An improved method for constructing tissue microarrays from prostate needle biopsy specimens

Abstract

Background: Prostate cancer diagnosis is routinely made by the histopathological examination of formalin fixed needle biopsy specimens. Frequently this is the only cancer tissue available from the patient for the analysis of diagnostic and prognostic biomarkers. There is, therefore, an urgent need for methods that allow the high-throughput analysis of these biopsy samples using immunohistochemical (IHC) markers and fluorescence in situ hybridisation (FISH) analysis based markers.

Methods: A method that allows the construction of tissue microarrays (TMAs) from diagnostic prostate needle biopsy cores has previously been reported. However, the technique only allows the production of low-density biopsy TMAs with a maximum of 20 cores per TMA. Here two methods are presented that allow the rapid and uniform production of biopsy TMAs containing between 54 and 72 biopsy cores. IHC and FISH techniques were used to detect biomarker status.

Results: Biopsy TMAs were constructed from prostate needle biopsy specimens taken from 102 patients entered into an active surveillance trial and 201 patients in a radiotherapy trial. The detection rate for cancer in slices of these biopsy TMAs was 66% and 79% respectively. Slices of a biopsy TMA prepared from biopsies from active surveillance patients were used to detect multiple IHC markers and to score TMPRSS2-ERG fusion status in a FISH-based assay.

Conclusions: The construction of biopsy TMAs provides an effective method for the multiplex analysis of IHC and FISH markers and for their assessment as prognostic biomarkers in the context of clinical trials.

Figure 1

Knives used for cutting biopsy checkers. The knives each consist of parallel surgical blades fixed in translucent acrylic plastic. The figure shows knives with the blades 4 mm apart (A, B) and 2 mm apart (C, D).

Figure 2

Construction of biopsy checkers. For clarity the formalin fixed prostate cancer needle biopsy has been coloured green. The position of the 4 mm length of biopsy on the surface of the wax block selected for checker construction is marked (A). The knife containing parallel blades 4 mm apart (fig 1A, B) is positioned over the wax block (B, C) so that a 4 mm length of biopsy is cut (D, E). A scalpel blade is used to cut the section of wax containing the attached 4 mm biopsy segment from the block (F). For orientation the side of the checker is marked with red ink (G). The knife containing blades 2 mm apart (fig 1C, D) is then used to make two further cuts (H–J) and for orientation an additional face of the checker is marked with blue ink (K). The final 4 mm×2 mm×2 mm checker is shown (L). The marking of faces of the checker with red and blue ink is important because it is often difficult to visualise the biopsy specimen as the checker is being cut. The entire process of checker construction takes approximately 5 minutes.

Figure 3

Construction of wax templates. Wax templates were constructed to accommodate 4 mm×2 mm×2 mm wax checkers. Two formats were used. In the first format (A–D) individual wells were created into which a single checker could be placed. In the second format (E–H) three larger wells (6 mm×20 mm, 8 mm×20 mm and 10 mm×20 mm) were created which together accommodated 72 checkers. The rubber moulds used to construct each template are shown respectively in (B) and (F). The detailed methods for constructing the wax templates are shown in supplementary figs 1 and 2. The methods used to insert individual checkers into the templates are shown in supplementary fig 3. (C, G) Completed biopsy tissue microarrays. (D, H) H&E stained sections of each biopsy tissue microarray (TMA). A black cross and black circle denote the position of a blank checker and of a representative biopsy core, respectively. The construction of the entire biopsy TMA from formalin fixed biopsy specimens takes 2 days.

Figure 4

Analysis of sections obtained from a biopsy tissue microarray (TMA). Left panel: Serial sections (×20) from a single biopsy specimen that contained both cancer (surrounded by thick line) and non-neoplastic epithelium (surrounded by dashed line) stained by H&E, p63/AMACR Ki-67 and Hif-1α. The insert shows a magnification of the Ki67 staining. Right panel: FISH detection of ERG gene breakpoints. (A) Principle of detection of ERG gene status. Interphase nuclei are hybridised to probes that detect sequences 5′ to the ERG gene (green) and 3′ to the ERG gene (red). (B, C) Results from biopsy TMA slices. The red and green co-localise for normal ERG loci (B) and are separated (C) when an ERG gene rearrangement occurs.