|Year : 2017 | Volume
| Issue : 1 | Page : 21-25
Evaluation of role of alpha-methyl acyl-coenzyme A racemase/P504S and high molecular weight cytokeratin in diagnosing prostatic lesions
Deepika Jain, Sumiti Gupta, Nisha Marwah, Rajnish Kalra, Veena Gupta, Meenu Gill, Nikita Jain, Shubha Lal, Rajeev Sen
Department of Pathology and Anesthesia, Pt. B D Sharma PGIMS, Rohtak, Haryana, India
|Date of Web Publication||16-May-2017|
Department of Pathology, Pt. B D Sharma PGIMS, Rohtak, Haryana
Source of Support: None, Conflict of Interest: None
Background: In recent years basal cell markers (high molecular weight cytokeratin [HMWCK]) and prostate biomarker alpha-methyl acyl-coenzyme A racemase (AMACR) have been used as adjuvant to morphology in diagnostically challenging cases with a very high sensitivity and specificity. This has increased the diagnostic accuracy of prostate cancer worldwide.
Materials and Methods: In this prospective study, total of 50 cases including 37 cases of malignant lesions and 13 cases of benign lesions of the prostate were taken. Tumor grade was determined according to Gleason's grading system. AMACR and HMWCK expressions were determined by immunohistochemical staining. The obtained results were analyzed and evaluated using Chi-square statistical test (SPSS version 20).
Results: AMACR was not expressed in any of the 13 cases of benign lesions of the prostate while in malignant lesions of prostate it was expressed in 33 of 37 (89.18%) cases. All 4 (100%) cases of well-differentiated carcinoma were positive for AMACR expression. 21 of 25 (84%) moderately differentiated and all 10 (100%) cases of poorly differentiated tumors were positive for AMACR. There was statistically significant difference in expression of AMACR between benign and malignant lesions of the prostate, indicated byP = 0.001. In benign lesions, HMWCK was expressed in all the 13 (100%) cases while in malignant lesions of prostate it was not expressed in any of the (0%) case. All 13 benign lesions were positive for HMWCK only. AMACR expression was not seen in any of the benign lesion. Out of 37 malignant cases, 4 cases were negative for both, 33 cases were positive only for AMACR, but no case was positive only for HMWCK.
Conclusions: As an adjunct to biopsy, AMACR and HMWCK have value for resolving diagnostically challenging cases.
Keywords: Alpha-methyl acyl-coenzyme A racemase, cytokeratin, immunohistochemistry, malignancy, prostate
|How to cite this article:|
Jain D, Gupta S, Marwah N, Kalra R, Gupta V, Gill M, Jain N, Lal S, Sen R. Evaluation of role of alpha-methyl acyl-coenzyme A racemase/P504S and high molecular weight cytokeratin in diagnosing prostatic lesions. J Can Res Ther 2017;13:21-5
|How to cite this URL:|
Jain D, Gupta S, Marwah N, Kalra R, Gupta V, Gill M, Jain N, Lal S, Sen R. Evaluation of role of alpha-methyl acyl-coenzyme A racemase/P504S and high molecular weight cytokeratin in diagnosing prostatic lesions. J Can Res Ther [serial online] 2017 [cited 2020 Feb 18];13:21-5. Available from: http://www.cancerjournal.net/text.asp?2017/13/1/21/206239
| > Introduction|| |
The Prostatic disease is responsible for significant morbidity and mortality in men, throughout the world. The diagnosis of prostate adenocarcinoma is often challenging. Accurate diagnosis of the prostatic disease frequently requires simultaneous data from clinical chemistry, imaging techniques, and surgical pathology laboratory.
Clinical presentation of benign prostatic hyperplasia (BPH) and carcinoma prostate are the retention of urine, dysuria, frequency, urgency, backache, hematuria, etc., Per rectal examination may detect some early prostatic carcinomas because of their posterior location, although the test suffers from both low sensitivity and low specificity. Ultrasonography has some characteristic findings of BPH and carcinoma prostate, but poor sensitivity and specificity limits their diagnostic utility. Prostate specific antigen (PSA) has been useful in the diagnosis and management of prostate cancer. PSA is organ specific but not cancer specific.
Even following detailed histopathological examination of biopsy tissues, taken from precisely defined microanatomic sites within the prostate, informed opinions as to the diagnostic and prognostic significance of particular morphologic features, frequently remain controversial, to the extent that distinction of benign from malignant disease may not be possible. Before making a diagnosis of carcinoma, it is necessary to consider the various benign pattern and processes that can simulate prostatic adenocarcinoma. Most common lesions giving rise to false positive carcinoma diagnosis are atrophy, postatrophic hyperplasia, atypical adenomatous hyperplasia (AAH), and seminal vesicle type tissue.
Although most prostate needle biopsy specimens are diagnosed as benign or malignant based solely on routine hematoxylin and eosin (H and E) stained sections, immunohistochemical (IHC) analysis is not infrequently used as an adjunct in the interpretation of these specimens, especially to confirm the diagnosis of minimal (limited; <1 mm), and unusual variants of prostate carcinoma and to exclude benign mimickers of carcinoma. While a wide variety of IHC markers have been proposed for this purpose, antibodies against two classes of prostatic biomarkers are most commonly used; firstly, basal epithelial cell-specific markers and secondly, prostate carcinoma-specific markers. Lack of staining with basal cell markers (high molecular weight cytokeratin [HMWCK]) in the appropriate histologic context supports a diagnosis of carcinoma. In addition, positive staining for alpha-methyl acyl-coenzyme-A racemase/P504S (AMACR/P504S) seems to be of diagnostic value beyond that of a negative basal cell immunostain by adding further support to the diagnosis of malignancy.
| > Materials and Methods|| |
This study was conducted in the Department of Pathology during the period from 2012 to 2014. In this prospective study, total of 50 cases including 37 malignant lesions and 13 benign lesions of the prostate were taken. The tissue samples obtained during transurethral electro resection, enucleation or needle biopsies were considered. Inadequate biopsies and cases with marked inflammation were excluded. Brief clinical data were noted from case records, which included age, presenting symptoms, per rectal and ultrasound findings, serum PSA levels, and clinical diagnosis.
As researchers, we strive to ensure that our work should make a positive contribution to the welfare of those affected by it.
Following inclusion and exclusion criteria were adopted in this study:
- Inclusion criteria:
All types of prostatic specimens including transurethral resection of the prostate, needle biopsy, and prostatectomy were considered in this study.
- Exclusion criteria:
Inadequate biopsies and poorly preserved prostatic specimens and cases with marked inflammation were excluded.
Prostate fragments were fixed in 10% formalin, paraffin-embedded, sectioned and standard H and E stained sections were studied under the light microscope and classified into benign and malignant lesions. Carcinoma cases were histologically graded according to Gleason's grading system, and Gleason's score was noted (well differentiated 2–4, moderately differentiated 5–7, poorly differentiated 8–10). Associated prostatic tissue changes such as tumor invasion, prostatic intraepithelial neoplasia (PIN), prostatitis, and others if any were also analyzed. Special stains like Van Gieson, periodic acid-Schiff, Masson's trichrome, and reticulin were employed whenever required for histopathological diagnosis.
IHC profile of the tumor was assessed by subjecting one section each from a representative block to AMACR/P504S and HMWCK immunostain. Immunohistochemistry was performed on 4 μm thick sections from 10% formalin-fixed paraffin–embedded specimens, according to the streptavidin-biotin immunoperoxidase technique (Dako-cytomation). Positive and negative controls were run simultaneously.
Positive staining for AMACR pertained to dark diffuse or granular, cytoplasmic or luminal, but circumferential. The percentage positivity was graded from 0+ to 3+ as follows: 0% cells (0+, negative), 1–10% cells (1+, mild), 11–50% cells (2+, moderate), >51% cells (3+, strong). The adjacent benign glands should not show more than weak, partial (noncircumferential) staining if any. Negative staining pertained to no staining or focal, weak noncircumferential fine granular staining. Whereas in case of HMWCK: Cytoplasmic positivity, continuous/discontinuous.
The results thus obtained were tabulated and analyzed using statistical methods. All statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 20 for windows. Data were expressed as a mean ± standard deviation for quantitative variables, numbers, and percentage. Comparisons between multiple groups were made using Chi-square test and Fisher exact test whichever was appropriate. The correlation between Gleason's grade and IHC expression was analyzed using the Chi-square test with an accompanying P value. A value of P< 0.05 was taken as significant, and P< 0.01 was taken as highly significant, whereas P value of more than 0.05 was considered nonsignificant.
| > Results|| |
Total cases with age group
A total of 50 cases were taken including 37 malignant and 13 benign lesions of the prostate. Patients with benign lesions of the prostate were in the age range of 41–80 years with a mean age of 68.06 ± 10.49 years. Patients with malignant lesions of the prostate were in the age range of 41–85 years with a mean age of 68.16 ± 10.58 years.
In malignant lesions of the prostate, cases were further graded according to Gleason's grading system. Gleason score of 6 was seen in the majority of cases (32.43%), followed by Gleason score 7 (27% cases) and 8 (18% cases). Out of 37 cases, 25 (67.56%) were moderately differentiated [Figure 1]a (Gleason score 5–7), 10 (27%) were poorly differentiated (Gleason score 8–10), and 2 (5.4%) were well differentiated (Gleason score 2–4).
|Figure 1: Carcinoma prostate - Moderately differentiated with cribriform pattern (a) (H and E, ×200); (b) alpha-methyl acyl-coenzyme A racemase: 3+ positivity (IHC, ×100); (c) high molecular weight cytokeratin - Negative (IHC, ×100)|
Click here to view
Alpha-methyl acyl-coenzyme A racemase immunoreactivity
AMACR was not expressed in any of the 13 cases of benign lesions of the prostate while in malignant lesions of prostate it was expressed in 33 of 37 (89.18%) cases. AMACR expression was significantly up-regulated in malignant lesions of the prostate (P < 0.001) as compared to benign prostatic lesions. Out of 2 cases of well-differentiated tumors, one case (50%) showed 1+ positivity while other (50%) case showed 2+ positivity. Out of 25 cases of moderately differentiated tumor, 10 (40%) cases revealed 3+ positivity [Figure 1]b while 8 cases (32%) revealed 2+ positivity followed by 4 (16%) cases with negative staining, and 3 (12%) cases with 1+ positivity. Out of 10 cases of poorly differentiated tumors, 7 cases (70%) revealed 3+ positivity followed by 2 (20%) cases with 2+ positivity, and 1 (10%) case with 1+ positivity. No case among poorly differentiated tumors showed the absence of positivity. A statistically significant correlation was not observed between AMACR expression and Gleason's grade of malignant lesions of the prostate (P = 0.75) [Table 1].
|Table 1: Frequency of the amacr expression in relation to tumor differentiation and gleason's grade (n=37)|
Click here to view
High molecular weight cytokeratin immunoreactivity
In benign lesions, HMWCK was expressed in all the 13 (100%) cases while in malignant lesions of prostate it was not expressed in any of the (0%) case [Figure 1]c. There was statistically significant difference in staining of HMWCK between cases of benign and malignant lesions prostate, indicated by P = 0.001. Of 13 benign cases, BPH revealed continuous staining pattern in all cases except one. Basal cell hyperplasia (BCH) also showed continuous staining pattern in both cases. Other benign lesions such as AAH and atrophy showed discontinuous staining pattern in each individual case.
Prostatic intraepithelial neoplasia staining
In addition, low grade PIN (LPIN) associated with one case of BPH showed grade 2+ positivity with AMACR and revealed discontinuous staining pattern with HMWCK. Four cases of HPIN associated with malignant lesions of prostate revealed 1+ positivity, 2+ positivity and 3+ positivity in 1, 2, and 1 case, respectively, and discontinuous staining pattern with HMWCK. All the cases of high grade PIN (HGPIN) were associated with poorly differentiated carcinoma.
| > Discussion|| |
Prostate needle biopsy is the preferred method for diagnosing of early prostate cancer. It has low morbidity and provides specific information on the grade and extent of the tumor. However, histologic confirmation of prostate carcinoma sometimes remains challenging in biopsy samples. The difficulty with needle biopsy stems not only from the small amount of tissue available for histological examination, but also from the fact that biopsies often identify only a few malignant glands (small focus carcinomas) or several histological benign mimics of cancer. Diagnostic difficulty in indeterminate cases concerns 1.5–9% of prostate biopsy. AMACR/P504S is used as a confirmatory stain for prostate cancer in conjunction with morphology and a basal cell–specific marker. Using AMACR as a positive marker alone might be misleading because the weak expression of AMACR might be seen in benign glands and expression of AMACR is also seen in HGPIN and AAH. Therefore, using AMACR as a positive marker along with a basal cell-specific negative marker, HMWCK (HMWCK/34βE12) will enhance the diagnostic accuracy in prostate cancer and reduce the chance of misdiagnosis.
The results of this study of age distribution of benign and malignant lesions of prostate are almost similar to the results of previous studies, which reported that cases of malignant lesions of prostate progressively rise after the age of 50 years with a peak incidence at and above 70 years. Patients with malignant lesions were in the higher age group as compared to patients with benign lesions.
Results were similar to a study by Djavan et al. which states Gleason score 6 as the commonest pattern. In our study, maximum number of cases that is, 67.56% were moderately differentiated (Gleason score 5–7), followed by poorly differentiated that is, 27% (Gleason score 8–10), and well differentiated that is, 5.4% (Gleason score 2–4). The results were in the similar line with the observations of Gleason who noted that majority of cases of malignant lesions of the prostate were of intermediate grade.
AMACR expression was significantly up-regulated in prostate cancer (P = 0.001) as compared with benign prostatic lesions. This finding was in agreement with Rubin et al. and Jiang et al. who also found that this marker was highly expressed in prostate cancer as compared with benign lesions of prostate, (P < 0.001)., Jiang et al., Rashed et al. also observed statistical significant (P < 0.001) difference in AMACR index between benign and malignant prostatic lesions.,
Jiang et al. also reported strongly positive staining in HGPIN, and noted that when HGPIN partially involved a gland, staining was confined to the HGPIN and did not extend into the normal epithelial cells within the same gland. With respect to staining intensities, Luo et al. reported that both invasive carcinoma and HGPIN had higher IHC staining scores than normal prostate epithelium; however, the score for carcinoma was significantly higher than that for HGPIN. Similar results were also reported by Rubin et al.
So, this study revealed no statistically significant correlation between AMACR positivity and Gleason's grade (P = 0.75) and serum PSA levels (P = 0.856) which was in concordance with studies by Rubin et al. (P < 0.001) and Ozgur et al. (P > 0.05)., Molinié et al. concluded AMACR reacted with only 2% of normal glands (4/260) with a focal weak staining, and 97% of prostatic cancer showed AMACR overexpression with a heterogeneous staining pattern from weak (30%) or moderate (31%) to strong (36%) intensity, independently of the Gleason score (P = 0.29), and the fixative technique (P = 0.27). Luo et al. distributed AMACR mean IHC scores for stratified by Gleason score, and pathological stage and noted no relation between AMACR IHC score and Gleason grade, pathological stage, patient age, or preoperative serum PSA (Kruskal–Wall, all P = 0.05). Beach et al. also found 82% AMACR/P504S expression in prostate cancer whatever the morphological aspect that is, classical, intraductal, mucinous, pseudohyperplastic or atrophic, degree or differentiation, Gleason score or additional treatment such as hormone therapy or radiotherapy.
The most commonly used basal cell–specific markers in prostate cancer are HMWCK and clone 34βE12. HMWCK shows continuous, intact, circumferential staining of basal cells in benign and premalignant lesions but discontinuous staining in malignant lesions. Negative staining must be interpreted with caution because cytokeratin is formalin sensitive, and a progressive loss of immunoreactivity can be seen in prolonged formalin fixation. In our study of benign lesions, HMWCK was expressed in all the 13 (100%) cases while in malignant lesions of prostate it was not expressed in any of the (0%) case. There was statistically significant difference in expression of HMWCK between cases of benign and malignant lesions prostate, indicated by P = 0.001. Similar findings were seen in the study by Shah et al., Lakhtakia et al. and Kotakidou et al.,,
BPH revealed continuous staining pattern in all cases except one. BCH also showed continuous staining pattern in both cases. Other benign lesions including AAH and atrophy showed discontinuous staining pattern in each individual case. In a study by Manna et al., HMWCK showed continuous staining of basal cell in benign and premalignant lesions, whereas discontinuous staining in malignant prostatic lesions. Two cases of BPH showed discontinuous staining of basal cells. Similarly, two cases of AAH and one case of PIN showed discontinuous staining of basal cells. Our study on HMWCK also showed similar results with studies done by Zhou et al., Boran et al., In a study by Garg et al., HMWCK was positive in all cases of BPH (100%), 1 case of BPH with suspicious focus, 11 cases of BCH, 4 cases of AAH, and 2 cases of atrophy and completely negative in cases of prostate cancer. One case of LPIN associated with BPH and four cases of HPIN associated with malignant lesions of prostate revealed discontinuous staining pattern in all cases.
In our study, all 13 benign lesions were positive for only HMWCK. No benign lesion was positive for only AMACR or both. Out of 37 malignant cases, 4 cases were negative for both, 33 cases were positive only for AMACR, but no case was positive only for HMWCK [Table 2].
These data suggest that positive staining for AMACR can increase the level of confidence in establishing a definitive malignant diagnosis in atypical cases with negative basal cell staining. The use of AMACR and HMWCK in combination might not only make uncertain diagnoses less frequent but also obviate the need for a number of repeated biopsies.
| > Conclusion|| |
We conclude that proliferative activity and invasiveness increases from benign to the malignant end in the spectrum of prostatic lesions. IHC plays an important role in the diagnosis of prostatic lesions and helps to differentiate malignant from benign lesions, especially for gray zone lesions which are difficult to be diagnosed on the routine histopathological study. Using AMACR as a positive marker alone might be misleading because the expression of AMACR is also seen in HGPIN. Therefore, using AMACR/P504S as a positive marker along with basal cell–specific negative markers (HMWCK) will enhance the diagnostic accuracy in prostate cancer and reduce the chance of misdiagnosis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
”The Term ''Prostates'', Liddell and Scott, “A Greek-English Lexicon”, at Perseus”. Available from: http://www. Perseus.tufts.edu
. [Last retrieved on 2011 Jan 24].
Epstein JI. The lower urinary tract and male genital system. In: Kumar V, Fausto N, Abbas A, editors. Robbins and Cotran Pathologic Basis of Disease. 8th
ed. Philadelphia: Saunders; 2010. p. 993-1002.
Srigley JR. Benign mimickers of prostatic adenocarcinoma. Mod Pathol 2004;17:328-48.
Epstein JI. Diagnostic criteria of limited adenocarcinoma of the prostate on needle biopsy. Hum Pathol 1995;26:223-9.
Dabir PD, Ottosen P, Høyer S, Hamilton-Dutoit S. Comparative analysis of three- and two-antibody cocktails to AMACR and basal cell markers for the immunohistochemical diagnosis of prostate carcinoma. Diagn Pathol 2012;7:81.
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.
Kumaresan K, Kakkar N, Verma A, Mandal AK, Singh SK, Joshi K. Diagnostic utility of α-methylacyl CoA racemase (P504S) and HMWCK in morphologically difficult prostate cancer. Diagn Pathol 2010;5:83.
Rashed HE, Kateb MI, Ragab AA, Shaker SS. Evaluation of minimal prostate cancer in needle biopsy specimens using amacr (p504s), p63 and ki67. Life Sci J 2012;9:12-21.
Djavan B, Ravery V, Zlotta A, Dobronski P, Dobrovits M, Fakhari M, et al.
Prospective evaluation of prostate cancer detected on biopsies 1, 2, 3 and 4: When should we stop? J Urol 2001;166:1679-83.
Gleason DF. The veterans administration cooperative urologic research group histologic grading system. Histologic grading system of prostate cancer. Hum Pathol 1992;23:273-9.
Rubin MA, Zhou M, Dhanasekaran SM, Varambally S, Barrette TR, Sanda MG, et al.
alpha-Methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer. JAMA 2002;287:1662-70.
Jiang Z, Woda BA, Wu CL, Yang XJ. Discovery and clinical application of a novel prostate cancer marker: Alpha-methylacyl CoA racemase (P504S). Am J Clin Pathol 2004;122:275-89.
Jiang Z, Iczkowski KA, Woda BA, Tretiakova M, Yang XJ. P504S immunostaining boosts diagnostic resolution of “suspicious” foci in prostatic needle biopsy specimens. Am J Clin Pathol 2004;121:99-107.
Luo J, Zha S, Gage WR, Dunn TA, Hicks JL, Bennett CJ, et al.
Alpha-methylacyl-CoA racemase: A new molecular marker for prostate cancer. Cancer Res 2002;62:2220-6.
Ozgur T, Atik E, Hakverdi S, Yaldiz M. The expressions of AMACR and iNOS in prostate adenocarcinomas. Pak J Med Sci 2013;29:610-3.
Molinié V, Fromont G, Sibony M, Vieillefond A, Vassiliu V, Cochand-Priollet B, et al.
Diagnostic utility of a p63/alpha-methyl-CoA-racemase (p504s) cocktail in atypical foci in the prostate. Mod Pathol 2004;17:1180-90.
Beach R, Gown AM, De Peralta-Venturina MN, Folpe AL, Yaziji H, Salles PG, et al.
P504S immunohistochemical detection in 405 prostatic specimens including 376 18-gauge needle biopsies. Am J Surg Pathol 2002;26:1588-96.
Garg M, Kaur G, Malhotra V, Garg R. Histopathological spectrum of 364 prostatic specimens including immunohistochemistry with special reference to grey zone lesions. Prostate Int 2013;1:146-51.
Shah RB, Zhou M, LeBlanc M, Snyder M, Rubin MA. Comparison of the basal cell-specific markers, 34betaE12 and p63, in the diagnosis of prostate cancer. Am J Surg Pathol 2002;26:1161-8.
Lakhtakia R, Bharadwaj R, Kumar VK, Mandal P, Nema SK. Immunophenotypic characterization of benign and malignant prostatic lesions. Med J Armed Force India 2007;63:243-8.
Kotakidou R, Lakis S, Papamitsou T, Mavridou M, Varvaresou D, Koutsou A, et al
. Diagnostic utility of immunohistochemical marker 34βe12 (keratin 903) in prostate pathology. Aristotle Univ Med J 2007;34:31-6.
Manna AK, Pathak S, Gayen P, Sarkar DK, Kundu AK. Study of immunohistochemistry in prostatic lesions with special reference to proliferation and invasiveness. Indian J Surg 2011;73:101-6.
Zhou M, Aydin H, Kanane H, Epstein JI. How often does alpha-methylacyl-CoA-racemase contribute to resolving an atypical diagnosis on prostate needle biopsy beyond that provided by basal cell markers? Am J Surg Pathol 2004;28:239-43.
Boran C, Kandirali E, Yilmaz F, Serin E, Akyol M. Reliability of the 34ßE12, keratin 5/6, p63, bcl-2, and AMACR in the diagnosis of prostate carcinoma. Urol Oncol 2011;29:614-23.
[Table 1], [Table 2]