The use of serum prostate specific antigen (PSA) screening to facilitate early detection of prostate cancer has resulted in a dramatic increase in the number of prostate needle core biopsies for pathologists to examine (Table 2).

This has been accompanied by a notable increase in the number of biopsies with ambiguous lesions as atypical small acinar proliferation suspicious for cancer but not diagnoses (ASAP) and microfoci of adenocarcinoma, defined as a single focus of tumour less than 1 mm in greatest dimension (Table 3).

These ambiguous lesions represent a great challenge for pathologists who must try, with confidence, to distinguish benign from malignant prostate glands.

The diagnosis of small focus carcinoma on biopsy specimens is based on a constellation of features more commonly present in adenocarcinoma than benign glands.

The accepted major histological criteria for the diagnosis of prostatic adenocarcinoma include the following: (1) acini that infiltrate the prostatic stroma, particularly in between benign glands; (2) nuclear atypia, with or without prominent macronucleoli; (3) cytoplasmic features with basophilic cytoplasm, (4) intraluminal contents as crystalloid or pink necrosis, and (5) the absence of a basal cell layer.

In most cases, the diagnosis of minimal adenocarcinoma can be made easily using these criteria. Only a few features, such as glomerulations, mucinous fibroplasia (collagenous micronodules), and perineural invasion are diagnostic in and of themselves for prostate cancer 12.

However, there are instances in which morphological features fall short of those needed for an outright diagnosis of malignancy and additional support, with basal cell markers are required.

Immunohistochemistry may be a useful adjunct in the diagnosis of limited focus of prostatic carcinoma. One feature distinguishing benign mimickers from cancer is that benign glands contain basal cells which are absent in cancerous glands, and pathologists often use immunohistochemical markers to label basal cells 16.

Although as with any immunohistochemical studies, there are problems with both sensitivity and specificity. Since the first reported results of the high molecular weight cytokeratin 903 (34?E12) as a marker of prostatic basal cells by Vogel et al. 18 in 1984, numerous studies have shown that the systematic application of anti-CK 903 permitted diagnostic confirmation in 58% of the cases, restored a correct diagnosis in 18% of doubtful cases, and changed the diagnosis in 2% of the cases 17-19.

However, application of this antibody is not practical in 8% of the cases 20, and not all benign glands label uniformly with basal cell markers, and the disappearance of the layer of basal cells has been reported in 11% in cases of atrophy, 12% in cases of basal cell hyperplasia and in 10 to 90% in cases of atypical adenomatous hyperplasia 17.

Furthermore, certain mimickers of adenocarcinoma of the prostate are even less frequently labelled uniformly with these stains. Consequently, negative staining in a small focus of atypical glands for basal cell markers is not diagnostic of adenocarcinoma of the prostate.

Cytokeratins 5 and 6 (CK5/6) has been reported to be a more sensitive and specific marker of prostatic basal cells, with fewer unsatisfactory results 21. CK 5/6, normally expressed by complex epithelium, is a marker of mesothelial cells, malignant mesothelioma, as well as pancreatic, bile tract and mammary carcinomas 22, 23.

This antibody reacts with prostatic basal cells, but not with prostatic tumoral cells nor with high grade prostatic intraepithelial neoplasia, independently of the timing delay and the type of fixative (formalin vs Bouin?s solution) 21. CK 5/6 does not require complex antigenic restoration and presents a sensitivity of 97-100 % and a specificity of 62%, which is better than the 40 % observed with CK 903 21. In our experience, in the case of ambiguous lesions, CK 5/6 is more effective than CK 903 24, 25.

Despite the use of new methods applied to immunohistochemical techniques, including that of the immunostainer automates (Ventana, Dako), automation of heat antigenic restoration, these two antibodies remains difficult to use, especially with Bouin?s fixation which requires technical subtleties for antigenic restoration, such as protease digestion, heat recovery, developer systems, and even, in some cases, all three 19, 20, 26.

p63 is a new developed antibody to prostate basal cells 14. Protein p63, which shares homology with the suppressor gene of tumor p53, seems to play a critical role as a regulator of growth and development of cutaneous epithelium, uterine cervix, breast and the urogenital tract, and in particular, of prostate development 27.

Signoretti et al. first individualized the ?Np63 isoform in prostate basal cells and demonstrated the crucial role of p63 in development of the prostate 13. Subsequently, numerous authors showed that p63 was expressed by basal cells in normal prostatic glands 3, 11, 14. demonstrated that the vast majority (89 to 94%) of prostate carcinomas do not express p63 14 and that p63 immunostaining represents a novel adjuvant method for facilitating pathologic diagnosis in prostate needle biopsy.

Further studies indicate that p63 is not expressed by prostate tumor cells and can therefore be used in the same way as CK 903 in the diagnosis of atypical small acinar proliferation suspected to be, but not diagnosed as, malignant 16. As Zhou et al., in our experience, we found a total absence of basal cells after p63 immunostaining in prostate cancer 24, 25.

In normal glands p63 reacted with basal cells in 98% (254/259) of our cases, with weak (9%), moderate (11%) or strong (78%) nuclear staining. The intensity of nuclear staining was independent of the fixative technique (p = 0.1). In atrophic and benign lesions, 10 to 100% persistent basal cell staining was observed with p63.

In summary there a at least 3 separate useful immnostains for basal cells (CK903, CK5/6 and p63), all of which are regarded as "negative markers" for cancer, in the sense that when they stain positively, the atypical small acinar focus is negative for cancer. None of these markers are completely sensitive in the detection of basal cells, and furthermore rares cases of primary prostate cancer have been reported to demonstrate focal staining with CK903.

P504s / AMACR

For these reasons it is fortuitous that an additional useful marker for prostate cancer has been identified: ?-methylacyl-Co A racemase (AMACR). Using RNA subtraction hybridization and complementary DNA microarray analysis of prostatic carcinoma 15, 28, has been identified that AMACR along and its RNA messenger (p504s) was found to be over expressed in prostatic carcinoma. AMACR is known to be involved in the ?-oxidation of branched-chain fatty acids and fatty acid derivatives 10, 29, and has been located in peroxisomes and mitochondria in human cells. Expression of the enzyme is strongly expressed in normal liver and kidney cells 30, and over expression has been detected in many human tumours such as colon, breast, melanomas, and papillary renal cell carcinomas 8, 31, 32.

In prostate, with western blot analysis a 36-fold overexpression of p504s was observed in carcinomas when compared with benign glandular tissues 15. Using a highly specific antibody (p504s) directed against the enzyme, Jiang et al. found that strong immunostaining for the enzyme was consistently present in prostatic carcinoma and in high grade prostatic intraepithelial neoplasia of the peripheral zone of the prostate 33. In contrast, immunostaining was not detected in 88% of the 173 cases of benign hypertrophic hyperplasia, whereas in the remaining 12% only focal weak immunostaining was present 33. Therefore, the investigators concluded that p504s was a highly specific and sensitive marker of prostatic carcinoma. Over the past three years, many studies have reported extensive upregulation of AMACR at the protein and transcript levels in high grade prostatic intraepithelial neoplasia and prostate cancer 6, 9, 17.

Initial studies suggested that AMACR was uniformly and strongly positive in 97% to 100% of prostate cancer 4,6,7.

Further studies found that AMACR will label the cytoplasm of approximately 80% of limited prostatic carcinoma on needle biopsy 34, and that in positive cases, not all of the tumoral glands are positive and are often not intensely positive. Certain variants such as foamy glands, pseudo hyperplastic or atrophic carcinomas, were even less frequently positive for AMACR expression, with only a 60% to 80% positivity 35.

More recently at the 100th AUA congress in San Antonio, Schostak et al. Demonstrated the usefulness of quantitative AMACR RNA transcript detection in prostatic tissues, by PCR, as an alternative to immunohistochemical study specially in smallest amount of needle biopsy [AUA poster n° 531].

In our experience, we found that the positivity for p504s was observed in 97% of prostatic cancer independently of the Gleason?s score (p = 0.29), and the fixative technique (p = 0.27) with a greater sensitivity to p504s/p63 (95%) than to CK5/6 (57%) or p63 (86%), and higher specificity for p504s/p63 (95%) than for CK5/6 (88%) or p63 (81%), and could be heterogeneous 25, 36.

The diagnostic implication of these findings is that AMACR staining should be interpreted and used with caution. If we considered p504s intensity of immunostaining we found only 67% moderate and intense cytoplasmic staining and weak staining in 31% of our cases 25. This could explain our better score of immunostaining compared to the 80 to 85% p504s expression in prostate cancer usually reported. As Zhou et al. and Herawi et al. we though that a result of negative AMACR staining never downgrade a cancer diagnosis rendered on the hematoxylin and eosin-stained sections to atypical, or an atypical diagnosis to benign 34 [Herawi et al. Am J Surg Pathol 2005 Juillet].

In addition to problems with sensitivity, AMACR is yet considered as not entirely specific for adenocarcinoma, and will label almost all cases of high-grade prostatic intraepithelial neoplasia, some foci of adenosis, partial atrophy, crowned glands [Herawi am J surg pathol 2005] and even some morphological benign glands. In high grade prostatic intraepithelial neoplasia (HG PIN), prostatic cells expressed p504s in 20 to 99 % of cases, with a granular intracytoplasmic pattern 9, 25, with a weaker staining intensity for P504s in HG PIN than in prostatic carcinomas 37.

Several studies reported that p504s could be positive 21 to 36.4% of normal glands 9, 34. In our series, we found only a 2% positivity with p504s in normal prostatic glands. These 4 cases had a granular intracytoplasmic expression by rare normal prostatic cells, and with a focal and weaker positivity than tumoral glands 9.

Furthermore the application of coexistent antibody against basal cells (p63) demonstrated that these positive p504s normal glands have fewer basal-stained than non-positive p504s normal glands and led us to believe that they could be interpreted as low grade prostatic intraepithelial neoplastic glands.

We thought that the increased AMACR expression by some normal prostatic glands may represent an earliest preneoplastic change and could reflect upregulation of metabolic pathways of prostate glands during the carcinogenic process 6.

Increased AMACR expression confirmed the reflects the upregulation of metabolic pathways of normal glands 38 and for this reason p504s could be considered as a useful marker of neoplastic transformation in the prostate gland 38.

In our experience, we confirm the absence of expression of p504s in cases of atrophy, postatrophic hyperplasia (or partial atrophy), transitional metaplasia and basal cell hyperplasia 33. These results are quite different from those of Herawi et al.

These authors found a AMACR staining in 79% of partial atrophy and in 67% of crowded glands, two mimickers of prostate cancer, while HMWCK could be negative in respectively 13% and 17% of their cases. These differences could be explained by the fact that the cases were stained in a variety of institutions with varying antibodies and techniques. Moreover in their studies they didn?t give the results of both HMWCK/p504s staining. The high expression observed in more than 70% of the prostate cells in atypical adenomatous hyperplasia confirms the results reported by Yang et al. 39.

From the diagnostic standpoint, it is important to recognise that an alone positive AMACR staining does not equate to malignancy. Focal p504s staining have been shown in benign prostatic lesions : partial atrophy, adenosis, crowded glands. Usually the staining is lower than in prostate carcinoma, and we think that only a phenotype with both a negative basal cell marker (CK903, CK5/6 or p63) and a positive p504s is in favour of malignancy. This association of two kinds of markers have been pointed out by several authors.

In our studies we used p63 rather than CK5/6 or CK903 as basal cell marker for two reasons : we proved that p63 was a more sensitive basal cell marker than CK903 and CK5/6 [Ref 36]; we think that p63 as nuclear immunostaining is quite more easy to identify with a single avidin-biotin complex staining procedure than CK903 or CK5/6 which have cytoplasmic staining.

Generally, authors that published on coexistence of CK903 orCK5/6 staining with p504s needs to use a double-colour reactions with both avidin-biotin and alkaline phosphatase substrates, that is not a routine procedure.

The association of p504s with p63 as a useful marker of neoplastic transformation in the prostate gland and could be proposed in adjunct to morphology as a potential marker of prostatic tumoral cells in a diagnostic context, with increased sensitivity of up to 97% and specificity of up to 100% 5, 31, 40. Our 89.4% rate of resolved ASAP in a final diagnosis of benign lesions in 25% of cases, minimal cancer focus in 47.11%, HG PIN in 14.4% and AAH in 2.8% of our cases is higher than the 45% resolute cases observed with p504s immunostaining alone 2.

Furthermore, automatic immunostaining processor enabled us to carry out a successful immunostaining procedure with two-step primary antibody incubation and a secondary mixed anti-rabbit and anti-mouse antibody, with the same avidin-biotin complex staining procedure in the same run. This considerably reduced the time of processing and enabled this detection in a routine procedure.

This technique of coupling the two antibodies at the same time decreased the number of slides needed, the technical time, and in case of ambiguity, it reduced the percentage of inconclusive interpretations and additional biopsies. As Hemmed et al. [Am J Surg pathol 2005 29 ; 579-87] we have demonstrated that the application of a commercialised cocktail staining is an available routinely procedure, that impact the ability to conclusively recognise prostate cancer.

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