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1.
Curr Protoc Essent Lab Tech ; 21(1): e46, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33381282

ABSTRACT

Stored biological materials should have minimal pre-analytical variations in order to provide researchers with high-quality samples that will give reliable and reproducible results, yet methods of storage should be easy to implement, with minimal cost and health hazard. Frozen tissue samples are a valuable biological resource. Here we compare different methods, such as liquid nitrogen (LN) or dry ice (DI), to a cheap and safe alternative using an aluminum platform (AP). Murine fresh liver and pancreas tissues were used with varying lengths of warm ischemia time. Quality assessment was based on histological evaluation, DNA and RNA extraction and quantification, and RNA degradation analysis, as well preservation of antigens for immunofluorescence, in a blinded manner. Both in superficial and deep tissue sections, based on histological assessment, AP is superior to DI, or as good as LN techniques in terms of presence of ice crystals, cutting artifacts, and overall quality/structural preservation. DNA and RNA were successfully extracted in reasonable quantities from all freezing techniques, but RNA degradation was seen for pancreas samples across all techniques. Immunofluorescence with cytokeratin8 (CK-8), alpha smooth muscle actin (αSMA), CD3, and B220 shows equally good outcomes for AP and LN, which are better than DI. The aluminum platform is a cheap, yet reliable method to freeze samples, rapidly preserving histological, antigenic, and DNA/RNA quality. Wider testing is required across different sample types. © 2020 The Authors. Basic Protocol: Flash-freezing fresh tissue with aluminum platform Alternate Protocol 1: Freezing fresh tissue with liquid nitrogen Alternate Protocol 2: Freezing fresh tissue with dry ice.

2.
Ann Diagn Pathol ; 47: 151557, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32593808

ABSTRACT

Recently, stromal targeting, by agents such as All trans retinoic acid (ATRA), has been regarded as a promising avenue for the treatment of pancreatic ductal adenocarcinoma (PDAC). The intra-cellular transportation of ATRA to the nuclear receptors is performed by either: fatty acid binding protein 5 (FABP5) or cellular retinoic acid binding protein 2 (CRABP2), dictating the transcription of downstream genes and, thus, eventual cell phenotype. Here, we explored the levels of each protein, in pancreatic tissues of patients presenting with a range of pancreatic diseases (pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP), cholangiocarcinoma (CC)). We demonstrate that there is a significantly lower CRABP2 and FABP5 expression in activated fibroblasts or pancreatic stellate cells (PSC) in PDAC, as well as other diseased pancreas as in CC and CP, versus quiescent fibroblasts. The quiescent fibroblasts consistently show a pattern of high FABP5:CRABP2 ratio, whereas PSC in all non-PDAC tissues showed a low FABP5:CRABP2 ratio. PSC in PDAC patients had a range of FABP5:CRABP2 ratios (high, even and low). There was a lower CRABP2 expression in cancerous epithelial cells (PDAC) versus normal epithelial cells. This is also present in other disease states (CP, CC). Contrasting to the patterns seen for fibroblasts, the FABP5 expression in PDAC epithelial cells matched that of the normal epithelial cells. However, the normal epithelial cells had a high FABP5:CRABP2 ratio, compared to the PDAC epithelial cells. These ratios may have correlation with tumor progression, and overall survival. These findings could be confirmed in in vitro cell lysates. CRABP2 and FABP5 levels and ratios could serve as valuable biomarkers.


Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Fatty Acid-Binding Proteins/genetics , Pancreas/pathology , Receptors, Retinoic Acid/genetics , Tretinoin/pharmacokinetics , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bile Duct Neoplasms/pathology , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/pathology , Cholangiocarcinoma/metabolism , Cholangiocarcinoma/pathology , Disease Progression , Epithelial Cells/metabolism , Fatty Acid-Binding Proteins/drug effects , Fibroblasts/metabolism , Fluorescent Antibody Technique/methods , Gene Expression Regulation, Neoplastic/genetics , Humans , Pancreas/physiopathology , Pancreatitis, Chronic/metabolism , Pancreatitis, Chronic/pathology , Receptors, Retinoic Acid/drug effects , Survival Analysis , Tissue Array Analysis/methods , Tretinoin/pharmacology , Tretinoin/therapeutic use
3.
Gastroenterology ; 132(2): 527-30, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17258725

ABSTRACT

BACKGROUND & AIMS: We report a patient of Indian descent with parental consanguinity, who developed 10 carcinomas and 35 adenomatous polyps at age 23 and duodenal adenocarcinoma at age 25. He also had dysmorphic features, mental retardation, and café-au-lait spots but no brain tumor. We aimed to establish his molecular diagnosis. METHODS: Germ-line screening for APC and MYH/MUTYH mutations was normal as was immunohistochemistry for MLH1 and MSH2 proteins. Investigation by array-comparative genomic hybridization revealed deletion of a small region on chromosome 7. Using polymerase chain reaction, this region was refined to a 400-kilobase deletion, which included exons 9-15 of the PMS2 gene, and all coding regions of oncomodulin, TRIAD3, and FSCN1. RESULTS: The deletion was confirmed as homozygous, and both parents were carriers. Immunohistochemistry showed absent PMS2 expression in all tumors and normal tissue. Most tumors showed microsatellite instability, more marked at dinucleotide than mononucleotide repeats. The tumors harbored no somatic mutations in APC, BRAF, AXIN2, or beta-catenin, but KRAS2 and TGFBR2 mutations were found. CONCLUSIONS: Our patient represents a novel phenotype for homozygous PMS2 mutation and perhaps the most severe colorectal cancer phenotype-in terms of numbers of malignancies at an early age-described to date. PMS2 mutations-and perhaps other homozygous mismatch repair mutations-should be considered in any patient presenting with multiple gastrointestinal tumors, since our patient could not be distinguished clinically from cases with attenuated familial adenomatous polyposis or MUTYH-associated polyposis.


Subject(s)
Adenomatous Polyps/genetics , Adenosine Triphosphatases/genetics , Colorectal Neoplasms/genetics , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Gene Deletion , Homozygote , Intestinal Polyposis/genetics , Adenomatous Polyposis Coli/diagnosis , Adenomatous Polyps/diagnosis , Adenomatous Polyps/pathology , Adult , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/pathology , DNA Glycosylases/genetics , Diagnosis, Differential , Duodenal Neoplasms/genetics , Fatal Outcome , Gene Expression Regulation, Neoplastic , Humans , Intestinal Polyposis/diagnosis , Intestinal Polyposis/pathology , Male , Microsatellite Instability , Mismatch Repair Endonuclease PMS2 , Mutation , Pedigree , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/genetics , Severity of Illness Index , ras Proteins
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