Immunohistology of the Prostate

Principal Antibodies

Many different antibodies are used for the IHC evaluation of urologic neoplasms. The generally used epithelial, neuroendocrine, and mesenchymal antibodies are discussed in other chapters. In each section, we will summarize antibodies of particular importance for the neoplasms covered in that section.

Prostate-Specific Antigen

Prostate-specific antigen (PSA) is a serine protease member of the human glandular kallikrein family. PSA is a 34-kDa glycoprotein of 237 amino acids with high sequence homology with human glandular kallikrein 2 (HK2). It is almost exclusively synthesized in the prostate ductal and acinar epithelium and is found in normal, hyperplastic, and malignant prostate tissue. PSA liquefies the seminal fluid coagulum through proteolysis of the gel-forming proteins, thus releasing spermatozoa. It can reach the serum by diffusion from the luminal cells through the basal cell layer, glandular basement membrane, and extracellular matrix (ECM). Measuring total serum PSA levels remains the mainstay of PCa detection, and numerous studies have shown that patients with PCa have, in general, elevated serum PSA levels. The most commonly used cutoff for PSA is 4 ng/mL. When serum PSA concentrations are 4 to 10 ng/mL, the incidence of cancer detection on prostate biopsy in men with a normal digital rectal examination (DRE) is approximately 25%. With serum PSA levels higher than 10 ng/mL, the incidence of PCa on biopsy increases to approximately 67%. However, the risk of cancer is proportional to the serum PSA level, even at values less than 4 ng/mL. As large screening trials have demonstrated, clinically significant cancers occur in men with serum PSA levels of 2.5 to 4.0 ng/mL. Thus, some experts have proposed lowering the PSA cutoff to 2.5 ng/mL to improve early detection of cancer in younger men.

Once PSA gains access into the circulation, most remains bound to serine protease inhibitors. The three most recognizable inhibitors are α-1-antichymotrypsin (α1-AT), α-2-macroglobulin, and α-1-protein inhibitor. PSA bound to α1-AT is the most immunoreactive and the most clinically useful in diagnosing PCa. A smaller fraction (5% to 40%) of the measurable serum PSA is free (non-complexed) PSA. Therefore, the total serum PSA measured reflects both free and complexed PSA. It has been demonstrated that the percentage of free PSA can improve the specificity of PSA testing for PCa. A free PSA value of less than 10% is worrisome for cancer. Additional isoforms of free PSA have been discovered and were detailed in a review by Gretzer and Partin. PSA is first secreted in the form of a precursor termed pro-PSA . This inactive form of the enzyme constitutes the majority of free PSA in serum in men with PCa, making the relative increase of serum pro-PSA a risk marker of PCa. Benign PSA (BPSA) refers to a cleaved form of PSA from benign prostatic hyperplasia tissue. The measurement of the ratio of pro-PSA to BPSA has been proposed as a means of improving the accuracy of diagnosing cancer in men with a very low percentage of free PSA levels and who are at relatively high risk for cancer. ^,

Serum PSA tests may also be used to monitor patients after therapy to detect early recurrence. Following radical prostatectomy, the serum PSA should drop to undetectable levels. Elevated serum PSA levels following radical prostatectomy (>0.2 ng/mL) indicate recurrent or persistent disease. Following radiotherapy for PCa, serum PSA values will decrease to a nadir, although not to the same extent as those following radical prostatectomy. Three subsequent rises in serum PSA values after radiotherapy indicate treatment failure.

Although PSA expression in extraprostatic tissues and tumors other than PCa have been rarely demonstrated ( Box 16.1 ), for all practical purposes, PSA expression at the IHC level is a specific and sensitive marker of prostatic lineage of differentiation, with as much as 97.4% sensitivity found in a study from our group. Urethral, periurethral, and perianal glands are among normal tissues that have been rarely reported to show PSA reactivity. Extraprostatic neoplasms that occasionally express PSA include urethral and periurethral adenocarcinoma, cloacogenic carcinoma, pleomorphic adenoma of salivary gland, salivary duct carcinoma, and rare mammary carcinomas. Although a rare report indicated PSA expression in intestinal-type urachal adenocarcinoma of the bladder, we failed to reveal such expression in a study of villous adenoma and adenocarcinoma of the bladder. The latter is especially important from a differential diagnosis point of view, given the topographic proximity of the two organs. Although weaker intensity of PSA expression can be encountered in higher Gleason grade PCa, we were able to demonstrate a high degree of PSA immunostain sensitivity (97.4%) in high-grade prostate carcinoma. Likewise, PSA expression is very valuable in defining a prostatic primary site of origin during the evaluation of a poorly differentiated metastatic carcinoma.

Prostate-Specific Membrane Antigen

Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein expressed in prostate tissue and, to a lesser extent, in peripheral and central nervous system, small intestinal, and salivary gland tissues. PSMA expression has also been documented in endothelial cells of the neovasculature of many solid tumors, including renal cell carcinoma (RCC). In the prostate, PSMA is expressed by benign and malignant prostatic epithelial cells, with a higher extent of staining seen in the latter. It is also expressed by high-grade prostatic intraepithelial neoplasia (PIN). PSMA expression correlates with PCa stage and Gleason grade. The increase in both expression and enzymatic activity of PSMA in aggressive PCa points to a selective advantage imparted on cells that express PSMA, thereby contributing to the development and progression of PCa. Increased PSMA expression is an independent predictor of PCa recurrence, ^, and PSMA expression is maintained in hormone-refractory PCa, thus increasing its utility in such settings. ^, Several imaging strategies exploit PSMA specificity to PCa and are currently in use for PCa diagnostic imaging. Furthermore, PSMA is under investigation as a target of therapy in PCa and other solid tumors, given its expression by the neovasculature of extraprostatic tumors. Cytoplasmic and, to a lesser degree, membranous PSMA expression has been documented in 11% of analyzed urinary bladder adenocarcinomas, a fact worth noting when the differential diagnosis includes prostatic and bladder adenocarcinoma.

Prostatic Acid Phosphatase/Prostate-Specific Acid Phosphatase

Prostate-specific acid phosphatase (PSAP) is one of the earlier prostate lineage markers to be exploited for immunolabeling of PCa before the discovery of PSA. Currently, the use of PSAP as a marker of prostatic differentiation has declined, given its relative lack of specificity compared with PSA and the more variable staining of PSAP in higher grade PCa. ^,

Prostein/P501S

P501S is a 553-amino acid protein localized to the Golgi complex. It is expressed in both benign and neoplastic prostate tissues. Typical P501S stain has a perinuclear cytoplasmic (Golgi) location and a speckled pattern. Expression is retained in poorly differentiated and metastatic PCa. P501S demonstrated up to 99% sensitivity in an initial study from our group by Sheridan and colleagues. In rare metastatic lesions, P501S positivity may be encountered in the presence of PSA-negative expression, making it an advantageous addition to a prostate lineage immunopanel. To date, P501S expression has not been shown in extraprostatic carcinomas, which makes it of great utility in differentiating high-grade PCa from other high-grade carcinomas, including colorectal and urothelial carcinoma. ^{, ,}

α-Methylacyl–Coenzyme-A Racemase/P504s

α-Methylacyl-CoA racemase (AMACR) is mainly localized to peroxisomal structures and plays a critical role in peroxisomal beta oxidation of branched chain fatty acid. In their original detailed IHC analysis, Luo and associates demonstrated that both prostate carcinomas and high-grade PINs consistently revealed a significantly higher expression than that of matched normal prostate epithelium. Both untreated and hormone-refractory PCa metastases generally maintain a strong positive reactivity for AMACR. An overall PCa sensitivity and specificity of 97% and 92%, respectively, have been shown in a multi-institutional study by Jiang and Woda.

Cytoplasmic AMACR staining combined with the absence of basal cell markers, such as nuclear protein p63 and high-molecular-weight cytokeratins (HMWCKs), has proved to be of greatest utility in providing an added layer of assurance in establishing the diagnosis of PCa on small needle biopsy foci. However, AMACR expression has been repeatedly demonstrated in high-grade PIN and in some benign mimics of PCa, such as glandular and partial atrophy and adenosis. Therefore, AMACR is of limited utility as a single marker in resolving the differential diagnosis of PCa in such lesions. A panel of immunostains that includes AMACR, HMWCK, and p63 (positive AMACR immunostaining, along with negative basal cell markers; Table 16.1 ) is recommended in the interrogation of atypical prostatic glandular foci.

High-Molecular-Weight Cytokeratins

HMWCKs are helpful in highlighting the presence or absence of basal cells in a focus of atypical prostate glands. 34βE12 is currently the most widely used clone, both individually or as a component of a three-antibody cocktail, which includes a second basal cell marker, such as p63, and AMACR. Alternatively, CK5/6 can be used as the HMWCK marker, individually or in combination with p63 and AMACR. A study by Abrahams and colleagues suggested a superior sensitivity for CK5/6 as an HMWCK in prostate biopsies fixed in Hollande’s fixative. Following the initial examination of hematoxylin and eosin (H&E)-stained routine sections, the application of such IHC cocktail to previously prepared, unstained, intervening sections is recommended for biopsies in which establishing the presence or absence of basal cells in a questionable focus will lead to a definitive resolution of a benign or malignant diagnosis, respectively.

p63

The p53 homolog p63 encodes for different isotypes that can either transactivate p53 reporter genes (TAp63) or act as p53-dominant negatives (ΔNp63). p63 is expressed in the basal or myoepithelial cells of many epithelial organs; its germline inactivation in the mouse results in the agenesis of organs, such as skin appendages and the breast. In the prostate, p63 expression is limited to basal cells and is absent in secretory and neuroendocrine cells, and ΔNp63α isotype is the most abundantly represented isotype in normal prostate basal cells. Experimental evidence suggests that the p63 gene is essential for normal stem cell function in the prostate. Several studies have confirmed the clinical utility of p63 immunostain as a prostate basal cell marker, and some studies suggest a slight sensitivity advantage for p63 over HMWCK alone. Additionally, the use of basal cell HMWCK and p63 cocktails may reduce the staining variability that may be encountered in basal cells, and may also further decrease the false-negative and false-positive rates of basal cell labeling by either marker alone ( Table 16.2 ). ^,

Finally, given the fact that immunostains for basal cell markers are typically used in a “negative” diagnostic mode, to show the absence of basal cells in PCa, sole reliance on such markers is not advocated, and the identification of a combination of major and minor histologic features of PCa is crucial for achieving clinical diagnostic accuracy. In this regard, consideration should be given to the fact that benign prostatic glands from the transition zone are subject to basal cell staining variability, which may result in occasional negative basal cell staining in such benign glands. Furthermore, basal cells can be retained, albeit very rarely, in individual glands in otherwise typical acinar PCa focus, and the constellation of diagnostic features are to be relied on in such rare cases. We have described an intriguing p63-positive, HMWCK-negative variant of PCa in which nuclear p63 staining is seen in secretory PCa cells in a nonbasal distribution.

NKX3-1

NKX3-1 is a prostate-specific androgen-regulated homeobox gene required for tissue differentiation, whose loss of function leads to carcinogenesis. In the normal prostate, NKX3-1 controls differentiation and protects against oxidative damage by regulating gene expression in conjunction with other transcription factors. In cancer, a relative loss of NKX3-1 expression occurs due to loss of heterozygosity (LOH), promoter methylation, or alterations in NKX3-1 degradation. Downregulation of NKX3-1 protein leads to increased prostate epithelial cell proliferation, differentiation, and susceptibility to DNA damage, thereby furthering oncogenic insults. In addition to benign and malignant prostatic epithelium, NKX3-1 expression is found in normal testis, bronchial mucous glands, and infiltrating lobular carcinoma of the breast. Sensitivity of NKX3-1 for PCa has ranged from 68% to 94.7%. In poorly differentiated PCa, NKX3-1 appears to be superior to PSA, which may show a relatively focal weaker staining in that subset. ^,