Prostate adenocarcinomas aberrantly expressing p63 are molecularly distinct from usual-type prostatic adenocarcinomas

Abstract

We have described a rare group of prostate adenocarcinomas that show aberrant expression of p63, a protein strongly expressed in prostatic basal cells and absent from usual-type acinar prostate cancers. The partial basal-like immunophenotype of these tumors is intriguing in light of the persistent debate surrounding the cell-of-origin for prostate cancer; however, their molecular phenotype is unknown. We collected 37 of these tumors on radical prostatectomy and biopsy and assessed subsets for a diverse panel of molecular markers. The majority of p63-expressing tumors were positive for the ΔNp63 isoform (6/7) by immunofluorescence and p63 mRNA (7/8) by chromogenic in situ hybridization. Despite p63 positivity, these tumors uniformly expressed luminal-type cytokeratin proteins such as CK18 (13/13), CK8 (8/8), and markers of androgen axis signaling commonly seen in luminal cells, including androgen receptor (10/11), NKX3.1 (8/8), and prostein (12/13). Conversely, basal cytokeratins such as CK14 and CK15 were negative in all cases (0/8) and CK5/6 was weakly and focally positive in 36% (4/11) of cases. Pluripotency markers including β-catenin, Oct4, and c-kit were negative in p63-expressing tumors (0/11). Despite nearly universal expression of androgen receptor and downstream androgen signaling targets, p63-expressing tumors lacked ERG rearrangements by fluorescence in situ hybridization (0/14) and ERG protein expression (0/37). No tumors expressed SPINK1 or showed PTEN protein loss (0/19). Surprisingly, 74% (14/19) of p63-expressing tumors expressed GSTP1 protein at least focally, and 33% (2/6) entirely lacked GSTP1 CpG island hypermethylation by bisulfite sequencing. In contrast to usual prostatic adenocarcinomas, prostate tumors with p63 expression show a mixed luminal/basal immunophenotype, uniformly lack ERG gene rearrangement, and frequently express GSTP1. These data strongly suggest that p63-expressing prostate tumors represent a molecularly distinct subclass and further study of this rare tumor type may yield important insights into the role of p63 in prostatic biology and the prostate cancer cell-of-origin.

Figure 1

p63-expressing prostate cancers are positive for p63 mRNA by chromogenic in situ hybridization (CISH) and the ΔNp63 isoform by immunohistochemistry (A) p63-expressing prostate carcinoma (top row) expresses p63 protein in a non-basal cell distribution (using the 4A4 antibody which detects both the ΔNp63 andTAp63 isoforms, 400×magnification) as well as p63 mRNA by CISH (arrowheads), at levels similar to or higher than surrounding benign basal cells (arrow, 630× magnification). The concurrent usual-type adenocarcinoma in this case (bottom row) does not express p63 protein or mRNA, however nearby benign basal cells are positive for both (arrows). (B) Dual ΔNp63 and CK8 staining in prostatic tissues. These markers are expressed in separate compartments in benign prostatic tissue, with basal cells expressing ΔNp63 and luminal cells expressing CK8 (left panel). In p63-expressing tumors, these two markers are expressed in the same cells (middle panel). In contrast, usual-type acinar carcinomas express CK8 and are negative for ΔNp63. All images at 400× magnification.

Figure 2

p63-expressing prostate tumors are generally positive for low molecular weight cytokeratins and negative for high molecular weight cytokeratins. Most p63-expressing tumors diffusely and strongly express CK18 at levels similar to benign luminal cells (case 1 and 3), although some show weaker diffuse expression (case 2). Similarly, most are entirely negative for CK5/6 (cases 2 and 3), although a minority weakly and focally express CK5/6 (case 1). All images are at 200 × magnification.

Figure 3

p63-expressing prostate tumors express androgen receptor (AR) and prostatic differentiation markers (NKX3.1). Two representative cases showing AR levels in tumor cells similar to those in surrounding benign luminal cells (200 × magnification). NKX3.1, a prostatic differentiation marker and marker of downstream androgen signaling is also expressed in p63-positive tumors (400 × magnification).

Figure 4

p63-expressing prostate tumors do not rearrange the ERG gene locus or express ERG protein. ERG break-apart FISH assay in a usual-type carcinoma occurring concurrently with a p63-expressing tumor demonstrates rearrangement involving TMPRSS2 locus (green probe) which is spatially separated from the red and orange probes (centromeric and telomeric relative to the ERG gene, respectively) (630 × magnification). In contrast, the p63-expressing tumor from the same patient is negative for any rearrangement at this locus. Notably, neither tumor expresses ERG protein, despite positivity in endothelial cell nuclei which provide an internal positive control (arrowheads). Lack of ERG staining in the TMPRSS2-rearranged usual-type acinar carcinoma could be consistent with a rearrangement involving another ETS-family gene (400 × magnification).

Figure 5

p63-expressing prostate tumors do not invariably silence GSTP1. (A) A representative p63-expressing tumor with high GSTP1 protein expression by immunohistochemistry and concomitant lack of hypermethylation at the CpG island of the GSTP1 gene by bisulfite sequencing (case A, bottom). (B) This p63-expressing tumor shows lack of GSTP1 expression, consistent with hypermethylation at the GSTP1 locus by bisulfite sequencing at levels comparable to usual-type adenocarcinoma controls (case B, bottom). (C) This p63-expressing tumor is admixed with usual-type p63-negative carcinoma and shows mixed GSTP1 protein expression. Bisulfite sequencing in this case shows hypermethylation of the GSTP1 locus at levels similar to usual-type adenocarcinomas (case C, bottom). Red bar indicates genomic location of the bisulfite sequencing amplicon; yellow bar indicates region of differential methylation between normal prostatic epithelium and usual-type adenocarcinoma; green bar indicates promoter associated CpG island. All images at 100 × magnification.