Genetic heterogeneity in a prostatic carcinoma and associated prostatic intraepithelial neoplasia as demonstrated by combined use of laser-microdissection, degenerate oligonucleotide primed PCR and comparative genomic hybridization

Abstract

We combined laser-assisted microdissection from H&E-stained paraffin sections, degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR), and comparative genomic hybridization (CGH) to analyse chromosomal imbalances in small tumour areas consisting of 50-100 cells. This approach was used to investigate intratumour genetic heterogeneity in a case of metastatic prostatic adenocarcinoma and chromosomal changes in areas of prostatic intraepithelial neoplasia (PIN) adjacent to the invasive tumour. In four microdissected invasive tumour areas with different histological patterns (acinar, cribriform, papillary and solid) marked intratumour heterogeneity was found by CGH. Recurrent chromosomal imbalances detected in at least two microdissected tumour areas were gains on 1p32-->p36, 2p22, 3q21, 7, 8q21-->q24, 11q12-->q13, 16p12-->p13, 17, 19 and loss on 16q23. Additional chromosomal changes were found in only one of the microdissected areas (gains on 16q21-->q23, 20q22 and losses on 8p21-->p23, 12p11-->q12, 12q21-->q26, 13q21-->q34, 16q12, and 18q22). In PIN, gains on chromosomes 8q21-->q24 and 17 were found in both samples investigated (low and high grade PIN), while gains on chromosomes 7, 11q, 12q, 16p, and 20q and losses on 2p, 8p21-->p23, 12q were found only in one PIN area. Controls to ensure reliable CGH results consisted in CGH analyses of (i) approximately 80 microdissected normal epithelial cells, which showed no aberrations after DOP-PCR and (ii) larger cell numbers (approximately 10(5) or 10(7) cells) of the primary tumour investigated without DOP-PCR and partially displaying the chromosomal imbalances (gain on 16p12-->p13, losses on 2p25, 8p21-->p23, 12p11-->p12, 12q21-->q26, 18q22) found in the small microdissected areas. Microsatellite and FISH analyses further confirmed our CGH results from microdissected cells. The combined approach of laser-assisted microdissection, DOP-PCR and CGH is suitable to identify early genetic changes in PIN and chromosomal imbalances associated with the particular histological patterns of invasive prostatic adenocarcinoma.

High Expression of a New Marker PCA-1 in Human Prostate Carcinoma

“…Whereas PCA-1 was not detected by immunohistochemistry in either benign prostatic hyperplasia or normal prostatic tissue, many but not all atypical cells in high-grade PIN do frequently overexpress PCA-1. PIN has been well-described using microdissection techniques (27,28), with the hope that any genetic alterations developing in a continuum from normal epithelium to malignant tumor will provide a clearer understanding of the molecular history and clinical future. Whereas it remains to be proven, it is possible that these atypical PIN cells expressing PCA-1 have a higher potential to undergo malignant progression and that expression of PCA-1 may correlate to the earlier stages of prostate cancer development; however, we found no significant correlation between PCA-1 expression and tumor grades or pathologic stages.…”

Chromosomal changes during development and progression of prostate adenocarcinomas

“…At the histological level, PINs outside the invasive carcinoma are considered as premalignant lesions of prostate cancer (Bostwick and Brawer, 1987;Bonkhoff and Remberger, 1996). In the PIN-carcinoma-metastasis sequence, PIN areas are poorly characterized for chromosomal alterations because they appear as very small cell compartments which can be only studied utilizing either microdissection and subsequent molecular genetic techniques like CGH (Weber et al, 1998;Zitzelsberger et al, 1998;Kim et al, 1999) or FISH on paraffin sections (Alers et al, 1995;Qian et al, 1996;Jenkins et al, 1997). In this study, we were able to demonstrate cytogenetic changes in 5 lymph node metastases and 12 PIN areas from 6 different cases.…”

“…DOP-PCR was performed according to a published procedure (Weber et al, 1998;Zitzelsberger et al, 1998). PCR reaction was carried out in a 50 µl reaction volume (3.5 mM MgCl 2 , 50 mM KCl, 20 mM Tris/HCl, pH 8.4) containing the microdissected and pretreated cells in 20 µl laser buffer, 0.2 mM primer UW4B (5'-CCGACTCGAGNNNNNNATGTGG-3 '), and 4 units Taq polymerase.…”

“…Application of CGH and fluorescence in situ hybridization (FISH) to primary tumours of prostatic adenocarcinomas revealed consistent changes on chromosomes 7, 8p, 10, 13q, 16, 17 and 18q (Brothman et al, 1994;Macoska et al, 1994;Matsuyama et al, 1994;Joos et al, 1995;Qian et al, 1995;Visakorpi et al, 1995;Bova and Isaacs, 1996;Cher et al, 1996;Huang et al, 1996;Deubler et al, 1997). However, cytogenetic changes in prostatic intraepithelial neoplasias (PIN) which are considered as premalignant lesions and which are often present besides the invasive tumour are only poorly characterized cytogenetically (Alers et al, 1995;Qian et al, 1995;Zitzelsberger et al, 1998). Methodological improvements of approaches combining microdissection and CGH analysis (Kuukasjärvi et al, 1997) were prerequisites for the analysis of such early chromosomal aberrations in premalignant cells of other tumours like cervical carcinoma (Heselmeyer et al, 1996), breast cancer (Aubele et al, 2000b) and oral malignant lesions (Weber et al, 1998).…”

“…Methodological improvements of approaches combining microdissection and CGH analysis (Kuukasjärvi et al, 1997) were prerequisites for the analysis of such early chromosomal aberrations in premalignant cells of other tumours like cervical carcinoma (Heselmeyer et al, 1996), breast cancer (Aubele et al, 2000b) and oral malignant lesions (Weber et al, 1998). Their application to premaligant lesions in prostate cancer has recently been described (Zitzelsberger et al, 1998;Kim et al, 1999), but for an improved understanding of mechanisms of tumour development and tumour progression more data on cytogenetic changes in thePIN-adenocarcinoma-metastasis sequence are needed.…”