Expression of Oncogenic Antigen 519 (OA- 519) in Prostate Cancer Is a Potential Prognostic Indicator

Transcription

1 ANATOMIC PATHOLOGY Original Article Expression of Oncogenic Antigen 519 (OA- 519) in Prostate Cancer Is a Potential Prognostic Indicator MAHAMMAD S. SHURBAJI, M.D., 1 " 2 FRANCIS P. KUHAJDA, M.D., 3 GARY R. PASTERNACK, M.D., PH.D;, 3 AND THANE S. THURMOND, M.S. 2 Predicting the prognosis of patients with prostate cancer is a clinically important problem. Previous studies have indicated that the expression of haptoglobin-related protein epitopes in samples of breast cancer in early stages was associated with earlier relapses and higher risk for tumor recurrence. Oncogenic antigen 519 (OA-519) is the new marker designation for mole cules expressing haptoglobin-related protein epitopes. The ob jective of this immunohistochemical study was to examine OA- 519 expression in prostate cancer samples and its relationship to the established prognostic indicators of tumor grade, tumor volume, and clinical stage. Forty-two consecutive tissue samples of prostate adenocarcinoma were examined using an affinitypurified anti-oa-519 antibody. Twenty specimens (48%) tested positive, whereas 22 (52%) tested negative. No staining was ob served in normal or hyperplastic prostate tissue. Staining oc curred in 6 of 9 (67%) grade III, 14 of 23 (61%) grade II, and in none of 10 (0%) grade I cases (I vs. II and/or III: Fisher exact test, P < 0.006). Twenty-three of the 42 samples were trans urethral resection specimens with cancer; 11 (48%) of these tested positive. The mean percentage of tissue chips with tumor, a mea sure of tumor volume, was significantly higher in the positive group (57%) than in the negative group (15%) (P = 0.004). The proportion of positively stained cases increased with advancing clinical stage, with 25% of Stage A cases expressing OA-519, and 46%, 67%, and 64% of Stages B, C, and D, respectively, expressing OA-519. OA-519 expression correlates with higher tumor grades, larger tumors, and possibly with advanced stage, and thus, it is potentially of prognostic value in prostate cancer. (Key words: Prostate adenocarcinoma; Haptoglobin-related pro tein; OA-519; Prognosis; Immunohistochemistry) Am J Clin Pathol 1992; 97: Prostate cancer is the most common newly diagnosed not become clinically manifest during the lifetime of the cancer in men and is the second leading cause of cancer individual and underscores the importance of identifying death after lung cancer, killing approximately 28,000 men factors that help predict the biologic behavior of these per year. The incidence of Stage A (clinically inapparent, incidentally discovered tumors. incidentally discovered) disease increases with age; how The prognosis of a patient with prostate cancer worsens ever, the clinical incidence of Stage A prostate cancer is progressively with advancing clinical stage and histopath about one half of that predicted by autopsy studies. 2 " 5 ologic grade, but even within a given stage and grade group This suggests that a large proportion of these lesions do there remain differences in the duration of survival and the pattern of tumor dissemination. 2,6 An additional re liable prognostic indicator would help predict the outcome From the Laboratory Service, VA Medical Center, the department of Pathology, East Tennessee State University and 2 James H. Quillen of patients with prostate cancer and aid in the selection College of Medicine, Johnson City, Tennessee, and the ^Department of therapy. of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland. Received May 29, 1991; manuscript accepted for publication July 12, Presented in part at the Annual Meeting of the United States and Canadian Academy of Pathology in Chicago, Illinois, March 18, Supported in part by an R.D.C. Major Research Grant from East Tennessee State University. Address reprint requests to: Dr. Shurbaji, Department of Pathology, J. H. Quillen College of Medicine, East Tennessee State University, P.O. Box 70568, Johnson City, Tennessee Previous studies have shown that some breast cancer specimens express the haptoglobin-related protein (Hpr) or proteins that share epitopes with Hpr. 7 Expression of Hpr epitopes in breast cancer specimens has been asso ciated with a significantly increased risk of recurrence and early relapse. 7 The acquisition of Hpr-epitope expression also parallels the acquisition of increased malignant po tential by the tumor, and once acquired, Hpr-epitope expression tends to be a permanent characteristic of the 686

2 SHURBAJI ET AL. 687 OA-519 and Prognos is in Prostate Cancer tumor. 8 Prostate carcinoma shares with breast carcinoma the distinction of being a tumor that responds to steroid hormone therapy. It is thus a logical extension of this work to determine if similar proteins are expressed by prostate carcinoma samples, and if so, whether they are prognostically significant. Haptoglobin-related protein is the product of the haptoglobin-related protein (HPR) gene, which is located 2.2 kilobases downstream from the conventional haptoglobin locus. 9 In term pregnant women, Hpr is detectable as a plasma protein that is similar in structure to haptoglobin l. 10 In breast carcinoma specimens, however, proteins bearing Hpr epitopes may not necessarily be the product of the HPR gene, but rather distinct proteins sharing only structural epitopes. To avoid confusion until these mol ecules are characterized further, proteins that bear relevant Hpr epitopes have been designated as Oncogenic Antigen 519 (OA-519). The goals of this study were to examine the expression of OA-519 by prostatic adenocarcinoma and to determine its relationship to the established prognostic indicators of tumor grade, tumor volume, and clinical stage. 2 6 " Case Selection MATERIALS AND METHODS Forty-two consecutive cases of prostate adenocarci noma were selected from the files of the Mountain Home VA Medical Center. The specimens consisted of 23 trans urethral resections (TUR), 14 needle biopsies, and 5 pros tatectomy specimens. Clinical stage information was obtained from the tumor registry abstracts or by review of the clinical records. Histopathologic Studies Tumor grading. All slides were reviewed and a Gleason score was determined by adding the numbers for the two most predominant patterns. 6 Gleason scores 2 to 4 were assigned grade I, scores 5 to 7 were assigned grade grade II, and scores 8 to 10 were assigned grade III. Tumor volume estimation in TUR specimens. All sub mitted slides from the TUR cases were examined and the ratio of tumor chips with cancer to the total number of chips submitted was determined as described by Hum phrey and Vollmer," expressed as a percentage, and used as a measure of tumor volume in TUR specimens. Immunohistochemical Studies A single representative tissue block was selected from each cancer specimen for immunohistochemical staining. An affinity-purified polyclonal anti-oa-519 antibody was used in this study. This antibody was raised against a synthetic peptide corresponding to the 34 N-terminal residues of the predicted HPR gene product. 7,810 Staining was performed on routinely processed, formalin-fixed, paraffin-embedded tissue. The avidin-biotin complex immunoperoxidase technique using unlabeled primary an tibody was employed. 78 Briefly, 6-^m deparaffinized and rehydrated tissue sections were incubated in 3% hydrogen peroxide in methanol for 15 minutes to block endogenous peroxidase activity and then in a 1:20 dilution of normal goat serum in phosphate-buffered saline for 30 minutes. Slides were incubated with affinity-purified polyclonal anti-oa-519 at 5 Mg/mL in 1% bovine serum albumin in phosphate-buffered saline at ph 7.2 for 1 hour at room temperature. (Alternatively, an overnight incubation at 4 C can be used with comparable results.) With intervening washes with phosphate-buffered saline, the sections were incubated successively with biotinylated goat anti-rabbit immunoglobulin diluted 1:500 in 1% bovine serum albumin in phosphate-buffered saline (Vector Labora tories, Burlingame, CA) and avidin-horseradish peroxi dase complex (VectaStain, Vector Laboratories), both for 30 minutes at 22 C. Aminoethylcarbazole (Vector Lab oratories) was used as the chromogen with Mayer s he matoxylin counterstain. For negative controls, phosphate*- buffered saline was substituted for primary antibody for each case. A known OA-519-positive case was used as a positive control with every run. Staining was defined as positive for OA-519 if (1) immunoreactivity was discernible at low power (loox), (2) granular cytoplasmic staining was present without ob servable nuclear staining (Fig. 1), and (3) staining was heterogenous (i.e., the level of reactivity varied from cell to cell or from region to region). Tumors were scored as testing positive or negative. 8 RESULTS OA-519 Expression and Tumor Grade Twenty (48%) of the 42 prostate cancers tested positive for OA-519 (Fig. 1), whereas 22 (52%) tested negative. No staining was noted in normal or hyperplastic prostate tissue. OA-519 expression occurred in 6 of 9 (67%) grade HI, 14 of 23 (61%) grade II, and in none of 10 (0%) grade I cases. The differences in the proportion of the positively stained cases was highly statistically significant when grade I was compared to grade II or grade III and when grade I was compared to combined grades II and III (Fisher exact probability, P < 0.002, p < 0.006, p < , re spectively). The difference in the proportion of positively stained cases between grades II and III was not statistically significant. Vol. 97 No. 5

3 688 ANATOMIC PATHOLOGY Original Article FIG. 1. Immunohistochemical staining with polyclonal anti-oa-519 in a case of moderately differentiated prostatic adenocarcinoma. Note the granular cytoplasmic staining (immunoperoxidase with AEC chromogen and Mayer s hematoxylin counterstain; original magnification, X945). OA-519 Expression and Tumor Volume 100n 90(_ nee Twenty-three TUR specimens were included in this study. Of these, 11 (48%) stained positive for OA-519, whereas 12 (52%) tested negative. The similarity of the proportion of positive cases to that of the entire group was striking and provided further assurance that this was a representative group. Tumor volume was estimated in all of these TUR specimens by the ratio of tissue chips with cancer to the total number of chips examined." The mean percentage of chips with tumor in the positive group was 57% (range, 2% to 100%), whereas that in the negative group was 15% (range, 1 % to 74%) (Fig. 2). The difference between the two means was highly statistically significant (t = 2.9, P = 0.004). 80- ro o n 4-> en a. c u rr 3 H c 20- Q> <L> OA-519 Expression and Clinical Stage flflfllP NNNNNNNNNNNNPPPPPPPPPPP OA-519 Staining FIG. 2. Percentage of TUR chips with prostate cancer (an estimate of tumor volume) versus OA-519 staining in 23 TUR specimens. Each bar represents a single case (N = negative, mean = 15%; P = positive, mean = 57%; t = 2.9, P = 0.004). A.J.C.P. Mav o a. The specimens examined in this study consisted of 12 Stage A, 13 Stage B, 6 Stage C, and 11 Stage D cancers. A clear trend toward a higher proportion of positive cases with advancing clinical stage was observed (Fig. 3). Only 25% of Stage A cases expressed OA-519 with 46%, 67%, bo40-

4 SHURBAJI ET AL. 689 OA-519 and Prognosis in Prostate Cancer Clinical Stage OA-519 Negative OA-519 Positive FlG. 3. OA-519 staining versus clinical stage in 42 cases of prostate cancer. and 64% for Stages B, C, and D, respectively. These dif ferences were not, however, statistically significant. DISCUSSION Histopathologic grading of prostatic adenocarcinoma has a striking correlation with the local extent of disease, 12 the incidence of lymph node and bone metastases, 314 survival rate, and response to therapy. 14 " 7 The histologic grade not only correlates with the clinical stage but also shows a strong correlation with mortality rates within each clinical stage, and thus, a combination of grading and staging has the highest predictive value. 6 However, even within a given stage and grade group there remain differ ences in survival and in rate and pattern of disease pro gression. 2,6 This has stimulated research efforts to identify additional prognostic indicators that will help stratify fur ther the prognosis of patients with prostate cancer. To this end, various morphometric measurements have been proposed to improve the prognostic value of grading and some provided a statistically significant separation of pa tients on the basis of outcome.. 8 Studies using DNA quantitation by image analysis and flow cytometric eval uation have shown that aneuploid prostate cancers are more likely to progress than neoplasms with diploid DNA content. 19 ~ 21 Nativ and associates 21 found that a combi nation of histologic tumor grade and nuclear DNA ploidy gave the strongest association with prognosis in the group of Stage C patients they studied. Prostatic acid phosphatase was the first urologic serum marker developed, and it has been used extensively in the evaluation and follow-up of patients with prostate can cer. 22 " 24 Whitesel and co-workers 25 have demonstrated that patients with clinically localized prostate cancer and persistently elevated prostatic acid phosphatase levels are very likely to have occult disseminated disease. Others have shown that a correlation exists between prostatic acid phosphatase levels in the upper limits of the normal range in patients with localized cancer and histologic ev idence of extraprostatic tumor extension. 26 Oesterling and colleagues 27 noted that the serum prostatic acid phospha tase level and the Gleason grade correlated well with cap sular penetration, seminal vesicle involvement, and lymph node metastasis in patients with clinically localized pros tate cancers. Since it was first described in 1979, 28 prostate-specific antigen (PSA) has been investigated extensively for its po tential in the diagnosis, prognosis, and follow-up of pa tients with prostatic cancer. 22 It is a glycoprotein with a molecular weight of 34,000 kd that is immunologically and biologically distinct from prostatic acid phosphatase. 23 The greatest value of the PSA serum assay appears to be to detect residual and recurrent tumor after therapy for localized and metastatic disease, including after radical prostatectomy, 29 radiation therapy, orchiectomy, or other hormonal manipulations. 29 " 31 Efforts to use PSA to detect clinically occult cancers have been largely unsuccessful, 31 perhaps due to the low positive and negative predictive value of the test because PSA levels are elevated in up to 20% of men with benign prostatic hyperplasia, 3233 whereas 20% to 50% of men with documented prostate cancer do not have elevated PSA levels. 34 In addition, preoperative levels of PSA are not predictive of the final pathologic stage and therefore have limited value in staging these lesions. 29 Nearly 80% of tumors of the prostate respond to hor monal manipulation. 35 The relative success with which the response of breast cancer to endocrine treatment can be predicted by assay of female sex steroid receptors has led to attempts to use androgen receptor assays in prostate tumors to predict the success of hormonal therapy. Some investigators reported no correlation between cytosol content of androgen receptor from prostate tumors and the response of the patient to estrogen therapy, whereas others determined that those patients with the lowest cy tosol androgen receptor content uniformly failed to re spond to hormonal therapy In a recent study, Trachtenberg and Walsh 38 compared the levels of total tissue, cytosol, and nuclear androgen receptors of the tumor with the patient response to hormone therapy and their sub sequent survival. These authors found no correlation be tween total or cytosol androgen receptor content and clinical response; however, those patients with prostatic tumor nuclear androgen receptor levels greater than 110 fmoles/mg DNA responded to endocrine therapy for a significantly longer period than did patients with lower nuclear androgen receptor levels, and consequently had a better survival rate. 38 Other investigators have also shown that a high nuclear androgen receptor content cor- Vol. 97 No. 5

5 690 ANATOMIC PATHOLOGY Original Article relates with enhanced survival of patients who receive hormonal therapy. 39 Certain clinical features of prostate cancer have limited the utility of such receptor studies. This may be due to the heterogeneity of the tumor itself, the presence ofaccompanying areas of hyperplasia, or the presence of androgen receptors in stromal cells. Another problem is the difficulty in obtaining samples for receptor studies because most initial diagnoses are made using needle biopsy, which yields only a small amount of tissue. Specimens obtained by transurethral resection yield suf ficient amount of tissue; however, the use of the electro cautery may result in heat denaturation of the receptor. 40 The role of growth factors and oncogene expression in the determination of prognosis of prostate cancer remains unclear. Nag and Smith 41 studied the structure and expression of the c-myc protooncogene in DNA isolated from the LNCaP human prostate cancer cell line. They found a 10-fold amplification of c-myc accompanied by a 50-fold increase in c-myc RNA transcripts. An earlier study had examined the expression of both c-myc and c- Ha-ras protooncogenes. 42 Northern blot analysis showed a considerable amount of c-myc transcripts in six cancers, whereas three other tumors and all the benign tissue showed little or no detectable c-myc transcripts. The tu mors with the elevated levels of c-myc transcripts were found to have a Gleason score of 5 or more, whereas the tumors with little or no c-myc transcripts all had Gleason scores of 4 or less, suggesting a link between over-expres sion of c-myc transcripts and poor prognosis. None of the tumors, and only one of the benign prostatic hyperplasia specimens, examined in this study showed appreciable amounts of c-ha-ras RNA transcripts. 42 Viola and associates 43 used an immunohistochemical assay and monoclonal antibodies against the ras oncogene protein p21. Normal and hyperplastic prostate tissue samples were all negative for p21 protein. The antigen was detected in two of six cases with grade I carcinoma, 4 of 6 grade II, and all 17 with higher grade tumors. Expression of p21 antigen correlated with histologic grade, leading the au thors to conclude that ras oncogene P21 protein may be a relevant prognostic marker. Therefore, among the on cogenes studied thus far, the early results of immunohis tochemical detection of ras p21 protein show the most promise as a practical prognostic marker. As was found in breast carcinoma, the current study has demonstrated that proteins with Hpr epitopes (OA- 519) are expressed by some prostate cancers. Furthermore, OA-519 expression (1) is not seen in normal or benign hyperplastic prostate tissues, (2) is significantly associated with higher tumor grades and larger tumors, and (3) is associated with advancing clinical stage. Because high tu mor grades, large tumor volumes, and advanced stage are proved indicators of poor prognosis, we conclude that OA-519 expression is of potential prognostic significance in prostate cancer. Further clinical studies examining OA- 519 expression and prognosis within single Gleason grades and comparing OA-519 expression to that of ras p21 will further determine the role of OA-519 as an independent prognostic indicator in prostate cancer. REFERENCES 1. Boring CC, Squires TS, Tong T. Cancer statistics, CA 1991;41: Whitmore WF. Natural history and staging of prostate cancer. Urol Clin N Am 1984;11: Halpert B, Sheehan EE, Schmalhorst WR, et al. Carcinoma of the prostate: A survey of 5000 autopsies. Cancer 1963; 16: Halpert B, Schmalhorst WR. Carcinoma of the prostate in patients 70 to 79 years old. Cancer 1966; 19: Hanash KA, Utz DC, Cook EN, et al. Carcinoma of the prostate: A 15 year follow up. J Urol 1972; 107: Gleason DF, The Veterans Administration Cooperative Urologic Research Group. Histological grading and clinical staging of prostatic carcinoma. In: Tannenbaum M, ed. Urologic Pathology: The Prostate. Philadelphia: Lea and Febiger, 1977, pp Kuhajda FP, Piantadosi S, Pasternack GR. Haptoglobin-related protein (Hpr) epitopes in breast cancer as a predictor of recurrence of the disease. N Engl J Med 1989;321: Shurbaji MS, Pasternack GR, Kuhajda FP. Expression of haptoglobin related protein in primary and metastatic breast cancers: A lon gitudinal study of 48 fatal cancers. Am i Clin Pathol 1991;96: Maeda N. Nucleotide sequence of the haptoglobin and haptoglobinrelated gene pair: The haptoglobin-related gene contains a retrovirus-like element. J Biol Chem 1985;260: Kuhajda FP, Katumuluwa Al, Pasternack GR. Expression of hap toglobin-related protein and its potential role as a tumor antigen. Proc Natl Acad Sci USA 1989; 86: Humphrey P, Vollmer RT. The ratio of prostate chips with cancer: A new marker of tumor extent and its relationship to grade and prognosis. Hum Pathol 1988; 19: Cantrell BB, deklerk DP, Eggleston JC, Boitnott JK, Walsh PC. Pathological factors that influence prognosis in stage A prostatic cancer: The influence of extent versus grade. J Urol 1981; 125: Zincke H, Farrow GM, Myers RP, et al. Relationship between grade and stage of adenocarcinoma of the prostate and regional pelvic lymph node metastases. J Urol 1982; 128: Fan K, Peng CF. Predicting the probability of bone metastasis through histological grading of prostate carcinoma. A retrospective correlative analysis of 81 autopsy cases with antemortem trans urethral resection specimen. J Urol 1983; 130: Utz DC, Farrow GM. Pathologic differentiation of and prognosis of prostatic carcinoma. JAMA 1969; 209: Perez CA, Bauer W, Garza R, Royce RK. Radiation therapy in the definitive treatment of localized carcinoma of the prostate. Cancer 1977;40: Gibbons RP, Correa RJ, Brannen GE, Mason JT. Total prostatec tomy for localized prostatic cancer. J Urol 1984; 131: Partin AW, Walsh PC, Pitcock RV, et al. A comparison of nuclear morphometry and Gleason grade as a predictor of prognosis in Stage A2 prostate cancer: A critical analysis. J Urol 1989,142: Peters JM, Crissman JD. Histopathologic diagnosis and classification of prostate adenocarcinoma: Biologic significance. Henry Ford HospMedJ 1989;37: Winkler HZ, Rainwater LN, Myers RP, et al. Stage DI prostatic adenocarcinoma. Significance of nuclear DNA ploidy pattern studied by flow cytometry. Mayo Clin Proc 1988;63: Nativ O, Winkler HZ, Raz Y, et al. Stage C prostatic adenocarcinoma: A.J.C.P.-May 1992

6 SHURBAJI ET AL. 691 OA-519 and Prognosis in Prostate Cancer Flow cytometric nuclear DNA ploidy analysis. Mayo Clin Proc 1989;64: Gutman AB, Gutman EB. An acid phosphatase occurring in the serum of patients with metastasizing carcinoma of the prostate gland. J Clin Invest 1938; 17: , 23. Ban Y, Wang MC, Chu TM. Immunologic markers and the diagnosis of prostatic cancer. Urol Clin N Am 1984; 11: Heller JE. Prostatic acid phosphatase: Its current clinical status. J Urol 1987; 137: Whitesel JA, Donohue RE, Man JH, et al. Acid phosphatase: Its influence on the management of carcinoma of the prostate. J Urol 1984;131: Brahnson RR, Catalona WJ. Adverse implications of acid phos phatase in the upper range of normal. J Urol 1987; 137: Oesterling JE, Brendler CB, Epstein Jl, et al. Correlation of clinical stage, serum prostatic acid phosphatase and preoperative Gleason grade with final pathological stage in 175 patients with clinically localized adenocarcinoma of the prostate. J Urol 1987; 138: Wang, MC, Valenzuela LA, Murphy GP, Chu TM. Purification of a human prostatic specific antigen. Invest Urol 1979;17: Oesterling JE, Chan DW, Epstein Jl, et al. Prostate specific antigen in the preoperative and postoperative evaluation of localized prostatic cancer treated with radical prostatectomy. J Urol 1988;139: ,. 30. Stamey TA, Yang N, Hay AR, et al. Prostate specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med 1987;317: Morse RM, Resnick ML. Detection of clinically occult prostate can cer. Urol Clin N Am 1990; 17: Ercole CJ, Lange PH, Mathesin M, et al. Prostate specific antigen and prostatic acid phosphatase in the monitoring and staging of patients with prostatic cancer. J Urol 1987; 138: Guinan P, Bhatti R, Ray P. An evaluation of prostate specific antigen in prostatic cancer. J Urol 1987; 137: Williams RD. Prostate specific antigen. J Urol 1988; 140: Perez CA, Fair WR, lhde DC. Carcinoma of the prostate. In: Devita VT, Hellman S, Rosenberg SA, eds. Cancer: Principles and Prac tice of Oncology. Philadelphia: JB Lippincott, 1989, pp de Voogt HJ, Dingjam P. Steroid receptors in human prostate cancer. Urol Res 1978;6: Wagner RK, Schulze KK. Clinical relevance of androgen receptor content in human prostatic carcinoma. Acta Endocrinol (Suppl) 1978;215: Trachtenberg J, Walsh PC. Correlation of prostatic nuclear androgen receptor content with duration of response and survival following hormonal therapy in advanced prostatic cancer. J Urol 1982; 127: Conolino C, Marocchi A, Margiotto G, et al. Steroid receptors and hormone responsiveness of human prostate carcinoma. Prostate 1982;3: Buttyan R, Olsson CA. Androgen receptor assay in advanced prostatic cancer. Urol Clin N Am 1984;11: Nag A, Smith RG. Amplification, rearrangement, and elevated expression of c-myc in the human prostatic carcinoma cell line LNCaP. Prostate 1989;15: Buttyan R, Sawczuk IS, Benson MC. Enhanced expression of the c-myc proto-oncogene in high-grade human prostate cancers. Prostate 1987;11: Viola MV, Fromowitz F, Oravez S, et al. Expression of ras oncogene p21 in prostate in prostate cancer. N Engl J Med 1986; 314: Vol. 97. No. 5