Molecular evidence that invasive adenocarcinoma can mimic prostatic intraepithelial neoplasia (PIN) and intraductal carcinoma through retrograde glandular colonization

Abstract

Prostate cancer often manifests as morphologically distinct tumour foci and is frequently found adjacent to presumed precursor lesions such as high-grade prostatic intraepithelial neoplasia (HGPIN). While there is some evidence to suggest that these lesions can be related and exist on a pathological and morphological continuum, the precise clonal and temporal relationships between precursor lesions and invasive cancers within individual tumours remain undefined. Here, we used molecular genetic, cytogenetic, and histological analyses to delineate clonal, temporal, and spatial relationships between HGPIN and cancer lesions with distinct morphological and molecular features. First, while confirming the previous finding that a substantial fraction of HGPIN lesions associated with ERG-positive cancers share rearrangements and overexpression of ERG, we found that a significant subset of such HGPIN glands exhibit only partial positivity for ERG. This suggests that such ERG-positive HGPIN cells either rapidly invade to form adenocarcinoma or represent cancer cells that have partially invaded the ductal and acinar space in a retrograde manner. To clarify these possibilities, we used ERG expression status and TMPRSS2-ERG genomic breakpoints as markers of clonality, and PTEN deletion status to track temporal evolution of clonally related lesions. We confirmed that morphologically distinct HGPIN and nearby invasive cancer lesions are clonally related. Further, we found that a significant fraction of ERG-positive, PTEN-negative HGPIN and intraductal carcinoma (IDC-P) lesions are most likely clonally derived from adjacent PTEN-negative adenocarcinomas, indicating that such PTEN-negative HGPIN and IDC-P lesions arise from, rather than give rise to, the nearby invasive adenocarcinoma. These data suggest that invasive adenocarcinoma can morphologically mimic HGPIN through retrograde colonization of benign glands with cancer cells. Similar clonal relationships were also seen for intraductal carcinoma adjacent to invasive adenocarcinoma. These findings represent a potentially undervalued indicator of pre-existing invasive prostate cancer and have significant implications for prostate cancer diagnosis and risk stratification.

Keywords: ERG; PTEN; clonality; ductal spreading; prostate cancer; prostatic intraepithelial neoplasia.

Figure 1

ERG protein expression in carcinoma and HGPIN. Sections were subjected to immunohistochemical staining for ERG (brown) and basal cell-specific markers (p63/903, red) (original objective magnification 20×). (A) Representative micrograph of ERG-expressing tumour. (B) HGPIN (tufting type) lesion showing ERG expression. (C) Partial ERG-positive PIN lesion with ERG-expressing neoplastic cells (arrows) adjacent to normal prostatic epithelium (arrowheads). (D) H&E stain of adjacent section showing distinct HGPIN morphology of the ERG-positive cells.

Figure 2

Detailed analysis of representative case 808. Case 808 was immunohistochemically stained for ERG, PTEN, and basal cell markers. The locations of lesions that were microdissected and further analysed are indicated with dotted blue lines within each numbered lesion. (A) Slides were immunostained with a two-colour combination for ERG (brown) and basal cell markers (first and third columns of images; ERG in brown and a cocktail of 34BE12/ck903 and p63 in red), or PTEN and basal cell markers (second and fourth columns of images; PTEN in brown and 34BE12/ck903 and p63 in red). Individual lesions (1–9) are shown at 20× original objective magnification. The numbers in the upper right corner correspond to the lesion identifier. Lesions 1 and 8 are invasive adenocarcinomas. Lesion 7 represents cribriform adenocarcinoma. Lesions 2, 5, and 6 represent partial involvement by PIN. Lesion 4 represents PIN, tufting and focally cribriform pattern that does not expand the acinus. Lesions 3 and 9 contain normal prostate epithelium. The relative location and H&E-stained low- and high-magnification images of each lesion (1–9) are presented in Supplementary Figure 5. (B) Representative micrographs of cell nuclei with intact and homozygous deleted PTEN locus and schema of four colour FISH probes used to interrogate the PTEN locus. Note the homozygous loss of the PTEN probe (orange signal) in the right panel. (C) Gel image of PCR products amplified with either TMPRSS2–ERG rearrangement-specific or TMPRSS2 control genomic primers from DNA microdissected from individual lesions. PTEN FISH status is indicated in blue for homozygous loss and grey for no deletion. (D) Sequence chromatogram for the genomic rearrangement junction amplified from ERG-positive lesions. AC denotes adenocarcinoma; PIN denotes prostatic intraepithelial neoplasia.

Figure 3

Summary of ERG and PTEN status in analysed cases.

Figure 4

Proposed model of retrograde glandular colonization. All invasive adenocarcinoma cells are ERG-positive due to an early TMPRSS2–ERG fusion event, yet only part of the invasive adenocarcinoma shows PTEN loss and is therefore considered subclonal loss. In such an ERG-positive invasive adenocarcinoma with subclonal loss of PTEN, TMPRSS2–ERG genomic breakpoint analysis establishes the shared clonal origin of lesions. Subclonal PTEN loss can be used as a temporal vector. The observation that ERG-positive PTEN-negative cells populate normal acinar and ductal structures strongly suggests a retrograde spreading pattern of invasive carcinoma cells into benign glandular structures. PTEN-proficient normal prostate epithelial cells and adenocarcinoma are shown in brown. PTEN-deficient cells are drawn in grey. ERG overexpression is indicated by blue nuclei. Intact basal cells are highlighted in red.