P504S, a-methylacyl-CoA racemase, AMACR

504S is a 382 amino acid cytoplasmic protein which has been recently identified by microarray screening of prostatic carcinomas. It has been identified with a-methylacyl-CoA racemase (AMACR), an enzyme that catalyses the racemization of a-methylacyl branched carbolic coenzyme A thioesters17. It is located in mitochondria and peroxisomes. Within peroxisomes, it plays a role in the oxidation of branched-chain fatty acids. It is also responsible for the racemization of an intermediate in the synthesis of bile acids. AMACR deficiency in humans has been linked to adult-onset sensory motor neuropathy and to infantile-onset liver dysfunction. Intriguingly, in neoplasia AMACR is most often expressed in tumours associated with a high fat diet, such as colonic and prostatic carcinoma.

Immunohistochemical expression

A range of non-neoplastic tissues express AMACR14:

prostate: expression is only occasional and at a lower level than in malignant glands, with greater expression in young men14.
periurethral glands show a consistent moderate level of positivity14.
proximal renal tubules22
liver
salivary gland
renal tubular epithelium: there is granular cytoplasmic plus brush border staining restricted to the proximal convoluted tubule.
pancreas

There is over-expression in a range of carcinomas (studied using tissue micro-arrays7,20):

lung	4/2820
papillary carcinoma of lung	2/1322
colorectum	20/247,13/2920
breast	23/52(invasive ductal carcinoma)7, 3/9420
papillary carcinoma of breast	0/622
renal papillary carcinoma	41/4122
renal clear cell carcinoma	13/52(focal or weak positivity)22
renal oncocytoma	3/2022
renal chromophobe carcinoma	0/1822
renal sarcomatoid carcinoma	0/1522
renal collecting duct carcinoma	0/222
urothelial carcinoma	2/17(diffuse weak or strong)22
prostatic adenocarcinoma	5/67, 34/3820, 52/5721
ovary	0/2620
papillary carcinoma of ovary	0/622
endometrium	2/2720
papillary carcinoma of endometrium	0/622
cervical	0/6(squamous cell carcinoma)7
melanoma	2/2120
papillary carcinoma of thyroid	2/1422
papillary carcinoma of pancreas	0/622

By prostatic tissue. Since benign prostatic glands produce some AMACR, careful antibody titration is essential to maximize the diagnostic efficacy of AMACR18. There have been two major studies which used slightly different categories for grading the degree of staining.

Jiang et al1:

		strongly positive					weakly positive	negative
		>75% of cells	51-75% of cells	6-50% of cells	<5% of cells	total	weakly positive	negative
benign prostate (biopsy)						0/541	9/541	45/541
benign prostatic hyperplasia						0/131,0/206, 25/4421	2/131	11/131,20/206
benign, near carcinoma, biopsy						0/541	8/541	46/541
benign, near carcinoma, prostatectomy						0/731	4/731	69/731
benign, total						0/1941	23/1941	171/1941
high grade prostatic in situ neoplasia						45/5221
M a l i g n a n t	Gleason grade 2	10/106
	Gleason grade 3	10/106
	Gleason 5	2/21	0/21	0/21	0/21	2/21	0/21	0/21
	Gleason 6	77/801	2/801	1/801	0/801	80/801	0/801	0/801
	Gleason 7	28/321	1/321	1/321	2/321	32/321	0/321	0/321
	Gleason >=8	19/231	3231	0231	1231	23/231	0/231	0/231
	Total	126	6	2	3	137/1371	0/1371	0/1371

The small benign glands seen in atrophy, basal cell hyperplasia, inflamed glands, urothelial metaplasia and most adenoses were negative1.

Beach et al found less clear-cut results, with some benign glands showing staining and some carcinomas which failed to stain2:

		positive				negative
		>51% of cells	11-50% of cells	1-10% of cells	total	negative
benign	benign prostate, NOS	14/2702		64/270(non-circumferential luminal staining)2	78/2702	192/2702
	benign prostatic small gland proliferations				0/282	28/282
	benign prostate, atrophic			3/79(focal, faint and non-circumferential)2	3/792	76/792
	benign, total	142		672	812	2962
Atypical adenomatous hyperplasia					7/406	33/406
malignant	needle biopsies	60/1862	75/1862	18/1862	153/1862	33/1862
	treated carcinoma				12/132	1/132
	unusual morphological patterns in prostatectomies				14/162	2/162

The prostatic stroma shows weak diffuse staining, which does not impede the interpretation of the staining of the epithelium2. Invasive urothelial carcinoma is immunoreactive (6/62). Seminal vesicles are negative (0/102).

Both studies agree that the staining of prostatic carcinoma is irrespective of Gleason grade1,2.

The question of the utility of P504S in core biopsies with small samples of carcinoma has been specifically addressed.

	positive					negative
	>75%	51-75%	25-50%	<25%	total	negative
benign glands						142/1425
percentages of malignant glands immunoreactive	177/2093 , 63/735	1/2093, 3/735	4/2093, 3/735	2/2093	184/2093 (88%), 69/735	25/2093, 4/735

3Malignant glands were considered positive if the degree of staining was greater than that of the benign glands. Sensitivity in malignant glands varied from 80% to 100%, depending on the source institution for the biopsies. 3 of the 4 cases of Gleason grade 7 were negative for P504S, as were both of the prostatic carcinomas with ductal features. 31 of the 46 cases of high-grade PIN were positive. In 17 of the 209 cases, benign glands adjacent to the malignant glands showed focal apical border staining.

One study considered 793 prostatic needle cores that had been considered negative for carcinoma on initial H/E sections: 84 of these proved positive for P504S: nine of these cases were recognised as showing carcinoma, ten as showing atypia and fifty four as showing high-grade intra-epithelial neoplasia on review, while eleven cases were considered to be benign despite the positivity for P504S16.

Variants of prostatic carcinoma, including foamy gland cancer, pseudohyperplastic adenocarcinoma and atrophic adenocarcinoma show a lower sensitivity at 62-77%4,9.

The monoclonal antibody P504S has been compared with polyclonal anti-AMACR. P504S has a slightly lower sensitivity but higher specificity for carcinoma compared with AMACR:

			weakly positive	moderately or strongly positive
prostatic adenocarcinoma using tissue microarrays		P504S	21/6915	43/6915
		p-AMACR	7/7615	69/7615

needle biopsies	unequivocal minute carcinoma	P504S	5/2015	11/2015
	unequivocal minute carcinoma	p-AMACR	3/2015	13/2015
	"atypical" converted to carcinoma on basal marker	P504S	4/1715	8/1715
	"atypical" converted to carcinoma on basal marker	p-AMACR	2/1715	11/1715
	"atypical" converted to carcinoma on AMACR	P504S	1/515	4/515
	"atypical" converted to carcinoma on AMACR	p-AMACR	2/515	3/515
	"atypical" considered benign on the bases of positivity for basal markers	P504S	0/4
		p-AMACR	0/4
combined sensitivity for TMA and needle biopsies		P504S	87%
combined sensitivity for TMA and needle biopsies		p-AMACR	90%
			weakly positive	moderately or strongly positive
high-grade PIN using tissue microarrays		P504S	13/4315	20/4315
high-grade PIN using tissue microarrays		p-AMACR	12/5415	40/5415
benign prostatic glands		P504S	8/120	0/120
benign prostatic glands		p-AMACR	25/134	0/134

Nephrogenic adenoma may be positive for P504S, especially when the lesion occurs in the urethra.

P504S is commonly expressed in adenocarcinomas of the colorectum but not those of the small intestine:

	Small intestinal carcinoma		Large intestinal carcinoma
P504S	Ampullary	other	primary colorectal	secondary colorectal
	2/3413	1/2513	29/4213	12/2413

			mucinous	non-mucinous
			1/1213	40/5413

			well or moderately differentiated	poorly differentiated
			40/6013	1/613

			MSI	MSS
			5/1413	35/4913
CK7+/CK20+	17/3413	17/2513	4/6613
CK7+/CK20-	15/3413	8/2513	0/6613
CK7-/CK20+	2/3413	0/2513	58/6613
CK7-/CK20-	0/3413	0/2513	4/6613

Normal colorectal mucosa is negative for P504S13.

There is increasing expression of AMACR with increasing degrees of dysplasia in both Barrett's oesophagus and inflammatory bowel disease17:

Barrett's oesophagus	no dysplasia	0/36
	indefinite for dysplasia	3/14
	low-grade dysplasia	6/16
	high-grade dysplasia	26/32
	adenocarcinoma	26/36
Inflammatory bowel disease	no dysplasia	0/17
	indefinite for dysplasia	1/7
	low-grade dysplasia	25/26
	high-grade dysplasia	8/10
	adenocarcinoma	10/14

Diagnostic utility

Diagnosis of prostatic carcinoma, particularly on needle biopsy and the identification of minimal residual carcinoma, in conjunction with 34bE12, p63 and CK5/6, all of which stain basal cells in benign glands but not carcinoma. Positivity for AMACR appears to provide additional information to that of negativity for basal cell markers18. There appear to be significant variations in the sensitivity and specificity of P504S between institutions, depending on fixation and immunohistochemical methodology, in particular protease digestion, heat recovery and developer system8.

Positivity both for P504S and for either 34bE12 or p63 helps to identify high-grade prostatic intraepithelial neoplasia1 and atypical adenomatous hyperplasia6.

Since p63 is a nuclear stain and P504S is a cytoplasmic stain, they can be applied as a cocktail to the same section. The use of a cocktail is advantageous for minute lesions which may cut out8:

	p63	P504S
benign	+	-
atypical small acinar proliferation	-	-
high-grade prostatic intra-epithelial neoplasia	+	+
prostatic carcinoma	-	+

The sensitivity and specificity of both P504S and p63 have been shown to be as good in a cocktail as when used alone9:

positivity for P504S defined as granular cytoplasmic staining of the entire circumference of the gland.		P504S, when used alone	P504S, in cocktail with p63
High grade PIN	prostatectomy	25/27	24/27
High grade PIN	needle biopsy	45/47	45/47
Minimal prostatic carcinoma	prostatectomy	18/18	18/18
Minimal prostatic carcinoma	needle biopsy	23/25	23/25
Prostatic carcinoma	prostatectomy	29/30	28/30
Prostatic carcinoma	needle biopsy	107/116	107/116
The pattern of p63 staining was the same using the cocktail as using p63 in isolation.

The P504S/p63 cocktail proved useful in identifying and differentiating foci of minimal prostatic carcinoma (P504S+/p63-), HGPIN (P504S+/p63+), atrophic glands (a few cases showed focal P504S+ but were p63+) and basal cell hyperplasia (P504S-/p63+)9. In order to avoid spurious cytoplasmic staining by p63, it should be used at a sufficient dilution (1 in 500 for the human recombinant clone from Neomarkers)9.

Based on several papers:

	sensitivity for carcinoma	sensitivity for high grade PIN	specificity for carcinoma	specificity for high grade PIN
P504S+/p63- in combination	97.2%10, 97.6%8	86.2%10	99.7%10, 95.2%8	81.6%10
34bE12 negativity in isolation			67% to 94%11,12
p63 negativity in isolation			91% to 98%11,12
negativity for a 34bE12/p63 cocktail			98%12

If AMACR is used in a cocktail with 34bE12 (with or without p63), both being cytoplasmic antigens, two chromogens need to be used19.

Diagnosis of papillary renal cell carcinoma

As a member of a panel to differentiated primary ovarian endometrioid/mucinous carcinoma of the ovary from metastatic colorectal carcinoma.

Along with the cytokeratin profile (small intestine CK7+/CK20 variable, large intestine CK7-/CK20+), to differentiated between adenocarcinomas of the small intestine (negative for P504S) and the colorectum (positive for P504S).

The detection of dysplasia in Barrett's oesophagus and in inflammatory bowel disease.

References

1Jiang, Z., Woda, B. A., Rock, K. L. P504S: a new molecular marker for the detection of prostate carcinoma. Am J Surg Pathol 2001;25:1397-1404.

2Beach, R., Gown, A.M., De Peralta-Venturina, M.N., Folpe, A.L., Yaziji, H., Salles, P.G., Grignon, D.J., Fanger, G.R. and Amin, M.B. P504S immunohistochemical detection in 405 prostatic specimens including 376 18-gauge needle biopsies. Am J Surg Pathol 2002;26:1588-96.

3Magi-Galluzzi, C., J. Luo, et al. (2003). "Alpha-methylacyl-CoA racemase: a variably sensitive immunohistochemical marker for the diagnosis of small prostate cancer foci on needle biopsy." Am J Surg Pathol 27(8): 1128-33.

4Zhou, M., Z. Jiang, et al. (2003). "Expression and diagnostic utility of alpha-methylacyl-CoA-racemase (P504S) in foamy gland and pseudohyperplastic prostate cancer." Am J Surg Pathol 27(6): 772-8.

5Jiang, Z., C. L. Wu, et al. (2002). "P504S/alpha-methylacyl-CoA racemase: a useful marker for diagnosis of small foci of prostatic carcinoma on needle biopsy." Am J Surg Pathol 26(9): 1169-74.

6Yang, X. J., C. L. Wu, et al. (2002). "Expression of alpha-Methylacyl-CoA racemase (P504S) in atypical adenomatous hyperplasia of the prostate." Am J Surg Pathol 26(7): 921-5.

7Zhou, M., A. M. Chinnaiyan, et al. (2002). "Alpha-Methylacyl-CoA racemase: a novel tumor marker over-expressed in several human cancers and their precursor lesions." Am J Surg Pathol 26(7): 926-31.

8Molinie, V., G. Fromont, et al. (2004). "Diagnostic utility of a p63/alpha-methyl-CoA-racemase (p504s) cocktail in atypical foci in the prostate." Mod Pathol 17(10): 1180-90.

9 Hameed O, Sublett J,Humphrey PA Immunohistochemical stains for p63 and alpha-methylacyl-CoA racemase, versus a cocktail comprising both, in the diagnosis of prostatic carcinoma: a comparison of the immunohistochemical staining of 430 foci in radical prostatectomy and needle biopsy tissues. Am J Surg Pathol 2005; 29:579-87

10 Sanderson SO, Sebo TJ, Murphy LM, et al. An analysis of the p63/alpha-methylacyl coenzyme A racemase immunohistochemical cocktail stain in prostate needle biopsy specimens and tissue microarrays. Am J Clin Pathol 2004; 121:220-5