Analytical Cellular Pathology - Volume 36, issue 3-4

Authors: Wu, Shuai | Cheng, Zhongliang | Liu, Guohua | Zhao, Xinfeng | Zhong, Liang | Zhu, Yingjian | Zhu, Jiang

Article Type: Research Article

Abstract: Human umbilical cord-derived mesenchymal stromal cells (hUCMSCs) are the most primitive of those isolated from other post-natal tissue source. The hUCMSCs possess the capability of differentiating along multi-lineage. This study aimed to investigate whether hUCMSCs can differentiate into urothelium-like cells. The hUCMSCs were isolated from fresh human umbilical cord postpartum and expanded at least to passage 3 in vitro. Subsequently, they were cultured with conditioned medium from urothelial cells (UC-CM) supplemented with 20 ng/ml exogenous epidermal growth factor (EGF). Urothelial cell specific marker uroplakin II (UPII) and cytokeratins were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence technology. During …culture, hUCMSCs started to express UPII and cytokeratins weakly at 7 days and were significantly up-regulated at 2 weeks post-induction. Additionally, morphology of hUCMSCs changed from spindle-shape to a polygonal epithelial-shape similar to that of urothelial cells after 7 days. The study results indicated that hUCMSCs can differentiate into urothelium-like cells in a defined micro-environment in vitro constituted by UC-CM and exogenous EGF. Show more

Keywords: Cell differentiation, mesenchymal stromal cells, umbilical cord, urothelium

DOI: 10.3233/ACP-130081

Citation: Analytical Cellular Pathology, vol. 36, no. 3-4, pp. 63-69, 2013

Authors: Mimmi, Maria Chiara | Finato, Nicoletta | Pizzolato, Gloria | Beltrami, Carlo Alberto | Fogolari, Federico | Corazza, Alessandra | Esposito, Gennaro

Article Type: Research Article

Abstract: It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many “targeted” approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1 H NMR spectra of biological samples. Here …we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio. Show more

Keywords: Metabolomics, breast cancer, choline phospholipid metabolism, $^1$H-NMR, LC–ESI-MS, hydrophilic interaction chromatography

DOI: 10.3233/ACP-130082

Citation: Analytical Cellular Pathology, vol. 36, no. 3-4, pp. 71-83, 2013

Authors: Vaniawala, Salil | Acharya, Arpan | Parekh, Harsh | Mukhopadhyaya, Pratap N.

Article Type: Research Article

Abstract: OBJECTIVE: The primary objective of this work was to confirm the occurrence of rare BCR ABL fusion variant involving the a3 region of the ABL gene in a patient positive for t(9;22) translocation but negative for common major and minor breakpoint cluster regions and the challenges and threats that it poses in a routine laboratory setting which use commercial kits for monitoring the minimal residual disease. METHODS: A patient with elevated white blood cell count was subjected to classical cytogenetics, FISH as well as RT-PCR testing using commercial kits as well as published primers and in house testing protocol. PCR …amplicon generated from in the process was sequenced and analyzed. RESULTS: The translocation event in chromosome 9 and 22 could be successfully detected. BCR/ABL dual color, dual fusion probe generated a classical balanced translocation scenario within the nucleus of affected cells and presented a ‘1O1G2F’ signal pattern. RT-PCR with probes from commercial kit designed to detect common breakpoints within the M- and m-BCR regions involving e13a2, e14a2 and e1a2 fusion variants respectively failed to generate any signal. Further investigation revealed presence of the rare e14a3 (b3a3) fusion. DISCUSSION: This is the first report of rare e14a3 fusion in the BCR ABL gene in a CML patient from India. The observation indicates the need for interrogating rare BCR ABL fusions when common breakpoint cluster regions are absent such that minimal residual disease (MRD), critical for disease monitoring, can be performed and false positive remission cases can be avoided. It also emphasizes the utility and significance of cytogenetics and FISH techniques in primary diagnosis of CML and use of RT-PCR based assays only for generating secondary information within special reference to MRD. CONCLUSION: The rare e14a3 (b3a3) fusion of the BCR ABL gene is present in Indian population as demonstrated from this first report and clinical laboratories using commercial kit that do not cover such rare fusions are likely to generate false result thereby declaring complete molecular remission in CML patients under therapy while conducting MRD assay using RT-PCR technology. Show more

Keywords: CML, Leukemia, breakpoint, b3a3, e14a3

DOI: 10.3233/ACP-130083

Citation: Analytical Cellular Pathology, vol. 36, no. 3-4, pp. 85-92, 2013

Authors: Wang, Lin | Huang, Juxiang | Jiang, Minghu | Diao, Haizhen | Zhou, Huilei | Li, Xiaohe | Chen, Qingchun | Jiang, Zhenfu | Feng, Haitao | Wolfl, Stefan

Article Type: Research Article

Abstract: BACKGROUND: To understand cartilage oligomeric matrix protein (COMP) mechanism network from human normal adjacent tissues to lung adenocarcinoma. METHODS: COMP complete different activated (all no positive correlation, Pearson CC < 0.25) and uncomplete (partly no positive correlation except COMP, Pearson CC < 0.25) network were identified in higher lung adenocarcinoma compared with lower human normal adjacent tissues from the corresponding COMP-stimulated (≥0.25) or inhibited (Pearson CC ≤ -0.25) overlapping molecules of Pearson correlation coefficient (CC) and GRNInfer, respectively. COMP complete different activated and inhibited (all no positive correlation, Pearson CC < 0.25) mechanisms networks of higher lung adenocarcinoma and lower …human normal adjacent tissues were constructed by integration of Pearson CC, GRNInfer and GO. As visualized by integration of GO, KEGG, GenMAPP, BioCarta and Disease, we deduced COMP complete different activated and inhibited network in higher lung adenocarcinoma and lower human normal adjacent tissues. RESULTS: As visualized by GO, KEGG, GenMAPP, BioCarta and disease database integration, we proposed mainly that the mechanism and function of COMP complete different activated network in higher lung adenocarcinoma was involved in COMP activation with matrix-localized insulin-like factor coupling carboxypeptidase to metallopeptidase-induced proteolysis, whereas the corresponding inhibited network in lower human normal adjacent tissues participated in COMP inhibition with nucleus-localized vasculogenesis, B and T cell differentiation and neural endocrine factors coupling pyrophosphatase-mediated proteolysis. However, COMP complete different inhibited network in higher lung adenocarcinoma included COMP inhibition with nucleus-localized chromatin maintenance, licensing and assembly factors coupling phosphatase-inhibitor to cytokinesis regulators-mediated cell differentiation, whereas the corresponding activated network in lower human normal adjacent tissues contained COMP activation with cytolplasm-localized translation elongation factor coupling fucosyltransferase to ubiquitin-protein ligase-induced cell differentiation. CONCLUSION: COMP different networks were verified not only by complete and uncomplete COMP activated or inhibited networks within human normal adjacent tissues or lung adenocarcinoma, but also by COMP activated and inhibited network between human normal adjacent tissues and lung adenocarcinoma. Show more

Keywords: Cartilage oligomeric matrix protein (COMP), cell differentiation to proteolysis mechanism networks, from human normal adjacent tissues to lung adenocarcinoma

DOI: 10.3233/ACP-130084

Citation: Analytical Cellular Pathology, vol. 36, no. 3-4, pp. 93-105, 2013

Authors: Meng, Zhilan | Shi, Jie | Zhu, Chenyan | Gu, Jiangang | Zhou, Chen

Article Type: Research Article

Abstract: DNA aneuploidy is a cancer biomarker, which may have a potential diagnostic value in body effusion specimen. DNA aneuploidy is determined by measuring the DNA content of tested cells and comparing them with diploid cells (2c). In order to assess the value of automated DNA image cytometry (DNA-ICM) in the cytologic diagnosis of effusion, we measured DNA ploidy using an automated DNA-ICM analysis system in 126 consecutive effusion specimens and followed the cases for histologic diagnosis. Half of each effusion specimen was used to prepare cytologic smears for conventional cytologic diagnosis, while the other half was used to prepare a …monolayer slide stained by Feulgen stain for automated ICM. By using Youden index, we found that 4 cells exceeding 2.5c is the optimal cut off value for aneuploidy, which has a sensitivity of 88.3% and specificity of 100% for diagnosis of malignant effusion. We also found that the DNA aneuploidy thresholds used for other types of cytologic specimens cannot be used in the diagnosis of effusion specimens. Our study demonstrated that automated DNA image cytometry is a simple, practical and cost-effective method for adjunct diagnosis of malignant effusion. Show more

Keywords: Aneuploidy, DNA image cytometry, cytology, effusion

DOI: 10.3233/ACP-130085

Citation: Analytical Cellular Pathology, vol. 36, no. 3-4, pp. 107-115, 2013