Kenji Warigaya

Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

Tumor budding is an independent risk factor for lymph node metastasis in cutaneous squamous cell carcinoma: a single center retrospective study.

Although tumor budding is acknowledged as a risk factor for lymph node metastasis in certain types of carcinoma, it is not well investigated in cutaneous squamous cell carcinomas (SCCs). In this study, we analyzed the correlation between tumor budding and nodal metastasis in cutaneous SCC.

Significant Correlation between Chromosomal Aberration and Nuclear Morphology in Urothelial Carcinoma

We aimed to identify whether there is any correlation between chromosomal/genetic changes, nuclear morphology and the histological grade of urothelial carcinomas of the urinary bladder. Morphometry and multicolour fluorescence in situ hybridisation (FISH) techniques were applied to 250 cells in five low-grade cases and 350 cells in seven high-grade cases of urothelial carcinoma. Compared with low-grade carcinomas, most high-grade cases showed larger and more variable nuclear size, more frequent polysomy of centromere enumeration probes (CEPs) 3, 7 and 17, and the loss of the 9p21 locus. The number of CEP signals in cells was increased as the nuclear area of the cells became larger. Cells with gains in two or more types of CEP had significantly larger nuclei than cells with normal FISH signal patterns. In conclusion, the present study indicates that there was a correlation between nuclear morphology and chromosomal/genetic changes which were related to histological grading. Thus, we show that differences in the chromosomal/genetic aberrations present in low- and high-grade tumours can affect not only nuclear morphology but also the histopathological and clinical behaviour of urothelial carcinomas.

Adipophilin expression in cutaneous malignant melanoma

The lipogenic pathway is upregulated in cancer cells, including melanomas. However, the pathological significance of cellular lipids in melanocytic lesions has yet to be determined. In this study, we evaluated intracytoplasmic lipid droplets in melanocytic nevi (MNs) and malignant melanomas via immunohistochemical analysis of adipophilin (ADP), which coats lipid droplets.

Effective localization in tumor-induced osteomalacia using 68Ga-DOTATOC-PET/CT, venous sampling and 3T-MRI

Summary Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome characterized by renal phosphate wasting leading to hypophosphatemia due to excessive actions of fibroblast growth factor 23 (FGF23) produced by the tumors. Although the best way of curing TIO is complete resection, it is usually difficult to detect the culprit tumors by general radiological modalities owing to the size and location of the tumors. We report a case of TIO in which the identification of the tumor by conventional imaging studies was difficult. Nonetheless, a diagnosis was made possible by effective use of multiple modalities. We initially suspected that the tumor existed in the right dorsal aspect of the scapula by 68Ga-DOTATOC positron emission tomography/computed tomography (68Ga-DOTATOC-PET/CT) and supported the result by systemic venous sampling (SVS). The tumor could also be visualized by 3T-magnetic resonance imaging (MRI), although it was not detected by 1.5T-MRI, and eventually be resected completely. In cases of TIO, a stepwise approach of 68Ga-DOTATOC-PET/CT, SVS and 3T-MRI can be effective for confirmation of diagnosis. Learning points: TIO shows impaired bone metabolism due to excessive actions of FGF23 produced by the tumor. The causative tumors are seldom detected by physical examinations and conventional radiological modalities. In TIO cases, in which the localization of the culprit tumors is difficult, 68Ga-DOTATOC-PET/CT should be performed as a screening of localization and thereafter SVS should be conducted to support the result of the somatostatin receptor (SSTR) imaging leading to increased diagnosability. When the culprit tumors cannot be visualized by conventional imaging studies, using high-field MRI at 3T and comparing it to the opposite side are useful after the tumor site was determined.

Macrocystic ductal adenocarcinoma of prostate: A rare gross appearance of prostate cancer

Macroscopic cyst-formation by prostatic adenocarcinoma, herein referred to as macrocystic prostatic adenocarcinoma (MPA), is extremely rare. To date, no studies of prostate cancer performed based on gross cystic appearance have been reported. MPA can include various diseases, one of which is cystadenocarcinoma. Several cases of ductal adenocarcinoma exhibiting a macrocystic appearance have recently been reported; however, the histological and clinicopathological characteristics of MPAs have yet to be characterized and established. Therefore, we aimed to determine the histological and clinicopathological characteristics of MPA, via a multi-institutional investigation. We discovered five patients with MPA out of 1559 treated patients (0.32%); all cases were ductal adenocarcinomas. MPA was found to have three growth patterns: Two cases showed a prevalence of exuberant papillary proliferation with a fibrovascular core in the macroscopic multilocular cysts. Two others predominantly exhibited multilocular cysts lined by flat epithelium with foci of low papillae, and the fifth had a cystic lesion with intracancerous necrosis. Three of the cases showed extraprostatic invasion; however, none of the patients experienced recurrence during the follow-up period. Each predominant growth pattern tended to exhibit unique clinicopathological characteristics with respect to serum prostate specific antigen level and tumor size and location. In conclusion, we demonstrated that MPA is a ductal adenocarcinoma that is composed of intracystic exuberant papillary proliferation and flat proliferation with foci of low papillae, both of which might exhibit different clinicopathololgical appearances.

Refractory Ascites with Liver Fibrosis Developed in Late Phase Allogeneic Hematopoietic Stem Cell Transplantation: Report of Three Patients.

We report cases of three patients of refractory ascites without other fluid retention that occurred around five months after allogeneic hematopoietic stem cell transplantation (allo-HSCT). All three patients expired and postmortem examinations revealed unexpected liver fibrosis lacking histological evidences of graft-versus-host-disease (GVHD). The three patients showed normal hepatic function and size before transplantation. During their clinical courses, serum biochemistry test showed no elevation of hepatic enzymes and bilirubin; however, imaging studies demonstrated hepatic atrophy at the onset of ascites. One of the liver specimens showed bile obstruction, which could be seen in hepatic damage by GVHD. Although ascites resulting from venoocclusive disease in early phase allo-HSCT is well documented, ascites associated with hepatic fibrosis in late phase allo-HCST has not been reported. Further clinico-pathological studies on similar patients should be required to ascertain refractory ascites associated with liver fibrosis after allo-HSCT.

Correlation analysis of nuclear morphology, cytokeratin and Ki-67 expression of urothelial carcinoma cells

We aimed to delineate the morphogenesis of aberrant nuclear features of urothelial carcinoma (UC) cells in association with cytokeratin (CK) expression patterns and cell proliferation activity. Correlation analysis of the nuclear area by morphometry and the expression patterns of CK5, CK20 and Ki‐67 by triple immunofluorescence analysis was applied to 1699 cells from five low‐grade and seven high‐grade cases of UC. The majority of UC cells showed aberrant cellular differentiation represented by abnormal CK expression patterns of CK5(+)/CK20(+) (40.5%) or CK5(−)/CK20(+) (56.0%). CK5(+)/CK20(−) cells, a phenotype of cancer stem/progenitor cells, represented a very small population (1.9%) and showed a low proliferation activity. Ki‐67(+) cells showed a significantly different CK expression pattern compared with that of Ki‐67(−) cells. The nuclear areas of CK5(−)/CK20(+) cells (71.3 ± 25.9 μm2) were significantly larger than those of CK5(+)/CK20(+) cells (66.6 ± 25.5 μm2). Negativity for CK5 was related to the grade of UC and an increased number of CK5(−)/CK20(+)/Ki‐67(+) cells was related to a higher malignant potential. We conclude the nuclear morphology is related to cell differentiation represented by CK expression and cell proliferative activity.

Endoscopic ultrasound-guided biliary drainage using a newly designed metal stent with a thin delivery system: a preclinical study in phantom and porcine models

PurposeThis study was designed to evaluate the feasibility and safety of a newly designed self-expandable metal stent for endoscopic ultrasound-guided biliary drainage (EUS-BD) when it was delivered via three different stent delivery systems: a 7.5Fr delivery catheter with a bullet-shaped tip (7.5Fr-bullet), a 7Fr catheter with a bullet-shaped tip (7Fr-bullet), or a 7Fr catheter with a tee-shaped tip (7Fr-tee).MethodsThis experimental study utilized a porcine model of biliary dilatation involving ten pigs. In the animal study, technical feasibility and clinical outcomes of the stent when placed with each of the delivery systems were examined. In addition, a phantom model was used to measure the resistance of these delivery systems to advancement.ResultsPhantom experiments showed that, compared with 7Fr-bullet, 7Fr-tee had less resistance force to the advancement of the stent delivery system. EUS-BD was technically successful in all ten pigs. Fistulous tract dilation was necessary in 100% (2/2), 75% (3/4), and 0% (0/4) of the pigs that underwent EUS-BD using 7.5Fr-bullet, 7Fr-bullet, and 7Fr-tee, respectively. There were no procedure-related complications.ConclusionOur newly designed metal stent may be feasible and safe for EUS-BD, particularly when delivered by 7Fr-tee, because it eliminates the need for fistulous tract dilation.

HER2 is frequently overexpressed in hepatoid adenocarcinoma and gastric carcinoma with enteroblastic differentiation: a comparison of 35 cases to 334 gastric carcinomas of other histological types

Aims α-Fetoprotein (AFP)-producing gastric carcinoma (AFPGC) is one of the most aggressive GC subtypes. Frequent expression of human epidermal growth factor receptor 2 (HER2) has previously been reported in hepatoid adenocarcinoma (HAC), a major histological subtype of AFPGC originating from common-type GC (CGC). However, HER2 expression levels in other AFPGC histological subtypes are unknown. In this study, we analysed HER2 expression in GCs with primitive phenotypes in addition to HAC. Methods HER2 expression was evaluated in representative complete sections from 16 HACs, 19 GCs with enteroblastic differentiation (GCEDs) and 334 GCs of other histological types as controls. The Ruschoff/Hofmann method was used to score HER2 immunohistochemistry. Samples with a HER2 score of 2+ were further assessed using fluorescence in situ hybridisation. Oncofetal protein (OFP) expression in HAC and GCED was tested via immunohistochemical staining for AFP, glypican 3 and Sal-like protein 4. Results Thirty of 35 HAC/GCED cases comprised more than two histological patterns. The HER2 positivity rates of each histological component in the HACs/GCEDs were 25.0% for HAC (n=16), 34.6% for GCED (n=26) and 48.1% for CGC (n=27), which were higher than those of the control group (13.8%). Additionally, the majority of CGC components in HACs/GCEDs were positive for OFP (88.9%). Conclusions HER2 is frequently overexpressed not only in HAC but also in GCED and CGC components of HACs/GCEDs, which suggests an association between HER2 and OFP expression. Moreover, our findings suggest that HER2-positive CGC has a higher risk of progression to HAC/GCED than HER2-negative GC.