Assessing the order of critical alterations in prostate cancer development and progression by IHC: further evidence that PTEN loss occurs subsequent to ERG gene fusion

Abstract

Background: ERG rearrangements and PTEN (phosphatase and tensin homolog deleted on chromosome 10) loss are two of the most common genetic alterations in prostate cancer. However, there is still significant controversy regarding the order of events of these two changes during the carcinogenic process. We used immunohistochemistry (IHC) to determine ERG and PTEN status, and calculated the fraction of cases with homogeneous/heterogeneous ERG and PTEN staining in a given tumor.

Methods: Using a single standard tissue section from the index tumor from radical prostatectomies (N=77), enriched for relatively high grade and stage tumors, we examined ERG and PTEN status by IHC. We determined whether ERG or PTEN staining was homogeneous (all tumor cells staining positive) or heterogeneous (focal tumor cell staining) in a given tumor focus.

Results: Fifty-seven percent (N=44/77) of tumor foci showed ERG positivity, with 93% of these (N=41/44) cases showing homogeneous ERG staining in which all tumor cells stained positively. Fifty-three percent (N=41/77) of tumor foci showed PTEN loss, and of these 66% (N=27/41) showed heterogeneous PTEN loss. In ERG homogeneously positive cases, any PTEN loss occurred in 56% (N=23/41) of cases, and of these 65% (N=15/23) showed heterogeneous loss. In ERG-negative tumors, 51.5% (N=17/33) showed PTEN loss, and of these 64.7% (N=11/17) showed heterogeneous PTEN loss. In a subset of cases, genomic deletions of PTEN were verified by fluorescence in situ hybridization in regions with PTEN protein loss as compared with regions with intact PTEN protein, which did not show PTEN genomic loss.

Conclusions: These results support the concept that PTEN loss tends to occur as a subclonal event within a given established prostatic carcinoma clone after ERG gene fusion. The combination of ERG and PTEN IHC staining can be used as a simple test to ascertain PTEN and ERG gene rearrangement status within a given prostate cancer in either a research or clinical setting.

Figure 1

Homogeneously stained ERG positive tumor; (A) Whole tumor is stained with ERG (original magnification, ×10). (B) Higher power view of boxed area in A showing nuclear staining of ERG in tumor cells, negative benign glands (Nl), and vascular endothelial cells as positive internal control (original magnification × 200).

Figure 2

Heterogeneous staining of PTEN in a homogeneously ERG positive tumor; (A) Adenocarcinoma lesion with homogeneous staining for ERG (original magnification ×20). (B) The same tumor lesion as in A stained for PTEN from an adjacent section shows heterogeneous staining for PTEN with the majority of the tumor staining positively and a portion of the tumor in the center staining negatively (original magnification ×20). (C) Higher power view of boxed area in A showing nuclear staining for ERG in all tumor cells (original magnification ×200) and (D) Higher power view of boxed area in B showing PTEN positively staining cells (left) and PTEN negatively staining cells (*)(original magnification ×200).

Figure 3

(A) Double labeling of heterogeneous staining of PTEN (brown) in a homogeneously ERG positive (red) tumor (original magnification ×10). (Insets in A) show PTEN and ERG FISH. ‘PTEN intact’ is a representative nucleus from a tumor area which stained positively for PTEN by IHC and shows two intact sets of 4 colored signals. ‘PTEN del’ shows nuclei which belong to PTEN negatively staining tumor glands by IHC; one nucleus shows the loss of all 4 probes on one allele and the loss of PTEN (orange) and WAPAL (green) on the other allele with retention of one copy of the centromeric probe (red) and telomeric most probe (FAS, blue). The other nucleus shows apparent homozygous loss of PTEN (orange) and WAPAL (green) with retention of 2 centromere signals and one copy of FAS (blue). ‘ERG del’ is a representative FISH image of a cell from a region that was ERG positive by IHC which were encountered throughout the whole tumor. It shows one set of all 4 signals on one chromosome 21 and a loss of two probes (orange, which is slightly telomeric to ERG and spans HMGN1, and blue, which is more telomeric towards TMPRSS2 and spans DSCAM) between TMPRSS2 and ERG, consistent with an ERG rearrangement by deletion (original magnification ×60). (B) Higher power view from boxed region in A showing ERG positive glands which are heterogeneously stained with PTEN( ERG+/PTEN−, ERG+/PTEN+), and benign glands negative for ERG and positive for PTEN (Nl) (original magnification ×200).