Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Immunohistochemical markers in urinary bladder carcinomas from paraffin-embedded archival tissue after storage for 5-70 years.

Author(s): Litlekalsoy J, Vatne V, Hostmark JG, Laerum OD

Publication: BJU Int, 2007, Vol. 99, Page 1013-9

PubMed ID: 17437436 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if immunohistochemical staining of cancer biomarkers (p53 protein, p16 protein, epidermal growth factor receptor (EGFR), cytokeratin (CK7), high-molecular-weight cytokeratin (34βE12 cytokeratin)) is adversely affected by either postmortem interval (PMI, 5-72 h), the time between death and tissue preservation, or by the year of collection and the associated storage of formalin-fixed, paraffin-embedded (FFPE) blocks containing urinary carcinoma specimens or normal controls for 5 to 50 years (potentially confounding factors included differences in tissue fixation and processing protocols over the time period investigated). A total of 144 grade II or III urinary carcinoma tumors collected from the following periods were used for analysis: 1932-48 (18 autopsy specimens), 1950-59 (30 autopsy specimens), 1960-70 (41 autopsy specimens), 1990-2004 (42 autopsy specimens, 13 biopsies of surgically resected tumors).  Autopsy specimens were stored at 4-8°C for 5-72 h before preservation in 4% aqueous formaldehyde (if prior to 1985) or 4% formaldehyde that was buffered to a neutral pH (during or after 1985).  An additional 27 biopsies from normal tissue (connective tissue, fat, thyroid gland, smooth muscle, heart, lung, uterus, appendix, lymph node, endometrium, ureter, ovary) or normal tissue adjacent to the urinary carcinoma that collected between 1990 and 2004 were also included in the analysis as controls.  Some blocks necessitated re-embedding due to shrinkage or brittleness of the paraffin wax. Dehydration and clearing of fixed tissue were performed manually if collected prior to 1960 but was automated for specimens collected during or after 1960. FFPE blocks were stored at ambient temperature (18-20°C) under “relative high humidity due to the local climate.” No additional details on tissue processing, storage, or sectioning were provided. Immunohistochemistry was conducted on 5 µm-thick sections that were treated with proteinase K in 0.9% NaCl and or microwave heating in citrate buffer, depending on the antigen, then stained using a TechMate 500 machine and indirect streptavidin-biotin based methods.  Immunopositive staining for p53, p16, EGFR, CK7, and 34βE12 cytokeratin was scored as positive or negative. 

   Summary of Findings:

   The authors note that FFPE blocks were generally in good condition, although an unspecified number necessitated re-embedding in paraffin after the original paraffin, which was brittle, was trimmed away.  Tissue morphology was well preserved in all specimens although a limited degree of autolysis was present (a representative micrograph was provided but the prevalence of autolysis was not quantified). The authors note that there were clear demarcations between positive and negative cellular staining among bladder tumor specimens for all relevant antigens; weak positive immunostaining was not observed. Although data was not shown, the authors state that the duration of PMI was not correlated to the percentage of specimens that exhibited positive immunostaining when all antigens were considered together.  Appropriate and expected immunostaining was observed among tumor and normal adjacent tissue specimens that were collected across all collection/FFPE block storage time periods examined. For example, P53 staining was negative in all normal control tissues, P16 was positive in the cell nuclei of all specimens, and CK7 staining was absent in mesenchymal tissue but present in “bile ducts, bladder mucosa, and kidney and bronchial epithelium across collect time periods.  The percentage of a positive IHC score of autopsy urinary carcinoma specimens collected during different collection/storage time periods remained stable for EGFR, CK7, and P16, while the percentage of specimens that stained positive for P53 modestly increased over time and those that stained positive for HMW-cytokeratin decreased (by approximately 60%).  The authors postulate that these differences may reflect gradual changes in malignant phenotypes.

   In a follow-up study investigating the potential effect of postmortem collection, concordance in positive scores between the 27 case-matched autopsy and surgical resection urinary carcinoma specimens was >95% overall; however, differences between autopsy and resection specimens were more pronounced for P53 (autopsy: 16 positive/11 negative cases; biopsy: 23/4), P16 (autopsy: 12/15; biopsy 19/8), EGFR (autopsy: 17/10; biopsy: 14/13), CK7 (autopsy: 23/4; biopsy: 23/4), and HMW-cytokeratin (autopsy: 5/22; biopsy 4/23).

   Biospecimens

   • Tissue - Bladder

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Autopsy
   • Normal
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Postmortem interval	5-72 h
   Storage	Storage duration	5-70 y
   Biospecimen Acquisition	Method of tissue acquisition	Autopsy Surgical resection
   Immunohistochemistry Specific	Targeted peptide/protein	P53 P16 EGFR CK7 34βE12 cytokeratin

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