathological features or patient prognosis. CRC cases demonstrating high <i>CCAT2</i> expression were all microsatellite stable (<i>p </i>< 0.005). Together, this indicates that <i>CCAT2</i> expression was associated with microsatellite-stable CRC.</p

Supplementary Material for: Gastric Cancer Patients with High PLK1 Expression and DNA Aneuploidy Correlate with Poor Prognosis

<p>Gastric cancer is the fourth most common cancer worldwide. Although it is important to identify patients at high risk for a poor outcome, factors correlating with prognosis in gastric cancer are largely unknown. Here, we focus on the correlations among expression of Polo-like kinase 1 (PLK1), DNA ploidy, and clinical outcome in gastric cancer patients. Gastric cancer specimens were analyzed from 207 consecutive patients. Patients were classified into two groups according to tumor PLK1 expression and DNA content, and an analysis of their clinical outcomes was carried out. Prognoses of patients with PLK1-high tumors were worse than those of patients with PLK1-low tumors, but the differences were not statistically significant. In cell lines, overexpression of PLK1 induced centrosome amplification and multipolar spindles, potentially leading to DNA aneuploidy. Indeed, high expression of PLK1 was also associated with DNA aneuploidy in clinical gastric cancer specimens. Patients with both high PLK1 expression and DNA aneuploidy had poor recurrence-free survival, whereas PLK1 expression and DNA ploidy status alone were not significantly associated with outcome. Here, we provide clinical evidence that high expression of PLK1 could have detrimental effects in tumors with DNA aneuploidy, which may increase the risk of recurrence in gastric cancer patients.</p

Supplementary Material for: Protein Expression of Programmed Death 1 Ligand 1 and HER2 in Gastric Carcinoma

<p><b><i>Objectives:</i></b> Programmed death 1 (PD-1) is an immunoinhibitory receptor and has been identified as a new target for immunotherapy in cancer. Here we report the expression of PD-1 ligand 1 (PD-L1) in surgically resected gastric cancer. <b><i>Materials and Methods:</i></b> We examined formalin-fixed tumor samples from 144 gastric cancer patients with a primary diagnosis of gastric carcinoma. Immunohistochemistry was used to detect PD-L1. Human epidermal growth factor receptor 2 (HER2) expression and phosphatase and tensin homolog (PTEN) loss of heterozygosity were investigated in these patients. RNA interference was used to downregulate HER2 expression, and PD-L1 protein expression was assessed by flow cytometry using the gastric cancer cell line MKN45. <b><i>Results:</i></b> Overexpression of PD-L1 was significantly correlated with tumor invasion (<i>p</i> = 0.011) and associated with poor survival. The number of PD-L1-positive cases increased according to the HER2 score in clinical samples. siRNA-mediated downregulation of HER2 significantly decreased PD-L1 protein expression in MKN45 cells. <b><i>Conclusions:</i></b> PD-L1 expression was associated with poor survival of gastric cancer, and HER2 signaling affects the expression of PD-L1 in gastric cancer. In gastric cancer, PTEN and HER2 are potential candidate biomarkers for developing human antibodies that block PD-L1.</p

Supplementary Material for: Altered Expression of Hippo Signaling Pathway Molecules in Intrahepatic Cholangiocarcinoma

<p><b><i>Objective:</i></b> MOB1, a core component of the Hippo signaling pathway, suppresses cell proliferation, and MOB1 liver conditional knockout mice develop intrahepatic cholangiocarcinoma (ICC). However, its clinical significance in human ICC has not been established. The aim of this study was to characterize protein levels and the role of Hippo and TGF pathways in ICCs. <b><i>Methods:</i></b> The protein levels of yes-associated protein 1 (YAP1), MOB1, Smad2, and TGFβ2 in 88 ICC cases were analyzed. Protein level was graded by a scoring system; then, the clinicopathological factors, including prognosis, were analyzed based on protein level. <b><i>Results:</i></b> Nuclear overexpression of YAP1 was seen in 28 cases (31.8%), and it was significantly associated with a poor overall survival rate (<i>p</i> = 0.01). MOB1 expression decreased in 42 cases (47.7%) and was associated with a poor overall survival rate (<i>p</i> = 0.02). SMAD2 nuclear localization was significantly correlated with a high YAP1 level independent of TGFβ2. Multivariate analysis revealed that a high YAP1 level, a low MOB1 level, and lymphatic permeation were independent risk factors for overall survival. <b><i>Conclusions:</i></b> These results showed that key components of the Hippo signaling pathway are aberrantly expressed and associated with the malignant potential of human ICC.</p

Supplementary Material for: Prophylactic Effect of Dexamethasone on Regorafenib-Related Fatigue and/or Malaise: A Randomized, Placebo-Controlled, Double-Blind Clinical Study in Patients with Unresectable Metastatic Colorectal Cancer (KSCC1402/HGCSG1402)

<b><i>Background:</i></b> Regorafenib is an oral multikinase inhibitor with a proven survival benefit for metastatic colorectal cancer patients. The KSCC1402/HGCSG1402 study investigated the prophylactic effect of oral dexamethasone (DEX) on regorafenib-related fatigue and/or malaise. <b><i>Patients and Methods:</i></b> Patients who progressed after standard chemotherapy were randomized 1: 1 to a DEX group (2 mg/day; days 1–28) with regorafenib or a placebo group with regorafenib. The primary endpoint was the incidence of fatigue and/or malaise, based on version 4.0 of the National Cancer Institute’s CTCAE (Common Terminology Criteria for Adverse Events). One of the secondary endpoints was the in­cidence of fatigue and/or malaise based on the CTCAE assessed by patient-reported outcome (PRO). <b><i>Results:</i></b> The incidence of any grade of fatigue and/or malaise assessed by the investigators was 58.8% in the DEX group and 61.1% in the placebo group (<i>p</i> = 0.8101), and that assessed by PRO was 47.2 and 58.3%, respectively (<i>p</i> = 0.3450). The incidence of grade ≥2 fatigue and/or malaise, as assessed by the investigators, was 19.4% for the DEX group and 38.9% for the placebo group (<i>p</i> = 0.0695), and that assessed by PRO was 27.8 and 52.8%, respectively (<i>p</i> = 0.0306). <b><i>Conclusion:</i></b> Our results suggest that prophylactic oral DEX is clinically effective in improving regorafenib-related fatigue and/or malaise