Ryuichi Wada

“Wild type” GIST: Clinicopathological features and clinical practice

Gastrointestinal stromal tumor (GIST) is a mesenchymal tumor of the gastrointestinal tract. Mutation of KIT and PDGFRA genes is implicated in the tumorigenesis. Approximately 10% of GISTs do not harbor mutation of these genes, and they are designated as “wild type” GIST. They are classified into succinate dehydrogenase (SDH)‐deficient and non‐SDH‐deficient groups. SDH‐deficient group includes Carney triad and Carney Stratakis syndrome. The patients are young women. Tumors occur in the antrum of the stomach, and tumor cells are epithelioid. Lymph node metastasis is frequent. The non‐SDH‐deficient group includes neurofibromatosis (NF) type 1 and GISTs with mutations of BRAF, KRAS, and PIK3CA and with the ETV6‐NTRK3 fusion gene. GIST in NF occurs in the small intestine, and tumor cells are spindle shaped. GIST with BRAF mutation arises in the small intestine. Attention to the age, gender, family history and other neoplasms may raise the prediction of syndromic disease. Location of the tumor, morphology, and pleomorphism of the tumor cells are further informative. Lymphovascular invasion should be carefully evaluated. The determination of KIT expression is essential for the diagnosis. When wild type GIST is suspected, intensive genetic analysis is required. Further, a careful and long‐time observation is recommended.

Insulin-like growth factor 2 mRNA-binding protein-3 as a marker for distinguishing between cutaneous squamous cell carcinoma and keratoacanthoma

In the histopathological diagnosis of cutaneous tumors, the differential diagnosis of squamous cell carcinoma (SCC) with crateriform architecture and keratoacanthoma (KA) is often difficult so an accurate understanding of the biological features and the identification of reliable markers of SCC and KA are crucial issues. Insulin-like growth factor 2 mRNA-binding protein-3 (IGF2BP3, also known as IMP3) is thought of as a bona fide oncofetal protein, which is overexpressed and is involved in cell proliferation, migration, and invasion in several kinds of tumors. However, the role of IMP3 in cutaneous SCC and KA has not been well studied. Therefore, we focused on studying the biological functions of IMP3 in SCC and KA. In human skin SCC cell lines, HSC-1 and HSC-5, and the human keratinocyte cell line, HaCaT, IMP3 mRNA levels were significantly higher than that of normal human skin. The knockdown of IMP3 expression reduced the proliferation of HSC-1, and significantly reduced invasion by HSC-1 and HSC-5. In contrast, the knockdown of IMP3 did not significantly affect invasion by HaCaT cells. In immunohistochemical studies of SCC and KA tissues, the Ki-67 labeling index (LI) of the suprabasal cell layer was significantly higher in SCC, compared with KA tissues and the tumor-free margin (TFM) adjacent to SCC and KA. Most SCC tissues stained strongly positive for IMP3, but KA tissues and TFM were mostly negative for IMP3. The Ki-67 LI of the IMP3-positive group was significantly higher than that of the IMP3-negative group in the suprabasal cell layer of SCC. These results suggest that IMP3 plays an important role in proliferation and, more significantly, in the invasion of SCC, and may be a suitable marker for the histopathological diagnosis of SCC with a crateriform architecture and KA. Furthermore, IMP3 may potentially be a new therapeutic target for SCC.

Increased expression of PDIA3 and its association with cancer cell proliferation and poor prognosis in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) is unfavorable following complete tumor resection. The aim of the present study was to identify a molecule able to predict HCC prognosis through comprehensive protein profiling and to elucidate its clinicopathological significance. Comprehensive protein profiling of HCC was performed by liquid chromatography-tandem mass spectrometry. Through the bioinformatic analysis of proteins expressed differentially in HCC and non-HCC tissues, protein disulfide-isomerase A3 (PDIA3) was identified as a candidate for the prediction of prognosis. PDIA3 expression was subsequently examined in 86 cases of HCC by immunostaining and associations between PDIA3 expression levels and clinicopathological characteristics were evaluated. The Ki-67 index and apoptotic cell death of carcinoma cells were examined by immunostaining and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay in 24 cases. The results demonstrated that PDIA3 was expressed in all 86 HCC cases; 56 HCC cases (65%) exhibited high expression of PDIA3 and 30 (35%) exhibited low expression. The disease-free and overall survival times of HCC patients with high PDIA3 expression were significantly shorter than in HCC patients with low expression. Furthermore, increased expression of PDIA3 was associated with an elevated Ki-67 index, indicating increased cancer cell proliferation and a reduction in apoptotic cell death. Taken together, these results suggest that PDIA3 expression is associated with tumor proliferation and decreased apoptosis in HCC, and that increased expression of PDIA3 predicts poor prognosis. PDIA3 may therefore be a key molecule in the development of novel targeting therapies for patients with HCC.

Cystatin B as a potential diagnostic biomarker in ovarian clear cell carcinoma.

Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in EOCs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.

DNA damage response and its clinicopathological relationship in appendiceal tumors.

BackgroundAppendiceal tumors are rare, and their pathogenesis is not well known. DNA damage response (DDR) is a sequence from the detection of damaged DNA to the repair, and its impairment is implicated in the progression of cancers. The aim of the current study is to explore the expression and phosphorylation of checkpoint kinase 2 (Chk2) and TP53, which are key molecules in DDR, and their clinicopathological correlation in the appendiceal tumors.MethodsChk2, phosphorylated Chk2 (pChk2), and TP53 were immunostained in 4 cases of adenoma (AD), 5 non-mucinous adenocarcinomas (AC), 29 low-grade appendiceal mucinous neoplasms (LAMN), and 7 mucinous adenocarcinomas (MAC). Ki-67 labelingxa0index was also evaluated by immunostaining.ResultsChk2 was highly expressed in the nuclei of all the appendiceal tumors. While pChk2 was high in AD, LAMN, and MAC, it was reduced in AC. Nuclear positive reaction of TP53 was lower in LAMN compared with those of other tumors. The Ki-67 labeling index was slightly lower in LAMN than those in other tumors. The recurrence and death in LAMN is infrequent compared with those in AC and MAC.ConclusionsThe current study suggested the impairment of DDR in AC and MAC. DDR appeared to be preserved in LAMN, and it may account for low proliferating activity and a favorable clinical course in LAMN.

The Expression of Glucagon-Like Peptide-1 Receptor and Dipeptidyl Peptidase-IV in Neuroendocrine Neoplasms of the Pancreas and Gastrointestinal Tract

Neuroendocrine neoplasm (NEN) of the pancreas and gastrointestinal tract is infrequent but often produces hormones to cause distinct clinical features. Glucagon-like peptide-1 receptor (GLP1R) is a G-protein coupled receptor for GLP1, which is cleaved by dipeptidyl peptidase (DPP)-IV, a peptidase that regulates the activity of peptide hormones. Since these molecules are involved in the neuroendocrine function of NEN, they could serve molecular targets for diagnosis and therapy of NEN. However, the expressions of these molecules in NEN are not well studied. We therefore examined the expression of GLP1R and DPP-IV in 22 cases of pancreatic NEN (P-NEN) and 20 cases of gastrointestinal NEN (GI-NEN) by immunostaining. GLP1R was expressed in all eight insulinomas (100xa0%) but so in only four out of 14 cases (29xa0%) of non-insulinomas. In contrast to GLP1R, DPP-IV was detected in one out of eight insulinomas (13xa0%) and in 12 out of 14 cases (86xa0%) of non-insulinomas. In GI-NEN, GLP1R was negative in all 10 cases of the foregut NEN, whereas it was expressed in all three cases (100xa0%) of midgut NEN and four out of seven cases (57xa0%) of hindgut NEN. DPP-IV was expressed in five out of 10 cases (50xa0%) of the foregut NEN. The expression was detected in two out of three cases (67xa0%) of midgut NEN and in all seven cases (100xa0%) of hindgut NEN. In conclusion, we found distinct expression patterns of GLP1R and DPP-IV depending on the neuroendocrine cell types in P-NEN and the anatomical sites in GI-NEN.

Expression of DNA damage response proteins in gastric cancer: Comprehensive protein profiling and histological analysis

Gastric cancer is the third major cause of cancer-related mortality in Japan. The aim of this study was to identify a factor implicated in the biology of gastric cancer by comprehensive protein profiling. Protein profiling was carried out by liquid chromatography-tandem mass spectrometry, using formalin-fixed paraffin-embedded specimens of 17xa0gastric cancer cases. Pathway analysis and orthogonal partial least square-discriminant analysis suggested the significant expression of ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, interleukin binding factorxa02xa0(ILF2), KU70 and KU80, which are involved in DNA damage responsexa0(DDR). Thus, the expression and phosphorylation levels of KU70, ILF2, CHK1 and CHK2 were examined by immunohistochemistry in 42xa0cases of gastric cancer. The expressions of ILF2 and CHK1 were unaffected in all cases. The expression and phosphorylation of CHK2 were absent in 2 cases. Despite the expression of proteins, the phosphorylation of KU70 and CHK2 appeared to be impaired in 1 and 4xa0cases, respectively. In 7 out of 42xa0cases (17%), DDR appeared to be impaired. Recurrence was noted in 2 out of these 7xa0casesxa0(29%), whereas the recurrence was noted in 2 out of the remaining 35xa0casesxa0(6%). The expression levels of KU70, ILF2, CHK1, CHK2 and TP53 were further examined in 4xa0gastric cancer cell lines. The expression and phosphorylation levels following exposure to ultraviolet radiation were abnormal in the 3xa0cell lines. The normal consecutive phosphorylation of CHK1 and CHK2, the upregulation of TP53 and an increase in apoptotic cell death following exposure to ultraviolet radiation was detected only in one cell line, suggesting that the preserved functions of DDR and TP53 are necessary for the determination of cell fate. It is thus suggested that DDR plays an important role in the pathobiology of gastric cancers.

Uterine epithelioid leiomyosarcoma with c-kit expression and YWHAE gene rearrangement: a case report of a diagnostic pitfall of uterine sarcoma

BackgroundUterine sarcoma is a rare tumor that is often difficult to classify based on morphological and immunohistochemical analysis alone. Limited access to molecular biological analysis in routine practice would hinder making a definitive diagnosis.Case PresentationIn this report, we describe a case of a mesenchymal tumor arising from the uterine cervix in a 52-year-old woman. From microscopic morphology of the resected specimen, epithelioid leiomyosarcoma, high-grade endometrial stromal sarcoma, or uterine gastrointestinal stromal tumor (GIST) were considered as differential diagnoses. The immunophenotype of the tumor featured smooth muscle differentiation and hormone receptor expression. The cell membrane and cytoplasm were positive for c-kit, although no mutation was found in the c-kit or PDGFRA gene. Fluorescence in situ hybridization (FISH) analysis revealed a relatively low frequency of YWHAE rearrangement, whereas there were few NUTM2A and NUTM2B split signals.ConclusionsIn this case, the tumor was not typical of any three of the differential diagnoses mentioned above. However, insufficient frequency of YWHAE, NUTM2A, and NUTM2B gene rearrangement and absence of mutation in both the c-kit and PDGFRA genes suggested that this tumor should be categorized as epithelioid leiomyosarcoma. This is an instructive case showing a potential diagnostic pitfall of uterine sarcoma. Comprehensive approaches including molecular biological techniques are required for definitive diagnosis.

mRNA expression of delta-HER2 and its clinicopathological correlation in HER2-overexpressing breast cancer

Delta-human epidermal growth factor receptor 2 (HER2) is a splice variant of HER2, which lacks 16 amino acids in the extracellular domain. The aim of the present study was to elucidate the expression of delta-HER2 and its clinicopathological correlation in Japanese patients with HER2-overexpressing breast cancer. A total of 40 cases of HER2-overexpressing breast cancer were investigated. The mRNA expression levels of wild type (wt)-HER2 and delta-HER2 were quantitated by quantitative polymerase chain reaction using RNA extracted from formalin-fixed paraffin-embedded specimens. In addition, the activated form of phosphorylated-SRC (pSRC) and Ki-67 were detected by immunostaining. The mRNA expression levels of wt-HER2 varied largely, and the mRNA levels of delta-HER2 were correlated with those of wt-HER2. Furthermore, the enhanced immunostaining of pSRC was detected in 15 cases. Unsupervised hierarchal clustering of wt-HER2, delta-HER2 and pSRC was used to separate the cases into three clusters. In the first cluster, the levels of wt-HER2 and delta-HER2 were low, and pSRC immunostaining was low. Two other clusters were characterized by moderately and highly increased levels of wt-HER2 and delta-HER2 mRNA expression and enhanced pSRC. The delta/wt ratio was higher in the first cluster. Positive lymph node status and recurrence were more frequent in the first cluster compared with those in the two other clusters. Furthermore, the delta/wt ratio was significantly increased in lymph node-positive and recurrent cases, compared with in lymph node-negative and non-recurrent cases. The present study demonstrated that delta-HER2 was expressed in Japanese patients with HER2-overexpressing breast cancer. High mRNA expression levels of delta-HER2 were associated with pSRC and increased proliferation of tumor cells. A poor prognosis may be predicted by the increase in delta/wt ratio in HER2-overexpressing breast cancer.

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells

The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.