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Diagnostic utility of P504S/p63 cocktail, prostate-specific antigen, and prostatic acid phosphatase in verifying prostatic carcinoma involvement in seminal vesicles: a study of 57 cases of radical prostatectomy specimens of pathologic stage pT3b.

Identification of prostatic carcinoma invasion in the seminal vesicle is directly associated with tumor staging and is well regarded as an indication of poor prognosis. (1-3) Detection of seminal vesicle invasion can be difficult, especially with cribriform or papillary growth patterns. (4-6) Partial atrophy and crowded benign glands are also commonly encountered benign entities that result in diagnostic difficulty. (7) Immunohistochemical stains that use prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are instrumental in ambiguous cases in making the differential diagnosis between prostatic adenocarcinoma involving the seminal vesicle and seminal vesicle glands that are mimicking prostate carcinoma glands. To our knowledge, the use of P504S and basal cell markers (p63 and 34[beta]E12) in this situation has not been reported.

P504S, which is also referred to as the AMACR gene, encodes [alpha]-methylacyl-coenzyme A racemase. (8) P504S is overexpressed in most prostatic carcinomas and in highgrade prostatic intraepithelial neoplasia with cytoplasmic immunoreactivity and is not usually expressed in benign prostatic glands, small glandular proliferations, and atrophy, or, if expressed, it shows only focal or weak staining. (8) p63 stains the nuclei of basal cells of the benign prostatic glands. (9,10) The loss of a basal cell layer is an important diagnostic criterion used to differentiate carcinomatous from benign glands, which retain a basal cell layer. (10)

Placing the antibodies into a single cocktail does not alter the antigenic properties of the individual antibodies. (11) The use of antibody cocktails that have different colored stains, such as the triple-antibody cocktail, which includes the 34[beta]E12 high-molecular basal cell cytokeratin cytoplasmic stain in addition to P504S and p63, is effective in differentiating prostatic carcinoma from benign prostatic tissue. (11,12) The nuclear-staining pattern of basal cell nuclei of benign glands by p63, and the cytoplasmic staining pattern of P504S, in prostatic carcinoma makes the use of a single-color P504S/p63 cocktail stain possible. (13)


Previously, pathologists have used PSA and PAP to distinguish prostatic glands from the seminal vesicle in instances where morphology alone was inconclusive. Because P504S/p63 immunohistochemical stains provide the additional benefit of establishing whether prostatic glands are benign or malignant, we aimed to evaluate the usefulness of the P504S/p63 cocktail with one color to verify prostatic carcinoma within the seminal vesicle as an alternative to PSA and PAP immunostaining.



This study was conducted after approval from the institutional review board of The Methodist Hospital. We selected a total of 57 radical prostatectomy specimens of stage pT3b (invasion of the seminal vesicle by prostatic adenocarcinoma) from cases accessioned between 2005 and 2008. Currently, the total number of stage pT3b specimens at this institution since 2005 is 98, from a total of 1,487 radical prostatectomy specimens (6.6% incidence). No criteria, other than seminal vesicle invasion, were used to select cases, and representative blocks of seminal vesicle invaded by prostatic adenocarcinoma were included for this study.


Formalin-fixed, paraffin-embedded blocks were cut into 4-[micro]m, consecutive sections. Consecutive sections were individually stained with a hematoxylin-eosin (H&E) stain (Figure 1, A), an immunohistochemical stain using a cocktail containing antibodies against P504S (1:100; Dako, Carpinteria, California) and p63 (1:100; Neomarker, Fremont, California) (Figure 1, B), an immunohistochemical stain using antibodies against PSA (1:4000; Dako) (Figure 1, C), and an immunohistochemical stain using antibodies against PAP (1:2000; Dako) (Figure 1, D).

The P504S/p63 cocktail stain was prepared by combining 10 [micro]L of P504S antibody and 10 [micro]L of p63 antibody with 980 [micro]L of antibody diluent to achieve 1:100 ratio of antibody dilutions for P504S and p63, respectively. This cocktail was validated in our laboratory by preparing 3 separate dilutions of the cocktail stain (1:50, 1:100, and 1:150) and then, comparing them to the individual stains (P504S and p63) at similar dilutions. The cocktail-stained sections were compared with the sections stained with the individual antibodies against P504S and p63, and no aberrant staining was detected, and the staining patterns are highly consistent among them. A 1:100 dilution of the P504S/ p63 antibody cocktail and the individual P504S and p63 antibodies was determined to be appropriate because it is reproducible and provides the highest sensitivity and specificity in our laboratory.

Sections were immunostained using an autostainer (Dako). The sections were deparaffinized in xylene and sequentially washed in graded ethanol. Heat-induced epitope retrieval was performed with diluted Dako Target Retrieval Solution (sodium citrate buffer, pH 6.0) for 20 minutes inside a pressure cooker. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 8 minutes. Slides were incubated with primary antibodies for 20 minutes at room temperature with the aforementioned titers. The subsequent reactions were performed using components from EnVision + Dual Link System-HRP kit (Dako), including incubation with universal (rabbit and mouse) secondary antibody for 25 minutes, a biotin-free horseradish peroxidase enzyme-labeled polymer, and the signal visualization by 3,3'-diaminobenzidine hydrochloride. Then sections were counterstained with hematoxylin before being mounted on slides and examined by light microscopy. Proper positive and negative control sections were used.


The diagnosis of prostatic carcinoma was confirmed based on the morphology on H&E-stained sections by 2 pathologists without knowing the immunostain results. Because the sections were stained with a single-color cocktail for P504S/p63, the staining pattern was the key to determining the positivity and negativity of the particular stain. P504S stained the cytoplasm of epithelial cells, whereas p63 stained nuclei in the basal cell layer, without masking each other. p63 was considered positive if strong nuclear staining was present. P504S, PSA, and PAP were considered positive if strong cytoplasmic staining was present. Each marker was reported as positive (ranging from focal to diffuse and from weak to strong) or negative. Small foci of weak or nonspecific positive staining for P504S, including scant fine granular background staining of epithelial and stromal cells, were classified as negative. p63 was classified as negative when no immunostain was appreciated around the glands.


A summary of the findings of the immunohistochemical staining profile for PSA, PAP, and a cocktail of P504S/p63 within prostatic carcinoma, seminal vesicle, and benign prostatic glands is provided (Table).

Prostatic Carcinoma Staining

Glands that were unequivocal for prostatic carcinoma were identified on H&E-stained sections (Figure 2, A). The foci of prostatic carcinoma in all sections demonstrated positive cytoplasmic staining with P504S (57 of 57; 100%) and no nuclear staining of the basal cells with p63 (0 of 57; 0%) in sections stained with the P504S/p63 cocktail (Figure 2, B). These prostatic carcinoma foci stained positive for PSA (57 of 57; 100%) (Figure 2, C), and PAP (57 of 57; 100%) (Figure 2, D) with a cytoplasmic staining pattern.

Benign Prostatic Gland Staining

Benign prostatic tissue was identified on 27 H&E-stained sections (Figure 3, A). The basal cells around all benign glands showed positive nuclear staining with p63 (27 of 27; 100%) and no positive staining with P504S (0 of 27; 0%) within benign prostatic gland epithelial cells stained with the P504S/p63 cocktail (Figure 3, B). The benign glands were uniformly positive for PSA (27 of 27; 100%) (Figure 3, C) and PAP (27 of 27; 100%) (Figure 3, D) with cytoplasmic staining.

Seminal Vesicle Staining

Seminal vesicle was identified on all H&E sections (Figure 4, A). There was no cytoplasmic staining of seminal vesicle epithelium for P504S (0 of 57; 0%) and distinct positive nuclear staining of the basal cells of the seminal vesicle epithelium with p63 (57 of 57; 100%) in all specimens stained with the P504S/p63 cocktail (Figure 4, B). The seminal vesicle epithelium was uniformly negative for PSA (0 of 57; 0%) (Figure 4, C) and PAP (0 of 57; 0%) (Figure 4, D). The proteinous material within the seminal vesicle lumens was prone to show occasional, nonspecific, positive staining for PSA, P504S, and PAP, with PSA showing more intense staining than both P504S and PAP. However, this nonspecific staining was easily differentiated from that of the true positive staining in prostatic carcinomas because of the lack of cytoplasmic staining within the epithelial cells of the seminal vesicle.


Invasion of the seminal vesicle by prostatic carcinoma is an unfavorable prognostic factor and is associated with a high likelihood of local recurrence, lymph node metastasis, and distant metastasis, as well as increased mortality, when compared with prostatic carcinomas that do not involve the seminal vesicle. (3) Any microscopic involvement of the seminal vesicle results in an increase in cancer staging to pT3b. Previous studies have demonstrated that seminal vesicle invasion is present in as many as 20% to 26% of radical prostatectomy specimens. (3) For this reason, it is imperative that an accurate assessment of seminal vesicle involvement be made. To make a definitive diagnosis of seminal vesicle involvement, tumor glands should be present within the muscular layer of seminal vesicles; when tumor glands are present in the perivesicular fibroadipose tissue, the case should be interpreted as a case with extraprostatic extension (pT3a).

Traditionally, well-differentiated prostatic adenocarcinoma has been distinguished from benign glands by several well-accepted histologic criteria, including a small glandular proliferation with nuclear enlargement and prominent nucleoli, amphophilic cytoplasm, intraluminal crystalloids, and the absence of a basal cell layer. (14) Higher-grade tumors with a Gleason score of 7 or higher are generally easier to identify. Additionally, the presence of glomerulation, collagenous micronodules (mucinous fibroplasia), circumferential perineural invasion, and glands in fat have been shown to be unequivocal histologic findings for the diagnosis of prostatic carcinoma. (14)


With the increase in prostate biopsies because of early detection with screening tests, such as serum testing for PSA, there have been numerous studies detailing the diagnostic dilemma for detecting prostatic adenocarcinoma on H&E slides alone. It is common that small foci of prostatic adenocarcinoma can be underdiagnosed and that similarly small foci of benign mimickers can be overdiagnosed on needle biopsy of the prostate. (15) There are numerous benign mimickers of prostatic adenocarcinoma that can pose diagnostic dilemmas, such as complete atrophy, adenosis, granulomatous prostatitis, partial atrophy, and crowded benign glands. (7) The seminal vesicle and ejaculatory duct have also been identified as potential mimickers of carcinoma when the foci of interest are small, such as on a core needle biopsy. (7) Cribriforming and papillary growth patterns have been seen in as many as 86% of cases of prostatic adenocarcinoma detected by core needle biopsy. (4) The ejaculatory duct or seminal vesicle epithelium also commonly demonstrate similar cribriforming or papillary structures, as well as small, crowded gland proliferation, and it can be difficult to differentiate from prostatic carcinoma. (4)

One particularly difficult potential dilemma, although rare, is the diagnosis of ectopic prostatic glands within the seminal vesicle. (5) Ectopic prostatic glands within the seminal vesicle can easily be misdiagnosed as invasion of the seminal vesicle by prostatic carcinoma because it is unusual to see benign prostatic glands in the seminal vesicle and because there is usually only a small focus. (5,6) In that particular situation, PSA or PAP would be of no benefit because they do not differentiate benign from malignant prostatic glands. (5) However, the presence of a basal cell layer by nuclear staining with p63 and the absence of cytoplasmic staining with P504S would be helpful in differentiating this benign mimicker as well as seminal vesicle/ejaculatory duct epithelium from invasion by prostatic carcinoma. (5)

The presence of positive cytoplasmic staining with P504S has been shown to be a highly specific marker for prostate adenocarcinoma, with very few instances of false-positive staining within benign glands. (14,15) When positive staining with P504S was seen within benign glands, it was described as consistently focal and weak and typically nonspecific. (14) However, negative staining with P504S in suspicious foci does not necessarily indicate a benign diagnosis. (15) Focal nonreactivity of P504S within confirmed prostatic carcinoma has been reported. (15) Additionally, premalignant lesions, including high-grade prostatic intraepithelial neoplasia and atypical adenomatous hyperplasia, have also occasionally demonstrated positive staining with P504S. (15)

In addition to the high specificity of P504S in prostatic carcinoma, p63 is a sensitive and specific marker for staining the basal cells around benign prostatic glands. (14,16) Although the absence of positive nuclear staining with the basal cell marker p63 does not necessarily indicate that the suspicious foci is conclusively prostatic carcinoma, it does strongly support that diagnosis. When suspicious foci of glands demonstrate the lack of basal cells with the absence of positive nuclear staining by p63 and the glands show positive cytoplasmic staining with P504S, the combination of these antibodies is highly sensitive and specific for prostatic carcinoma. (14)


The nuclear staining pattern of basal cell nuclei of benign glands by p63 and the cytoplasmic staining pattern of P504S in prostatic carcinoma makes the use of the single P504S/p63 cocktail stain possible. (13) This cocktail allows fewer slides to be prepared, subsequently, reducing the technical time required to prepare them and, ultimately, improving the cost effectiveness of staining. (17) It may also eliminate the need for a repeat biopsy by allowing the pathologist to render a diagnosis on a small ambiguous lesion when minimal tissue is available. (17)

One study, using P504S stain on prostate tissue, reported negative staining by P504S in the ejaculatory duct or seminal vesicle epithelium. (8) However, there have been no studies using P504S/p63 in a single-color cocktail to distinguish prostatic cancer involvement in seminal vesicles versus cancer mimickers because of the architectural complexity of the seminal vesicles and ejaculatory ducts. Our study confirms the finding that the seminal vesicle epithelium stained uniformly negative for P504S (0 of 57; 0%), and there was uniform staining of the basal cells of the seminal vesicle with distinct positive nuclear staining by p63 (57 of 57; 100%). Therefore, the use of the P504S/p63 cocktail distinctively helped to identify prostatic carcinoma from a benign seminal vesicle by their opposite staining characteristics. The P504S/p63 cocktail also provides an advantage not found with PSA or PAP: its ability to distinguish benign prostatic glands within, or near, the seminal vesicle, from prostatic carcinoma on the same section. It is not uncommon that sections from the base of the prostate include seminal vesicle tissue because the so-called intraprostatic invagination of the seminal vesicle is located anatomically at the base of the prostate. Additionally, it is not uncommon for prostatic tissue to be included when specifically sampling the seminal vesicle.

In conclusion, we introduced a single-color cocktail of P504S/p63 in identifying invasive prostatic carcinoma within the seminal vesicle with a differential diagnosis from architectural patterns of seminal vesicle mimicking prostate carcinoma and potential benign ectopic prostatic tissue in seminal vesicles. The use of a single-color cocktail is cost-effective and conserves limited tissue. It also provides an advantage, not found with PSA or PAP, in its ability to distinguish benign prostatic glands from prostate carcinoma, which is particularly advantageous when benign prostatic glands are located adjacent to seminal vesicle epithelium and can potentially be mistaken for seminal vesicle invasion. Our data support the expanded use of this cocktail in seminal vesicle evaluation. Therefore, we recommend the use of the single-color P504S/p63 immunohistochemical stain cocktail for identifying prostatic carcinoma involving the seminal vesicle and for distinguishing benign prostatic glands from prostatic carcinoma when there is a question of seminal vesicle invasion.



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(11.) Ng VW, Koh M, Tan SY, Tan PH. Is triple immunostaining with 34pE12, p63, and racemase in prostate cancer advantageous?: a tissue microarray study. Am J Clin Pathol. 2007;127(2):248-253.

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(13.) Tacha DE, Miller RT. Use of p63/P504S monoclonal antibody cocktail in immunohistochemical staining of prostate tissue. Appl Immunohistochem Mol Morphol. 2004;12(1):75-78.

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Aaron M. Harvey, MD; Beverly Grice, HTL; Candice Hamilton, HTL; Luan D. Truong, MD; Jae Y. Ro, MD; Alberto G. Ayala, MD; Qihui "Jim" Zhai, MD

Accepted for publication August 17, 2009.

From the Department of Pathology, The Methodist Hospital, Weill Medical College of Cornell University, Houston, Texas (Drs Harvey, Truong, Ro, Ayala, and Zhai, and Mss Grice and Hamilton); Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea (Dr Ro).

The authors have no relevant financial interest in the products or companies described in this article.

Dr Zhai is now located at the University of Cincinnati, Ohio.

Presented in part at the annual meeting of the College of American Pathologists, San Diego, California, September 25-28, 2008.

Reprints: Qihui "Jim" Zhai, MD, Department of Surgical Pathology, University of Cincinnati, 231 Albert Sabin Way, Room 1358, ML 0529, Cincinnati, OH, 45267-0529 (e-mail:
Immunohistochemical Staining Profile in Seminal Vesicle, Prostatic
Carcinoma, and Benign Prostate Glands of 57 Radical Prostatectomy
Specimens of Pathologic Stage pT3b

Immunohistochemical Prostate Seminal Benign Prostate
Stain Cancer, Vesicle, Glands,
 No. (%) No. (%) No. (%)

PSA + (cytoplasm) 57 (100) 0 (0) 27 (100)
PAP + (cytoplasm) 57 (100) 0 (0) 27 (100)
p63 + (basal cell nuclei) 0 (0) 57 (100) 27 (100)
P504S + (cytoplasm) 57 (100) 0 (0) 0 (0)

Abbreviations: PAP, prostatic acid phosphatase; PSA, prostate-specific
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Title Annotation:Original Articles
Author:Harvey, Aaron M.; Grice, Beverly; Hamilton, Candice; Truong, Luan D.; Ro, Jae Y.; Ayala, Alberto G.;
Publication:Archives of Pathology & Laboratory Medicine
Date:Jul 1, 2010
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