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IMMUNOLOCALIZATION OF APOLIPOPROTEIN D, ANDROGEN RECEPTOR AND PROSTATE SPECIFIC ANTIGEN IN EARLY STAGE PROSTATE CANCERS
Vol. 159,548664, February 1998 Printed in USA.
IMMUNOLOCALIZATION OF APOLIPOPROTEIN D, ANDROGEN RECEPTOR AND PROSTATE SPECIFIC ANTIGEN IN EARLY STAGE PROSTATE CANCERS STEVEN X.D. ZHANG, JACQUELINE M. BENTEL, CARMELA RICCIARDELLI, DAVID J. HORSFALL, R. MARSHALL AND WAYNE D. TILLEY* DARROW E. HAAGENSEN, -IS From the Flinders Cancer Centre, Department of Surgery, Flinders University School of Medicine, Eedford Park,Australia, and the Department of Surgery, Methodist Hospital, Sacmmento, California
ABSTRACT
Purpose: To determine the cellular distribution and levels of immunohistochemicalstaining for apolipoprotein D (Apo-D), prostate specific antigen (PSA) and androgen receptor (AR)in early stage prostate cancers. Materials and Methods: Cellular distribution of Apo-D, PSA and AR in 30 stage A/B prostate cancers and in non-malignant glandular tissue contained in the same sections was detected immunohiatochemically, and staining was evaluated by computerized video image analysis. Results: Staining for Apo-D (percentage positive cellular area) was significantly increased in tumor cells of early stage prostate cancers compared with non-malignant glandular tissue. PSA and AR were present at high levels in both early stage prostate tumors and non-malignant prostate. Conclusions: Malignant transformation in the prostate is associated with increased cellular levels of Apo-D. Kcr WO-: prostatic neoplasms, immunohistochemistry, apolipoproteins, androgen receptor, prostate specific antigen
Hormonal therapies for the management of advanced pros- van& prostate cancers and outcome of subsequent In contrast, AR expression in early tate cancer were developed after Huggins and Hodges dem- hormonal onstrated prostatic tumor regression subsequent to removal stage prostate cancers has not been well characterized and of testicular andro ns by castration or administration of the relationship between AR levels and outcome of treatestrogens in 1941. While androgen ablation remains the ments of early stage disease is not known. The identity and function of androgen responsive proteins treatment of choice for metastatic (stage D,) disease, more recent clinical trials have evaluated hormonal treatments in in the normal prostate and in prostate cancer cells are poorly early stage tumors. These protocols have included androgen defined. Although expression of prostate specific antigen ablation as neoadjuvant hormonal therapy in combination (PSA) in glandular epithelial cells of the prostate appears to with radical prostatectomy for localized prostate tumor^^.^ be up-regulated by the availability of testosterone at puberand as chemopreventativeagents for development of prostate ty,14.15 prostate cancer cell expression and/or secretion of cancer in men with premalignant disease, such as low or high PSA may be regulated by factors other than testicular andrograde prostatic intraepithelial neoplasia (PIN).4To compre- gens. This is supported by studies demonstrating tumor cell hensively evaluate the outcomes of these protocols and to immunoreactivity for PSA and increasing serum PSA levels develop markers that will identify patients with localized in patients undergoing treatment with androgen ablation prostate cancers who w i l l respond most favorably to hor- therapies who have hormone refractory (stage D,) prostate monal manipulation, it is necessary to define the cellular cancers." Previous studies have reported that while the expression of the androgen receptor (AR)and other androgen majority of early, advanced and hormone refractory prostatic responsive proteins in early stage prostate tumors. tumors remain immunohistochemicallypositive for cellular The AR, a nuclear transcription fador that mediates an- PSA, staining is generally observed to be less than that seen drogen action in the prostate and in other androgen target in adjacent normal or benign hyperplastic gland^.'^.^^ tissues?' has been shown to be expressed in both glandular Apolipoprotein D (Apo-D), another androgen-regulated epithelial cells and in a proportion of interglandular fibro- protein, is a constituent of high density lipoprotein in plasma muscular stromal cells of the normal human pro~tate.6.~and is the major component of human breast cyst fluid." Several studies of advanced prostate cancers have demon- Apo-D is a member of the lipocalin superfamily of hydrophostrated that AR is expressed in tumor cells both prior to and bic protein transporters and is expressed in a variety of following hormonal therapies: with AR immunoreactivity steroid-responsive normal tissues including adrenal cortex homogeneously positive in a small number of tumors' and and corpus luteum" and in a proportion of steroid responsive hetero eneous in the majority of advanced prostate can- tumors (breast, prostate, ovarian, endometrial).". '' AlVideo image analysis studies have also demon- though its precise function in these tissues is unknown, cers?strated a relationship between AR immunostaining of ad- Apo-D specifically binds progesterone and pregnenolone and may be involved in steroid metabolism and/or processing in F p t e d for publication September 16,1997. Ftequ&a for r e p M h De ent of Surgery, Flindera Medical these tissues.20Androgen regulation of Apo-D has been demcentre.w d Park SA. 2. A ~ p t d i a . onstrated in prostate and breast cancer cell lines in in vitro Supportedby ants from the Antdancer Foundation of the Unihowever A D expression may also be reguveraihea of hug Australia, the National Health and Medical Re retinoic acidz4 and search Council of Australia and the Clive and Vera Ftamaciotti Foun- lated by glueoeorti~oids,~~strogen,22.~~ datiOIl8. interleukin-la." In light of these studies, the factors regu-
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548
549
IMMUNOHISTOCHEMISTRY OF PROSTATIC NEOPLASMS
lating Apo-D expression in specific tissues in vivo are unknown. Previous studies of Apo-D in non-malignant prostate and in advanced prostate cancers in our laboratory demonstrated cellular immunoreactivity for Apo-D in only occasional glandular epithelial cells and in cells shed into the glandular lumen of non-malignant prostatic tissues.15 In contrast, significantly increased Apo-D staining was evident in stage D, primary prostate tumors. To examine cellular levels of AR and the androgen-responsive proteins, Apo-D and PSA in early stage prostate tumors, and to determine whether cellular levels of Apo-D were increased early in tumor formation or whether this was a late event occurring during cancer progression, AR,Apo-D and PSA immunostaining were compared using computer assisted video image analysis in early stage primary prostate cancers and in non-malignant glandular tissue contained in the same slide. MATERIALS AND METHODS
Prostate tissues. Prostate cancer specimens were obtained from 30 patients referred to the urology units at Flinders Medical Center (Bedford Park, South Australia) and the Repatriation General Hospital (Daw Park, South Australia). The patients included in this study were clinically staged as A (10) or B (20) prostate carcinoma according to the modified Whitmore Jewett system.26All specimens used for immunohistochemical analysis were obtained at transurethral resection of the prostate (24) or radical prostatectomy (6)and had not been treated previously for prostate cancer. Following surgical resection, prostate specimens were immediately 6xed in formalin and then par& embedded within 48 hours. Immunohistochemistry. Five jun. sections were cut from paraffin-embedded tissues, mounted on poly-L-lysine coated glass microscope slides and baked at 60C for 1hour. Sections were deparaffinized in xylene and sequentially rehydrated in graded ethanol (loo%, 95%, 75% and 50%) for 2 minutes each. Endogenous peroxidase activity was blocked by incubation in phosphate buffered saline (PBS) containing 0.3% water for 5 minutes. Immunohistochemical staining for Apo-D, PSA and AR was carried out essentially as described previ~usly.'~*'~ Primary antibody for Apo-D was rabbit antiGCDFP-24,27and for AR was U402 (affinity purified rabbit antisera generated against amino acids 1to 21 of the human AR7)obtained from Drs. M. J. McPhaul, C. M. Wilson and J. D. Wilson, Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, TX. Apolipoprotein D. Slides were sequentially incubated at room temperature in blocking solution (PBS containing 10% normal goat serum (Vector Laboratories, Burlingame, CAI) for 15 minutes, primary antibody (1:1500 rabbit antiGCDFP-24 in blocking solution) for 30 minutes, secondary antibody (1:400 biotinylated goat anti-rabbit (Vector Laboratories) in blocking solution) for 30 minutes followed by avidin-biotin-peroxidase complex (ABC reagent) (1:400 in PBS, Vectastain ABC kit, Vector Laboratories) for 30 minutes. Slides were rinsed in PBS (2 X 5 min.) between incubations. Positive immunostaining was visualized with the chromogen 3'3'-diaminobenzidine tetrahydrochloride (DAB (Sigma Laboratories, St. Louis, MO) 0.05% DAB and 0.06% H,O, in 38 mM. Tris.HC1, pH 7.4) for 6 minutes. Slides were counterstained with weak Lillie Mayer's hematoxyh and mounted with Pix mounting medium. Specimens previously known to be reactive for the primary antibody were used as positive controls in each staining run.Primary antibody was replaced with non-immune sera at the same protein concentration for negative controls. Prostate specific antigen. Sections were incubated in block ing solution (10%normal goat serum in PBS) for 15 minutes primary antibody (1:1200 rabbit anti-human PSA (D&o Glostrup, Denmark) in blocking solution) for 30 minutes
iecondary antibody (1:400 biotinylated goat anti-rabbit Weeor Laboratories) in blocking solution) for 30 minutes and IBC reagent (1:400 in PBS) for 30 minutes. Slides were insed in PBS (2 x 5 min.) between incubations. The resultmt complexes were visualized as described above. Androgen receptor. Sections were immunohistochemically stained for AR following antigen unmasking in 10 mM. ci;rate buffer (pH 6.5). Slides were heated to boiling in a microwave, simmered for 8 minutes, then allowed to cool in che citrate buffer for 20 minutes. Sections were incubated in blocking solution (3% normal goat serum and 1% normal human serum in PBS) for 15 minutes, primary antibody :1:250 U402, in blocking solution) at 4C overnight, secondary antibody (1:400 biotinylated goat anti-rabbit in blocking solution) for 30 minutes, followed by ABC reagent (1:400 in PBS) for 30 minutes. Slides were rinsed in PBS (2 X 5 min.) between incubations. Positive immunostaining was visualized as described above. Video image analysis. The immunostained sections were magniexamined using an Olympus BH-2 microscope (~200 fication) coupled to a computer-assisted color image system (Video Pro 3 9 Leading Edge Pty. Ltd.,Adelaide, South Australia) based on methods described Color video image analysis measurements were generated independently for malignant foci and non-malignant epithelial components within the same section for each prostate specimen. Discrimination between malignant and benign tissue areas was based on standard cytological and pathological features. The majority of non-malignant glands analyzed exhibited histological features consistent with benign prostatic hyperplasia. As our previous studies demonstrated the absence of Apo-D immunoreactivity in the prostatic strorna,l6 this component was not included in the current analysis. In contrast to our previous study," epithelial cells sloughed into the glandular lumen also were not included in the analysis of Apo-D immunostaining. Video image measurements were made of the total area (pixels) of tissue analyzed (that is, positively and negatively stained cells) and the integrated optical density (IOD; arbitrary density units) of positively stained areas, for at least 20 fields per section. The following parameters of staining were derived for each sample: 1)the percentage area of positively stained cells (that is, positively stained aredtotal area XlOO%) and 2) the mean optical density (MOD; density unitdpixel) of positive staining, which is equal to the IOD/area of positively stained cells (that is, the density of staining in the positive immunoreactive area, which is stoichiometrically related to the cellular concentration of antigen). Statistical analysis. Data were presented as the mean and standard error of the mean (s.e.m.). Staining parameters of malignant and non-malignant tissue areas were analyzed using a student's t test. Statistical significance was established at the p <0.05 level. RESULTS
Immunohistochemical localization of Apo-D, PSA and AR. Apo-D was detected in all prostate cancer specimens as cytoplasmic immunoreactivity in tumor cells and in occasional non-malignant glandular epithelial cells. (fig. 1)No staining of stromal, endothelial or blood cells was observed. As reported previo~sly,'~ Apo-D staining was evident in only a few glandular epithelial cells and in a small number of cells shed into the glandular lumen in normal and benign hyperplastic glands. In contrast to nonmalignant glandular epithelial cells, prostate cancer cells in the same specimens exhibited extensive immunoreactivity for Apo-D. Apolipoprotein D staining of individual tumors ranged from weakly to strongly positive (fig. 1, A, B ) , with considerable heterogeneity of staining evident within tumor foci. Strongly positive A p D immunoreactivity was also detected in the majority
550
IMMUNOHISTOCHEMISTRYOF PROSTATIC NEOPLASMS
FIG.1. Immunohistochemicalstaining for Apo-D, PSA and AR in early stage prostate cancer specimens.A, cytoplasmic immunoreactivity for Apo-D in stage A tumor exhibiting strongly positive staining. Cells of Lon-malignant prostatic ducts localized among tumor foci were immunohistologicallynegative for Apo-D. B, re resentative immunostainingfor Apo-D in stage A tumor. Staining is heterogeneous with both positively and negatively immunoreactive ce&. C, positive Apo-D immunostaining in nerve fiber located in prostatic capsule of radical prostatectomy specimen. Inset negative control immunostaining of nerves in adjacent section of same tumor. D, strongly positive PSA immunostainingin tumor cells of stage B rostate tumor. Stromal cells within tumor are negative for PSA. E, immunostainingfor AR in same immunoreactivity is evident, with strongly and weakly staining cells present in tumor focus. tumor specimen as D. Heterogeneous Stromal cells within this area of the tumor are negative for AR,however, foci of positively staining stromal cells were evident in all tumors and in non-malignant are= of specimens. Magnification X200.
&
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IMMUNOHISTOCHEMISTRY OF PROSTATIC NEOPLASMS
(94.8+3.7%;mean+S.E.M) of the 155 nerve fibers identified in the prostatic capsules of the 6 radical prostatectomy specimens examined in this study (fig. 1,C ) . Consistent with previous s t u d i e ~ , ' ~cytoplasmic .~~ PSA staining was evident in glandular epithelial cells and in prostate tumor cells of all specimens (fig. 1, D ) . Nonepithelial cell types were not positively stained for PSA. Staining of tumor foci for PSA ranged from heterogeneous immunoreactivity with strongly and weakly stained cells, to uniform strongly positive PSA staining in a proportion of specimens (fig. 1,D). Nuclear AR immunoreactivity was observed in prostate cancer cells, non-malignant glandular epithelial cells and in a proportion of peritumoral and interglandular stromal cells (fig. 1,E ) . AR staining in tumor cells and benign glandular epithelial cells within the same section appeared similar, with heterogeneous staining composed of weakly to strongly positive nuclear immunoreactivity in the majority of cells. In contrast, stromal cells displayed focal immunoreactivity with cords of AR immunopositive stromal cells interspersed with regions of negatively staining stromal cells. Video image analysis. The immunostaining of prostate specimens for Apo-D, PSA and AR was quantitated using video image analysis. The mean percentage area of tumor cells positively stained for Apo-D was 28.3 2 4.3%, which is a significant increase compared with the almost negligible percentage area of glandular epithelial cells positively stained in non-malignant areas of the specimens 0.51 ? 0.19% (table; fig. 2, A). Six of the 30 tumor specimens analyzed exhibited less than 5% positive staining for Apo-D. The mean percentage area of PSA staining in tumor foci was statistically decreased compared with non-malignant glands (table); however, there was considerable overlap in the staining of individual tissues within the two groups (fig. 2, C).The percentage area positively stained for AR was not significantly different between tumor foci and adjacent nonmalignant glands in the specimens (p >0.05;table; fig. 2, E). Stromal AR staining was not quantitated by video image analysis. The cellular concentration of Apo-D staining in positively stained cells (MOD) was significantly decreased in tumor cells compared with non-malignant glandular epithelial cells; however, these values were based on very few positively stained cells in non-malignant specimens (table; fig. 2, B). In addition, Apo-D positive glandular epithelial cells may have been terminally differentiated or dying prior to sloughing into the glandular lumen at the time of fixation and this may have resulted in the apparent increased intensity of specific staining due to condensation of cellular contents andor the process of autolytic degradation?' The MODSof AR and PSA staining were not significantly different between nonmalignant and malignant epithelial cells in prostate specimens (table; fig. 2, D and 2, F).
ple as a consequence of gene mutation^).^' Relatively few cellular or molecular changes have been reported in association with prostatic carcinogenesis, and the majority of those alterations identified to date (for example mutations in the p53 and AR g e n e ~ ) ~appear * ~ l to occur more frequently in advanced disease?l In this study we have demonstrated a marked upregulation of cellular levels of Apo-D in early stage prostate cancers as compared with non-malignant prostatic epithelial cells. This finding, and our previous study demonstrating elevated Apo-D cellular levels in advanced primary prostate cancers,1s indicate that Apo-D may be a marker of malignant transformation in the prostate and may be involved in the initiation or maintenance of the transformed phenotype. The function of Apo-D in prostate cancer cells, and specifically the ligand(s) binding to Apo-D in tumors, are unknown. Due to the distribution of Apo-D in a wide variety of tissues, it has been suggested that different ligands may be associated with Apo-D in specific tissues.32 A previous report of induction of high levels of expression of Apo-D in regenerating rat sciatic nerve indicates that Apo-D may be involved in lipid transport andor metabolism in peripheral nerves?' Identification of Apo-D staining in the majority of nerve fibers of the prostatic capsules of the radical prostatectomy specimens analyzed in the current study suggests that it may be involved in normal cellular metabolism in nerves. In prostate cancer cells and in other steroid responsive malignant and non-malignant tissues where Apo-D is expre~sed,'~ pry gesterone or pregnenolone, which have been shown to s ically bind Apo-D, may be the predominant ligands.' Because the Apo-D ligand in prostate cancer cells has not yet been identified, the role of Apo-D in prostate tumors is not known. It seems likely, however, that Apo-D is involved in the local metabolism andor processing of steroids and may therefore constitute an appropriate target in the treatment of prostate cancers. Regulation of Apo-D expression and secretion in vivo is poorly characterized. A report of upregulation of Apo-D expression in quiescent fibr0b1ast.a:~ and our own observation of elevated Apo-D staining in prostatic glandular epithelial cells shed into the glandular lumen," suggest a correlation between cell senescence and increased cellular levels of Apo-D. In support of these findings, in vitro studies using prostate and breast cancer cell lines demonstrated that Apo-D secretion was upregulated by concentrations of androgens that inhibited cell pr~liferation.'~-'~ Retinoic acidz4and interleukin-la,26 which similarly decreased proliferation of breast cancer cell lines, were shown to increase Apo-D secretion. Conversely, growth stimulatory levels of 5adihydrotestosterone (DHT) decreased the secretion of Apo-D from the LNCaP prostate cancer cell linez1 and estradiol, which increased proliferation of breast cancer cell lines, resulted in decreased Apo-D ~ e c r e t i o n ? In ~ *contrast ~~ to these in vitro studies examining Apo-D secretion, it appears unDISCUSSION likely that the extensive expression of Apo-D in proliferating, The malignant transformation of cells is associated with invasive tumor foci observed in this and our previous study" aberrant expression or loss of expression of cell type specific and in human breast tumors'' is related to inhibition of cell proteins or alterations in the structure of proteins (for exam- proliferation. Further evaluation of the relationship between
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Video image analysis of Apo-D, PSA and AR immunostaining in 30 early stage prostate cancers Apolipoprotein D Prostate Specific Antigen
Androgen Receptor a
% Area positive staining
MODE % Area positive staining MOD % Area positive staining MOD
Non-malignantm
MaligIlaUP
p Valueb
0.5 2 0.2 59.0 ? 4.0 57.9 -t 3.7 50.2 -C_ 1.4 79.1 2 2.5 35.3 -c 0.9
28.3 2 4.3 44.1 -t 2.1 41.0 -C_ 3.3 49.8 ? 0.5 72.7 ? 3.7 37.2 2 0.6
0.003 0.001 0.804 0.163 0.091
Values are mean ? S.E.M.(standard error of the mean) for non-malignant glandular areas and malignant foci within the same tissue section. Student's t test. MOD = mean optical density, measured in density unitdpixel.
552
IMMUNOHISTOCHEMISTRY OF PROSTATIC NEOPLASMS
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Frc. 2. Video image analysis (percentage area positive staining (pixels) and mean optical density (MOD; density unitdpixel) of positive staining) of Apo-D, PSA and AFt m u n o s t a i n m g m prostate cancers. Immunostaining of 30 early stage prostate tumor foci and non-
malignant glandular tissue within same section was measured separately by image analysis following on-screen editing of captured images as described in Materials and Methods.
tumor cell expression, cellular accumulation and secretion of Apo-D and investigation of the in vivo function of Apo-D in tumor cells is required to explain the apparent disparity between in vitro and in vivo results and to define the conse-
quences of upregulation of Apo-D levels in prostate cancer cells. An association between cellular levels of Apo-D and expression of AR or PSA was not observed in this study. The finding
IMMUNOHISTOCHEMISTRYOF PROSTATIC NEOPLASMS
of no statistical differences in the distribution and/or intensity of AR immunostaining in early stage prostate cancers compared with the adjacent non-malignant tissues contrasts with results of studies of advanced prostate cancer from our own laboratory13 and where reduced AR immunoreactivity and increased heterogeneity of cellular staining was observed. The alterations in AR staining in advanced prostate cancers may represent greater genetic instability and development of altered androgen sensitivity arising during tumor progression or following administration of hormonal therapies. A reduced cellular area of PSA immunoreactivity was observed in malignant compared with benign glandular elements in early stage prostate cancer in this study. In an earlier study, Abrahamsson et al.17 reported a reduction in the number of PSA immunoreactive cells in prostate tumors in comparison to benign prostatic hyperplasia tissues, but the level of staining was not measured. The mechanisms that result in reduced cellular staining for PSA in prostate tumors prior to administration of hormonal therapies are unknown, but may result from reduced transcription and/or translation of the PSA gene and increased turnover or secretion of PSA protein by the tumor cells in response to alterations in the AR or other growth factor mediated pathways. The majority of prostate cancers detected by serum PSA screening are early stage, non-palpable tumors that are localized to the prostate land and are theoretically curable by radical prostatectomy!6 However, the associated morbidity of surgery and the prospects of disease recurrence following radical p r o s t a t e ~ t o m yhave ~ ~ shown the need for better cellular markers of (i) clinically unimportant disease that does not require definitive treatment and (ii) tumors with significant risk of progression. The present study has clearly demonstrated that the cellular content of Apo-D is markedly increased in tumor cells compared with non-malignant glands within the same section of early stage prostate cancer tissue. Expression of Apo-D in a proportion of human breast cancers and the demonstrated predictive value of cellular Apo-D levels on disease free interval and overall survival3s indicate the potential of further studies to determine whether cellular levels of Apo-D in early stage prostate cancers are indicative of clinically important tumors with an increased risk of progression. We acknowledge Dr. Jugen s a ,consultant PathOlo&t, Department of Anatomical Pathology, Flinders University School of Medicine, for his advice on nerve fibers of the prostatic capsule. REFERENCES
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IMMUNOHISTOCHEMISTRY O F PROSTATIC NEOPLASMS
Immunologic and steroid binding properties of the GCDFP-24 protein isolated from human breast gross cystic disease fluid. Breast Cancer Res. Treat., 1 6 253, 1990. 28. Epstein, J . I.: PSA and PAP as immunohistochemical markers in prostate cancer. Urol. Clin. North Am., 20: 757, 1993. 29. Rainbow, R. D.: Immunohistochemistry. In: Laboratory Histopathology; A Complete Reference. Edited by A E. Woods and R. C. Ellis. Churchill Livingstone., chapt. 8, pp. 8.2-52-53, 1994. 30. Renan, M. J.: How may mutations are required for tumorigenesis?: Implications from human cancer data. Mol. Carcinogen., 7: 139, 1993. 31. Rinker-Schaeffer. C. W., Partin, A. W., Isaacs, W. B., Coffey, D. S. and Isaacs, J. T.: Molecular and cellular changes associated with the acquisition of metastatic ability by prostatic cancer cells. Prostate, 2 5 249, 1994. 32. Milne, R. W., Rassart, E. and Marcel, Y. L.: Molecular biology of apolipoprotein D. Curr. Opin. Lipidol., 4 100, 1993. 33. Boyles, J. K, Notterpek, L. M. and Anderson, L. J.: Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. Identification of apolipoprotein D, apolipoprotein A-IV, apolipoprotein E, and apolipoprotein A-I. J. Biol. Chem., 265.17805, 1990.
34. Provost, P. R., Marcel, Y. L., Milne, R. W., Weech, P. K. and Rassart, E.: Apolipoprotein D transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures. FEBS Lett., 290: 139, 1991. 35. Pertschuk, L. P., Schaeffer, H., Feldman, J. G., Macchia, R. J., Kim, Y-D., Eisenberg, K, Braithwaite, L. V., Axiotis, C. A., F’rins, G. and Greene, G. L.: Immunostaining for prostate cancer androgen receptor in paraffin identifies a subset of men with poor prognosis. Lab. Invest., 7 3 302, 1995. 36. Lu-Yao, G. L., McLerran, D., Wasson, J . H. and Wennberg, J . E.: An assessment of radical prostatectomy. Time trends, geographic variation, and outcomes. The Prostate Patient Outcomes Research Team. JAMA, 269 2633, 1993. 37. Wasson, J . H., Cushman, C. C., Bruskewitz, R. C., Littenberg, B., Mulley, A. G., Jr. and Wennberg, J . E.: A structured literature review of treatment for localized prostate cancer. Prostate Disease Outcomes Research Team. Arch. Fam. Med., 2 487, 1993. 38. D i e z - h a , I., Vizoso, F., Merino, A. M., Sanchez, L. M., Tolivia, J., Fernandez, J., Ruibal, A. and Lopez-Otin, C.: Expression and prognostic significance of apolipoprotein D in breast cancer. Am. J. Pathol., 144 310, 1994.
IMMUNOLOCALIZATION OF APOLIPOPROTEIN D, ANDROGEN RECEPTOR AND PROSTATE SPECIFIC ANTIGEN IN EARLY STAGE PROSTATE CANCERS STEVEN X.D. ZHANG, JACQUELINE M. BENTEL, CARMELA RICCIARDELLI, DAVID J. HORSFALL, R. MARSHALL AND WAYNE D. TILLEY* DARROW E. HAAGENSEN, -IS From the Flinders Cancer Centre, Department of Surgery, Flinders University School of Medicine, Eedford Park,Australia, and the Department of Surgery, Methodist Hospital, Sacmmento, California
ABSTRACT
Purpose: To determine the cellular distribution and levels of immunohistochemicalstaining for apolipoprotein D (Apo-D), prostate specific antigen (PSA) and androgen receptor (AR)in early stage prostate cancers. Materials and Methods: Cellular distribution of Apo-D, PSA and AR in 30 stage A/B prostate cancers and in non-malignant glandular tissue contained in the same sections was detected immunohiatochemically, and staining was evaluated by computerized video image analysis. Results: Staining for Apo-D (percentage positive cellular area) was significantly increased in tumor cells of early stage prostate cancers compared with non-malignant glandular tissue. PSA and AR were present at high levels in both early stage prostate tumors and non-malignant prostate. Conclusions: Malignant transformation in the prostate is associated with increased cellular levels of Apo-D. Kcr WO-: prostatic neoplasms, immunohistochemistry, apolipoproteins, androgen receptor, prostate specific antigen
Hormonal therapies for the management of advanced pros- van& prostate cancers and outcome of subsequent In contrast, AR expression in early tate cancer were developed after Huggins and Hodges dem- hormonal onstrated prostatic tumor regression subsequent to removal stage prostate cancers has not been well characterized and of testicular andro ns by castration or administration of the relationship between AR levels and outcome of treatestrogens in 1941. While androgen ablation remains the ments of early stage disease is not known. The identity and function of androgen responsive proteins treatment of choice for metastatic (stage D,) disease, more recent clinical trials have evaluated hormonal treatments in in the normal prostate and in prostate cancer cells are poorly early stage tumors. These protocols have included androgen defined. Although expression of prostate specific antigen ablation as neoadjuvant hormonal therapy in combination (PSA) in glandular epithelial cells of the prostate appears to with radical prostatectomy for localized prostate tumor^^.^ be up-regulated by the availability of testosterone at puberand as chemopreventativeagents for development of prostate ty,14.15 prostate cancer cell expression and/or secretion of cancer in men with premalignant disease, such as low or high PSA may be regulated by factors other than testicular andrograde prostatic intraepithelial neoplasia (PIN).4To compre- gens. This is supported by studies demonstrating tumor cell hensively evaluate the outcomes of these protocols and to immunoreactivity for PSA and increasing serum PSA levels develop markers that will identify patients with localized in patients undergoing treatment with androgen ablation prostate cancers who w i l l respond most favorably to hor- therapies who have hormone refractory (stage D,) prostate monal manipulation, it is necessary to define the cellular cancers." Previous studies have reported that while the expression of the androgen receptor (AR)and other androgen majority of early, advanced and hormone refractory prostatic responsive proteins in early stage prostate tumors. tumors remain immunohistochemicallypositive for cellular The AR, a nuclear transcription fador that mediates an- PSA, staining is generally observed to be less than that seen drogen action in the prostate and in other androgen target in adjacent normal or benign hyperplastic gland^.'^.^^ tissues?' has been shown to be expressed in both glandular Apolipoprotein D (Apo-D), another androgen-regulated epithelial cells and in a proportion of interglandular fibro- protein, is a constituent of high density lipoprotein in plasma muscular stromal cells of the normal human pro~tate.6.~and is the major component of human breast cyst fluid." Several studies of advanced prostate cancers have demon- Apo-D is a member of the lipocalin superfamily of hydrophostrated that AR is expressed in tumor cells both prior to and bic protein transporters and is expressed in a variety of following hormonal therapies: with AR immunoreactivity steroid-responsive normal tissues including adrenal cortex homogeneously positive in a small number of tumors' and and corpus luteum" and in a proportion of steroid responsive hetero eneous in the majority of advanced prostate can- tumors (breast, prostate, ovarian, endometrial).". '' AlVideo image analysis studies have also demon- though its precise function in these tissues is unknown, cers?strated a relationship between AR immunostaining of ad- Apo-D specifically binds progesterone and pregnenolone and may be involved in steroid metabolism and/or processing in F p t e d for publication September 16,1997. Ftequ&a for r e p M h De ent of Surgery, Flindera Medical these tissues.20Androgen regulation of Apo-D has been demcentre.w d Park SA. 2. A ~ p t d i a . onstrated in prostate and breast cancer cell lines in in vitro Supportedby ants from the Antdancer Foundation of the Unihowever A D expression may also be reguveraihea of hug Australia, the National Health and Medical Re retinoic acidz4 and search Council of Australia and the Clive and Vera Ftamaciotti Foun- lated by glueoeorti~oids,~~strogen,22.~~ datiOIl8. interleukin-la." In light of these studies, the factors regu-
8"
f
P
548
549
IMMUNOHISTOCHEMISTRY OF PROSTATIC NEOPLASMS
lating Apo-D expression in specific tissues in vivo are unknown. Previous studies of Apo-D in non-malignant prostate and in advanced prostate cancers in our laboratory demonstrated cellular immunoreactivity for Apo-D in only occasional glandular epithelial cells and in cells shed into the glandular lumen of non-malignant prostatic tissues.15 In contrast, significantly increased Apo-D staining was evident in stage D, primary prostate tumors. To examine cellular levels of AR and the androgen-responsive proteins, Apo-D and PSA in early stage prostate tumors, and to determine whether cellular levels of Apo-D were increased early in tumor formation or whether this was a late event occurring during cancer progression, AR,Apo-D and PSA immunostaining were compared using computer assisted video image analysis in early stage primary prostate cancers and in non-malignant glandular tissue contained in the same slide. MATERIALS AND METHODS
Prostate tissues. Prostate cancer specimens were obtained from 30 patients referred to the urology units at Flinders Medical Center (Bedford Park, South Australia) and the Repatriation General Hospital (Daw Park, South Australia). The patients included in this study were clinically staged as A (10) or B (20) prostate carcinoma according to the modified Whitmore Jewett system.26All specimens used for immunohistochemical analysis were obtained at transurethral resection of the prostate (24) or radical prostatectomy (6)and had not been treated previously for prostate cancer. Following surgical resection, prostate specimens were immediately 6xed in formalin and then par& embedded within 48 hours. Immunohistochemistry. Five jun. sections were cut from paraffin-embedded tissues, mounted on poly-L-lysine coated glass microscope slides and baked at 60C for 1hour. Sections were deparaffinized in xylene and sequentially rehydrated in graded ethanol (loo%, 95%, 75% and 50%) for 2 minutes each. Endogenous peroxidase activity was blocked by incubation in phosphate buffered saline (PBS) containing 0.3% water for 5 minutes. Immunohistochemical staining for Apo-D, PSA and AR was carried out essentially as described previ~usly.'~*'~ Primary antibody for Apo-D was rabbit antiGCDFP-24,27and for AR was U402 (affinity purified rabbit antisera generated against amino acids 1to 21 of the human AR7)obtained from Drs. M. J. McPhaul, C. M. Wilson and J. D. Wilson, Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, TX. Apolipoprotein D. Slides were sequentially incubated at room temperature in blocking solution (PBS containing 10% normal goat serum (Vector Laboratories, Burlingame, CAI) for 15 minutes, primary antibody (1:1500 rabbit antiGCDFP-24 in blocking solution) for 30 minutes, secondary antibody (1:400 biotinylated goat anti-rabbit (Vector Laboratories) in blocking solution) for 30 minutes followed by avidin-biotin-peroxidase complex (ABC reagent) (1:400 in PBS, Vectastain ABC kit, Vector Laboratories) for 30 minutes. Slides were rinsed in PBS (2 X 5 min.) between incubations. Positive immunostaining was visualized with the chromogen 3'3'-diaminobenzidine tetrahydrochloride (DAB (Sigma Laboratories, St. Louis, MO) 0.05% DAB and 0.06% H,O, in 38 mM. Tris.HC1, pH 7.4) for 6 minutes. Slides were counterstained with weak Lillie Mayer's hematoxyh and mounted with Pix mounting medium. Specimens previously known to be reactive for the primary antibody were used as positive controls in each staining run.Primary antibody was replaced with non-immune sera at the same protein concentration for negative controls. Prostate specific antigen. Sections were incubated in block ing solution (10%normal goat serum in PBS) for 15 minutes primary antibody (1:1200 rabbit anti-human PSA (D&o Glostrup, Denmark) in blocking solution) for 30 minutes
iecondary antibody (1:400 biotinylated goat anti-rabbit Weeor Laboratories) in blocking solution) for 30 minutes and IBC reagent (1:400 in PBS) for 30 minutes. Slides were insed in PBS (2 x 5 min.) between incubations. The resultmt complexes were visualized as described above. Androgen receptor. Sections were immunohistochemically stained for AR following antigen unmasking in 10 mM. ci;rate buffer (pH 6.5). Slides were heated to boiling in a microwave, simmered for 8 minutes, then allowed to cool in che citrate buffer for 20 minutes. Sections were incubated in blocking solution (3% normal goat serum and 1% normal human serum in PBS) for 15 minutes, primary antibody :1:250 U402, in blocking solution) at 4C overnight, secondary antibody (1:400 biotinylated goat anti-rabbit in blocking solution) for 30 minutes, followed by ABC reagent (1:400 in PBS) for 30 minutes. Slides were rinsed in PBS (2 X 5 min.) between incubations. Positive immunostaining was visualized as described above. Video image analysis. The immunostained sections were magniexamined using an Olympus BH-2 microscope (~200 fication) coupled to a computer-assisted color image system (Video Pro 3 9 Leading Edge Pty. Ltd.,Adelaide, South Australia) based on methods described Color video image analysis measurements were generated independently for malignant foci and non-malignant epithelial components within the same section for each prostate specimen. Discrimination between malignant and benign tissue areas was based on standard cytological and pathological features. The majority of non-malignant glands analyzed exhibited histological features consistent with benign prostatic hyperplasia. As our previous studies demonstrated the absence of Apo-D immunoreactivity in the prostatic strorna,l6 this component was not included in the current analysis. In contrast to our previous study," epithelial cells sloughed into the glandular lumen also were not included in the analysis of Apo-D immunostaining. Video image measurements were made of the total area (pixels) of tissue analyzed (that is, positively and negatively stained cells) and the integrated optical density (IOD; arbitrary density units) of positively stained areas, for at least 20 fields per section. The following parameters of staining were derived for each sample: 1)the percentage area of positively stained cells (that is, positively stained aredtotal area XlOO%) and 2) the mean optical density (MOD; density unitdpixel) of positive staining, which is equal to the IOD/area of positively stained cells (that is, the density of staining in the positive immunoreactive area, which is stoichiometrically related to the cellular concentration of antigen). Statistical analysis. Data were presented as the mean and standard error of the mean (s.e.m.). Staining parameters of malignant and non-malignant tissue areas were analyzed using a student's t test. Statistical significance was established at the p <0.05 level. RESULTS
Immunohistochemical localization of Apo-D, PSA and AR. Apo-D was detected in all prostate cancer specimens as cytoplasmic immunoreactivity in tumor cells and in occasional non-malignant glandular epithelial cells. (fig. 1)No staining of stromal, endothelial or blood cells was observed. As reported previo~sly,'~ Apo-D staining was evident in only a few glandular epithelial cells and in a small number of cells shed into the glandular lumen in normal and benign hyperplastic glands. In contrast to nonmalignant glandular epithelial cells, prostate cancer cells in the same specimens exhibited extensive immunoreactivity for Apo-D. Apolipoprotein D staining of individual tumors ranged from weakly to strongly positive (fig. 1, A, B ) , with considerable heterogeneity of staining evident within tumor foci. Strongly positive A p D immunoreactivity was also detected in the majority
550
IMMUNOHISTOCHEMISTRYOF PROSTATIC NEOPLASMS
FIG.1. Immunohistochemicalstaining for Apo-D, PSA and AR in early stage prostate cancer specimens.A, cytoplasmic immunoreactivity for Apo-D in stage A tumor exhibiting strongly positive staining. Cells of Lon-malignant prostatic ducts localized among tumor foci were immunohistologicallynegative for Apo-D. B, re resentative immunostainingfor Apo-D in stage A tumor. Staining is heterogeneous with both positively and negatively immunoreactive ce&. C, positive Apo-D immunostaining in nerve fiber located in prostatic capsule of radical prostatectomy specimen. Inset negative control immunostaining of nerves in adjacent section of same tumor. D, strongly positive PSA immunostainingin tumor cells of stage B rostate tumor. Stromal cells within tumor are negative for PSA. E, immunostainingfor AR in same immunoreactivity is evident, with strongly and weakly staining cells present in tumor focus. tumor specimen as D. Heterogeneous Stromal cells within this area of the tumor are negative for AR,however, foci of positively staining stromal cells were evident in all tumors and in non-malignant are= of specimens. Magnification X200.
&
551
IMMUNOHISTOCHEMISTRY OF PROSTATIC NEOPLASMS
(94.8+3.7%;mean+S.E.M) of the 155 nerve fibers identified in the prostatic capsules of the 6 radical prostatectomy specimens examined in this study (fig. 1,C ) . Consistent with previous s t u d i e ~ , ' ~cytoplasmic .~~ PSA staining was evident in glandular epithelial cells and in prostate tumor cells of all specimens (fig. 1, D ) . Nonepithelial cell types were not positively stained for PSA. Staining of tumor foci for PSA ranged from heterogeneous immunoreactivity with strongly and weakly stained cells, to uniform strongly positive PSA staining in a proportion of specimens (fig. 1,D). Nuclear AR immunoreactivity was observed in prostate cancer cells, non-malignant glandular epithelial cells and in a proportion of peritumoral and interglandular stromal cells (fig. 1,E ) . AR staining in tumor cells and benign glandular epithelial cells within the same section appeared similar, with heterogeneous staining composed of weakly to strongly positive nuclear immunoreactivity in the majority of cells. In contrast, stromal cells displayed focal immunoreactivity with cords of AR immunopositive stromal cells interspersed with regions of negatively staining stromal cells. Video image analysis. The immunostaining of prostate specimens for Apo-D, PSA and AR was quantitated using video image analysis. The mean percentage area of tumor cells positively stained for Apo-D was 28.3 2 4.3%, which is a significant increase compared with the almost negligible percentage area of glandular epithelial cells positively stained in non-malignant areas of the specimens 0.51 ? 0.19% (table; fig. 2, A). Six of the 30 tumor specimens analyzed exhibited less than 5% positive staining for Apo-D. The mean percentage area of PSA staining in tumor foci was statistically decreased compared with non-malignant glands (table); however, there was considerable overlap in the staining of individual tissues within the two groups (fig. 2, C).The percentage area positively stained for AR was not significantly different between tumor foci and adjacent nonmalignant glands in the specimens (p >0.05;table; fig. 2, E). Stromal AR staining was not quantitated by video image analysis. The cellular concentration of Apo-D staining in positively stained cells (MOD) was significantly decreased in tumor cells compared with non-malignant glandular epithelial cells; however, these values were based on very few positively stained cells in non-malignant specimens (table; fig. 2, B). In addition, Apo-D positive glandular epithelial cells may have been terminally differentiated or dying prior to sloughing into the glandular lumen at the time of fixation and this may have resulted in the apparent increased intensity of specific staining due to condensation of cellular contents andor the process of autolytic degradation?' The MODSof AR and PSA staining were not significantly different between nonmalignant and malignant epithelial cells in prostate specimens (table; fig. 2, D and 2, F).
ple as a consequence of gene mutation^).^' Relatively few cellular or molecular changes have been reported in association with prostatic carcinogenesis, and the majority of those alterations identified to date (for example mutations in the p53 and AR g e n e ~ ) ~appear * ~ l to occur more frequently in advanced disease?l In this study we have demonstrated a marked upregulation of cellular levels of Apo-D in early stage prostate cancers as compared with non-malignant prostatic epithelial cells. This finding, and our previous study demonstrating elevated Apo-D cellular levels in advanced primary prostate cancers,1s indicate that Apo-D may be a marker of malignant transformation in the prostate and may be involved in the initiation or maintenance of the transformed phenotype. The function of Apo-D in prostate cancer cells, and specifically the ligand(s) binding to Apo-D in tumors, are unknown. Due to the distribution of Apo-D in a wide variety of tissues, it has been suggested that different ligands may be associated with Apo-D in specific tissues.32 A previous report of induction of high levels of expression of Apo-D in regenerating rat sciatic nerve indicates that Apo-D may be involved in lipid transport andor metabolism in peripheral nerves?' Identification of Apo-D staining in the majority of nerve fibers of the prostatic capsules of the radical prostatectomy specimens analyzed in the current study suggests that it may be involved in normal cellular metabolism in nerves. In prostate cancer cells and in other steroid responsive malignant and non-malignant tissues where Apo-D is expre~sed,'~ pry gesterone or pregnenolone, which have been shown to s ically bind Apo-D, may be the predominant ligands.' Because the Apo-D ligand in prostate cancer cells has not yet been identified, the role of Apo-D in prostate tumors is not known. It seems likely, however, that Apo-D is involved in the local metabolism andor processing of steroids and may therefore constitute an appropriate target in the treatment of prostate cancers. Regulation of Apo-D expression and secretion in vivo is poorly characterized. A report of upregulation of Apo-D expression in quiescent fibr0b1ast.a:~ and our own observation of elevated Apo-D staining in prostatic glandular epithelial cells shed into the glandular lumen," suggest a correlation between cell senescence and increased cellular levels of Apo-D. In support of these findings, in vitro studies using prostate and breast cancer cell lines demonstrated that Apo-D secretion was upregulated by concentrations of androgens that inhibited cell pr~liferation.'~-'~ Retinoic acidz4and interleukin-la,26 which similarly decreased proliferation of breast cancer cell lines, were shown to increase Apo-D secretion. Conversely, growth stimulatory levels of 5adihydrotestosterone (DHT) decreased the secretion of Apo-D from the LNCaP prostate cancer cell linez1 and estradiol, which increased proliferation of breast cancer cell lines, resulted in decreased Apo-D ~ e c r e t i o n ? In ~ *contrast ~~ to these in vitro studies examining Apo-D secretion, it appears unDISCUSSION likely that the extensive expression of Apo-D in proliferating, The malignant transformation of cells is associated with invasive tumor foci observed in this and our previous study" aberrant expression or loss of expression of cell type specific and in human breast tumors'' is related to inhibition of cell proteins or alterations in the structure of proteins (for exam- proliferation. Further evaluation of the relationship between
r*-
Video image analysis of Apo-D, PSA and AR immunostaining in 30 early stage prostate cancers Apolipoprotein D Prostate Specific Antigen
Androgen Receptor a
% Area positive staining
MODE % Area positive staining MOD % Area positive staining MOD
Non-malignantm
MaligIlaUP
p Valueb
0.5 2 0.2 59.0 ? 4.0 57.9 -t 3.7 50.2 -C_ 1.4 79.1 2 2.5 35.3 -c 0.9
28.3 2 4.3 44.1 -t 2.1 41.0 -C_ 3.3 49.8 ? 0.5 72.7 ? 3.7 37.2 2 0.6
0.003 0.001 0.804 0.163 0.091
Values are mean ? S.E.M.(standard error of the mean) for non-malignant glandular areas and malignant foci within the same tissue section. Student's t test. MOD = mean optical density, measured in density unitdpixel.
552
IMMUNOHISTOCHEMISTRY OF PROSTATIC NEOPLASMS
A.
Apolipoprotein D
Apolipoprotein D
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Frc. 2. Video image analysis (percentage area positive staining (pixels) and mean optical density (MOD; density unitdpixel) of positive staining) of Apo-D, PSA and AFt m u n o s t a i n m g m prostate cancers. Immunostaining of 30 early stage prostate tumor foci and non-
malignant glandular tissue within same section was measured separately by image analysis following on-screen editing of captured images as described in Materials and Methods.
tumor cell expression, cellular accumulation and secretion of Apo-D and investigation of the in vivo function of Apo-D in tumor cells is required to explain the apparent disparity between in vitro and in vivo results and to define the conse-
quences of upregulation of Apo-D levels in prostate cancer cells. An association between cellular levels of Apo-D and expression of AR or PSA was not observed in this study. The finding
IMMUNOHISTOCHEMISTRYOF PROSTATIC NEOPLASMS
of no statistical differences in the distribution and/or intensity of AR immunostaining in early stage prostate cancers compared with the adjacent non-malignant tissues contrasts with results of studies of advanced prostate cancer from our own laboratory13 and where reduced AR immunoreactivity and increased heterogeneity of cellular staining was observed. The alterations in AR staining in advanced prostate cancers may represent greater genetic instability and development of altered androgen sensitivity arising during tumor progression or following administration of hormonal therapies. A reduced cellular area of PSA immunoreactivity was observed in malignant compared with benign glandular elements in early stage prostate cancer in this study. In an earlier study, Abrahamsson et al.17 reported a reduction in the number of PSA immunoreactive cells in prostate tumors in comparison to benign prostatic hyperplasia tissues, but the level of staining was not measured. The mechanisms that result in reduced cellular staining for PSA in prostate tumors prior to administration of hormonal therapies are unknown, but may result from reduced transcription and/or translation of the PSA gene and increased turnover or secretion of PSA protein by the tumor cells in response to alterations in the AR or other growth factor mediated pathways. The majority of prostate cancers detected by serum PSA screening are early stage, non-palpable tumors that are localized to the prostate land and are theoretically curable by radical prostatectomy!6 However, the associated morbidity of surgery and the prospects of disease recurrence following radical p r o s t a t e ~ t o m yhave ~ ~ shown the need for better cellular markers of (i) clinically unimportant disease that does not require definitive treatment and (ii) tumors with significant risk of progression. The present study has clearly demonstrated that the cellular content of Apo-D is markedly increased in tumor cells compared with non-malignant glands within the same section of early stage prostate cancer tissue. Expression of Apo-D in a proportion of human breast cancers and the demonstrated predictive value of cellular Apo-D levels on disease free interval and overall survival3s indicate the potential of further studies to determine whether cellular levels of Apo-D in early stage prostate cancers are indicative of clinically important tumors with an increased risk of progression. We acknowledge Dr. Jugen s a ,consultant PathOlo&t, Department of Anatomical Pathology, Flinders University School of Medicine, for his advice on nerve fibers of the prostatic capsule. REFERENCES
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