Cytological features of signet‐ring cell carcinoma of the lung: Comparison with the goblet‐cell‐type adenocarcinoma of the lung

Signet‐ring cell carcinoma (SRCC) and goblet‐cell‐type adenocarcinoma (GCA) are mucin‐producing lung adenocarcinomas. Primary SRCC shows an aggressive clinical course, whereas GCA shows infrequent distant metastasis, but more frequent intrapulmonary metastases resembling lobar pneumonia. To distinguish SRCC from GCA, this study investigated the respective cytological features of these lesions. We selected 10 cases each of SRCC and GCA from the archival imprint smears. We assessed them for the following 10 cytological features. Necrosis/debris was observed in 60% of the SRCC and 90% of the GCA. A mucinous background was observed in 10% of the SRCC and 90% of the GCA. Significant inflammation was observed in none of the SRCC and 80% of the GCA. Stromal cluster was observed in 30% of the SRCC and 70% of the GCA. Nuclear overlapping was observed in 50% of the SRCC and in all of the GCA. Single tumor cells were observed in 80% of the SRCC and 10% of the GCA. Honeycomb‐like cluster was observed in none of the SRCC and 80% of the GCA. Prominent nucleolus was observed in 50% of the SRCC and 40% of the GCA. Nuclear membrane irregularity was observed in 70% of SRCC and 60% of the GCA. Nuclear pleomorphism was observed in all of the SRCC and none of the GCA. The cytological features of SRCC were the presence of single tumor cells and nuclear pleomorphism, whereas that of GCA were the presence of abundant mucin and significant inflammation in the background, and a honeycomb‐like cluster. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Expression of regulator of G protein signalling protein 5 (RGS5) in the tumour vasculature of human renal cell carcinoma

The gene expression profiles of tumour and normal vasculature are distinctively different. The altered expression of various angiogenesis-related genes in tumour-derived endothelial cells has been investigated intensively, but there may be as yet unidentified molecules that regulate tumour angiogenesis. In the present study, the distinctive expression of regulator of G protein signalling protein 5 (RGS5) in tumour vessels in human renal cell carcinoma (RCC) has been clarified. RGS5 is a member of the RGS superfamily and acts as a negative regulator of heterotrimeric G protein-mediated signalling through G protein-coupled receptors (GPCRs). RT-PCR showed strong expression of RGS5 in all RCCs examined, but expression was very weak or undetectable in normal kidneys. By real-time RT-PCR, the ratio of RGS5 mRNA in RCC to that in normal kidney was 6.6 : 1 (p = 0.0012). In situ hybridization showed strong expression of RGS5 in vessels within tumour cell nests. It was expressed neither in tumour cells nor in normal renal capillaries. Immunohistochemical staining using serial sections for endothelial cell markers (CD31 and CD34) and smooth muscle cell markers (alpha-SMA and desmin), as well as fluorescence double staining, strongly suggested that tumour endothelial cells were the main location of RGS5 in RCC. These findings suggest that RGS5 may be involved in G protein-mediated signalling in tumour vessels in human RCC.     

Correlation of clinicopathological features and leucine-rich repeat-containing G-protein-coupled receptor 5 expression in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. Previous studies have established leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) as a cancer stem cell marker in gastrointestinal cancers. However, few reports have examined LGR5 in PDAC. Here we examined LGR5 expression and its clinicopathological significance in PDAC. We evaluated LGR5 expression in 78 PDAC patients who underwent surgical resection in our institution using RNAscope, a newly described RNA in situ hybridization technique. All 78 PDAC cases expressed LGR5 in cancer tissues, and LGR5 expression was prominent in the gland-forming part. LGR5 expression was significantly higher in patients with low histological grade (G1–G2) (p < 0.001) and early clinical stage (p = 0.004). Univariate analysis showed that low LGR5 expression (p = 0.034) was significantly associated with worse overall survival. However, LGR5 expression did not remain a predictor of prognosis in multivariate analysis (p = 0.639). All PDAC cases showed LGR5 expression to varying degrees, indicating LGR5 might be a cancer stem cell marker of PDAC, as in gastrointestinal cancer. Reduced LGR5 expression in tumor cells was associated with worse prognosis in PDAC. Further studies are required to elucidate the relationship between tumor progression and LGR5 expression in PDAC.     

Cell kinetic study of the endometrium by nonisotopic in situ hybridization for histone H3 messenger RNA and immunohistochemistry for Ki‐67 and for estrogen and progesterone receptors

We investigated the cell kinetics of the endometrium in hysterectomy specimens taken for leiomyoma from 22 women with regular ovulatory menstrual cycles. Formalin‐fixed, paraffin‐embedded tissue sections were examined for proliferating activity using histone H3 messenger RNA in situ hybridization (H3 mRNA‐ISH) and immunostaining for the Ki‐67 antigen. The relationship of the proliferative activity of endometrial cells to the immunohistochemical expression of the estrogen receptor (ER) and the progesterone receptor (PR) was also examined. During the menstrual cycle, H3 mRNA expression was observed in both the epithelial cells and the stromal cells of the endometrium. In the functional layer, the labeling indices for H3 mRNA (H3 mRNA‐LIs) in the epithelial cells peaked in the late proliferative phase, decreased sharply in the early secretory phase, and remained unchanged thereafter. On the other hand, H3 mRNA‐LIs of stromal cells displayed two peaks: one in the midproliferative phase and the other in the late secretory phase, the former peak being the greater. In the basal layer, epithelial cells and stromal cells showed low H3 mRNA‐LIs and no significant variation throughout the menstrual cycle. The H3 mRNA‐LIs correlated well with the Ki‐67‐LIs and were lower than the corresponding Ki‐67‐LIs. The regression coefficient (H3 mRNA‐LIs against the Ki‐67‐LIs) was 0.33 for epithelial cells and 0.49 for stromal cells, suggesting that the cell cycle time was longer for epithelial cells than for stromal cells. The proliferative activity of endometrial cells showed close relationships with the expressions of ER and PR in the endometrium. When used in combination with other proliferative markers in paraffin‐embedded tissue sections, H3 mRNA‐ISH could open broader perspectives on the cell kinetics of the endometrium. Anat Rec 266:234–240, 2002. © 2002 Wiley‐Liss, Inc.     

Enrichment of Oct3/4‐positive cells from a human bronchial epithelial cell line

Most adult stem cells are in the G0 phase of the cell cycle, accounting for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. The anti‐tumor drug 5‐fluorouracil (5‐FU) selectively kills proliferating cells, sparing cells in the G0 phase. Thus, the objective of this study was to determine whether 5‐FU can be used to enrich stem cells in a human bronchial epithelial (HBE) cell population in vitro. Side population (SP) cells were isolated from untreated HBE cells or HBE cells treated with 5‐FU, and the resulting cells were subjected to colony formation assays, culturing of cell spheres, and tumorigenicity assays. Expression of Oct3/4, Sox2, PCK, and β‐catenin were examined by Western blot analysis and immunofluorescence. Treatment with 5‐FU increased the percentage of SP cells from 0.3% to 1.5%, and the clonogenic ability of 5‐FU‐treated cells was more than twofold higher than that of HBE cells. Cells that survived after 5‐FU treatment exhibited a higher capacity for sphere formation. Furthermore, spheres formed from 5‐FU‐treated cells possessed the capacity to generate differentiated progenies. Cells treated with 5‐FU also exhibited tumorigenic potential, based on tumor formation assays in nude mice, and Oct3/4‐positive cell aggregates were identified in the resulting tumors. In this study, we have shown that 5‐FU treatment enriched the population of cells expressing the putative embryonic markers Oct3/4 and Sox2 and exhibiting nuclear accumulation of β‐catenin. Furthermore, 5‐FU‐treated cells expressed low levels of the epithelial differentiation marker PCK. Analysis of epigenetic modifications suggested that Oct3/4‐positive cells possessed characteristics of stem cells. These results demonstrate that treatment with 5‐FU can enrich the stem cell population present in a human bronchial epithelial cell line, and implicate combined treatment with 5‐FU and serum‐free medium as a new method for isolation of stem‐like cells from the HBE cell line.     

Lgr5‐expressing stem cells are not the cells of origin of pyloric neuroendocrine carcinomas in mice

In intestinal and pyloric epithelia, leucine‐rich repeat‐containing G protein‐coupled receptor 5 (Lgr5)‐expressing cells represent long‐lived adult stem cells that give rise to all epithelial cell types, including endocrine cells. Ablation of the Apc gene in Lgr5‐expressing cells leads to intestinal and pyloric adenomas. To assess whether all epithelial tumours of the gastrointestinal tract are derived from LGR5‐positive stem cells, we crossed Lgr5–EGFP–IRES–creER^T2 mice, which express EGFP and Cre recombinase driven by the Lgr5 promoter, with CEA424–SV40–TAg mice, which develop pyloric neuroendocrine carcinomas of epithelial origin. In 19 day‐old mice, single SV40 T antigen (TAg)‐positive cells were identified preferentially at the the bases of pyloric glands, close to the stem cell compartment. However, contrary to previous publications describing subpopulations of LGR5‐positive cells in gastrointestinal neoplasia, we could not detect Lgr5–EGFP‐positive tumour cells in malignant lesions. The lack of expression of the Wnt target gene Lgr5 is probably not caused by suppression of Wnt signalling by TAg, since β‐catenin‐mediated Wnt signalling, as measured by the TOPflash assay, was not inhibited. To determine the cellular origin of CEA424–SV40–TAg tumours, we performed tracing experiments using Lgr5–EGFP–IRES–creERT2:CEA424–SV40–TAg:ROSA26–tdRFP mice. Following tamoxifen induction, it was possible to efficiently trace the progeny of Lgr5‐expressing cells in gastrointestinal tissue via red fluorescent protein (RFP) expression. No RFP‐positive tumour cells were detected, even when RFP gene activation occurred in 7 day‐old mice well before the appearance of TAg‐positive tumour cells. Hence, we conclude that Lgr5‐expressing stem cells probably do not constitute the cells of origin in CEA424–SV40–TAg mice. Consequently, not all epithelial tumours in the pyloric region are initiated by transformation of LGR5‐positive stem cells. Thus, additional long‐lived LGR5‐negative stem cells or progenitor cells with a low turnover rate might exist in the pyloric region, which could give rise to tumours. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Histopathological varieties of oral carcinoma in situ: Diagnosis aided by immunohistochemistry dealing with the second basal cell layer as the proliferating center of oral mucosal epithelia

To make reproducible diagnoses for oral carcinoma in situ (CIS), combined immunohistochemistry directed at the positioning of squamous cell proliferation (Ki‐67) and differentiation (keratin (K) 13 and K19) was used, both of which support histological evaluations by providing biological evidence. Normal/hyperplastic epithelia was defined by K19+ cells only in the first basal layer, K13+ cells in the third basal and upper layers, and sporadic Ki‐67+ cells in the second basal layer. These profiles indicated that a proliferating center of the oral epithelium is located in the parabasal cell layer, and K19 and K13 can be regarded as markers for basal and prickle cells, respectively. Epithelial dysplasia was characterized by irregular stratification of Ki‐67+ cells and the absence of K19/K13 in proliferating cells. Irregular emerging of K19+ and K13+ cells in proliferating foci with unique stratification of atypical Ki‐67+ cells indicated CIS. When the definition was applied, surgical margins in 172 recurrent cases were shown to contain CIS (39.4%) and squamous cell carcinoma (55.8%), indicating that the new diagnostic criteria for CIS reflected clinical behaviors of the cases. The results indicate that oral CIS contain more histological variations, especially those with definite keratinization, than what had been previously defined.