classification (grade I to IV) were analyzed by ... grade I tumors appeared similar. In grade II to. IV tumors ... Hospital grading system.7 The Gleason system,.
American Journal ofPathology, Vol. 150, No. 2, February 1997 Copyright X) American Society for Investigative Pathology
Short Communication Immunohistochemical Study Suggesting a Complementary Role of Kallikreins hK2 and hK3 (Prostate-Specific Antigen) in the Functional Analysis of Human Prostate Tumors
Roland R. Tremblay,* David Deperthes,* Bernard Tdtu,t and Jean Y. Dube* From the Department of Medicinel and Pathology,t Laval University, Quebec, Canada
The development of monoclonal antibodies directed against prostatic kaUikrein hK2 prompted us to evaluate its content, along witb that of bK3 (prostate-specific antigen), in buman prostate carcinoma. Seventy tumors categorized according to the M.D. Anderson Hospital classification (grade I to IV) were analyzed by immunohistocbemistry. The staining intensity or the kaUikrein content of benign prostatic byperplasia glandular tissue (used as control) and of grade I tumors appeared similar. In grade II to IV tumors, histochemical data revealed highly variable hK2 or hK3 content in approximately 25% of tumors. Such patterns are consistent with a current observation related to heterogeneity of prostate tumors. In addition, a few tumors did not express bK3 (n = 3), hK2 (n = 3), or botb (n = 3), indicating that some growth patterns of prostatic neoplasia are associated witb a lack of secretion or storage of hK3 or hK2for immunodetection. This statement also appears relevant to metastases. It was interesting to note that 4% of hK3-negative tumors had detectable hK2. Because of the importance of hK3 as a serum marker of prostate disorder, this study addresses for the first time the question of the relative importance of both hK3 and hK2 in the immunohistocbemical diagnosis of prostatic tumors. We conclude that hK2 may add new infor-
mation to prostate cancer diagnosis and cbaracterization. (Am JPathol 1997, 150:455-459)
Prostate-specific antigen (PSA or hK3) and its cousin protein (hK2) are both glycoproteins produced by the glandular cells of the human prostate. Their corresponding mRNA levels have been assessed by Northern blot analysis1 2 and by in situ hybridization3 in prostatic tissue. The hK2 mRNA estimates vary from 10 to 50% when hK3 mRNA is taken as reference.4 This observation could be indicative of significant variations of the enzymatic proteins either in the prostate gland or in biological fluids, such as seminal plasma or serum. The presence of a signal peptide5 associated with each kallikrein is consistent with the belief that hK2, to the same extent as hK3, is secreted by prostatic epithelial cells and released in the circulation. It is unknown, however, whether these gene products are subject to common regulatory mechanisms. After several years of effort, our group isolated the native hK2 protein6; this advance was associated with the development of specific monoclonal antibodies that allowed us to determine hK2 content in human prostatic cytosol and in seminal plasma. This study was undertaken to evaluate the correlation between tumor differentiation and the expresSupported by the Medical Research Council of Canada (MT-13416). Accepted for publication October 9, 1996. Address reprint requests to Dr. Roland R. Tremblay, Centre Hospital, Universit6 de Qu6bec, Pavilion CHUL, Hormonal Bioregulation Lab, Room T3-67m, 2705 Laurier Boulevard, Sainte-Foy, Qu6bec, Canada G1V 4G2.
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sion of both hK2 and hK3 in prostatic carcinoma and tumor metastases, but the main focus was to evaluate the respective contributions of either hK2 or hK3 to the identification of the remaining glandular tissue present in prostatic tumors. To some extent, this work contributes to a functional analysis of these tumors.
Materials and Methods A total of 70 prostatic carcinomas resected between 1970 and 1980 by various surgical approaches were selected from the files of the Pathology Department of l'Hotel-Dieu de Quebec. Blocks of tissues from these 70 tumors were available and constitute the basis of this study. All tumors were fixed in Bouin's fluid, embedded in paraffin, and stained with conventional hematoxylin and eosin (H&E). The sections were reviewed by one of us (B. Tetu), and each tumor was graded according to the M.D. Anderson Hospital grading system.7 The Gleason system, which recognizes five histological patterns of prostate adenocarcinoma, was not available for the purpose of this retrospective study. Two closely related histological sections of each block were deparaffinized in xylene, rehydrated in graded alcohols, and soaked in 1 % hydrogen peroxide in absolute methyl alcohol for 30 minutes to block endogenous peroxidase. After several washes in phosphate-buffered saline, the slides were incubated with monoclonal antibodies developed against hK3 (1A1 and 1A3) and hK2 (8A3) provided by Immunova (Quebec City, Canada). Commercial Vectastain ABC kits were subsequently used (Dimension Laboratories, Mississauga, Ontario, Canada), and the immunoperoxidase reaction was visualized by developing in
3-amino-9-ethylcarbazole. The specificity of both antibodies was rigorously tested; no cross-reactivity was observed with kallikrein hKl, and the specificity of the immunoreaction was verified by the staining of positive and negative control tissue sections in each assay. A negative control section was also included for each case in which the primary antibody (anti-hK3, anti-hK2) was substituted with nonimmune serum. Immunostaining for both kallikreins was evaluated on benign hyperplastic (BPH), well differentiated malignant (gland-forming) tissue, and poorly differentiated malignant (non-gland-forming) prostatic tissue. BPH was used as the positive control in each assay. Ten different specimens obtained by transurethral resection were treated along with
the tumors. Staining intensity was evaluated semiquantitatively by a scale of four (0, +, ++, and +++). Tumors with fewer than 10% positive cells were considered negative (rating 0). Clinical findings were obtained from patients' charts and included age, type of treatment, and survival. These data were previously published8 at the time of a similar study conducted with Zn-a2-glycoprotein and a prostatic secretory protein (PSP-94) of 94 amino acids. The patients ranged in age from 51 to 93 years (mean, 70.9 years); data were available for 68 patients in this study.
Results Grading of Tumors and Kallikrein Expression The grades of the 70 tumors and the corresponding immunostaining patterns for hK3 and hK2 provided general information. Grade I tumors (n = 15) presented a rather uniform pattern of staining for both kallikreins. Staining intensity was optimal (+ + +) and almost comparable to that observed when glandular tissue of BPH was used for comparison (Figure 1, A and B). Grade 11 (n = 27), grade Ill (n = 8), and grade IV (n = 18) tumors showed the whole spectrum of staining intensity for hK3 and hK2. Assessment of the immunoperoxidase reaction was performed by two different investigators.(R. R. Tremblay and J. Y. Dub6), and the rating of 0 to +++ was generally convergent.
Differential Expression of Kallikreins hK2 and hK3 in Prostatic Tumors Globally, the recourse to two sets of antibodies in evaluating tumor expression of both kallikreins was rewarding. In grade tumors, tissue expression of hK3 and hK2 was quite comparable according to our semiquantitative four-scale evaluation (Figure 1 C, hK3; Figure 1D, hK2). Starting with grade 11 tumors, similarities in staining intensity decreased to 66% (grade 11), 75% (grade 111), and 72%. (grade IV). Staining patterns were thus highly variable in 25% of the tumors. For example, some tumors (n = 3) were hK3 positive and hK2 negative; a mirror image also was observed with three other tumors. Going from one extreme to the other, it was illustrated that a given tumor may be positive for hK3 (Figure 1 E) and only slightly positive for hK2 (Figure 1 F), whereas another tumor was hK3 negative (Figure 1 G) but hK2 positive (Figure 1 H). Of the 70 tumors, 3 showed no staining for either hK3 or hK2 (Table 1). Similar
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hK3 STAINING (Panel ACEG)
hK2 STAINING
(Panel BDFH)
1z B
'0*46aj -.
i.,
A.'
.-FeA_
Figure 1. Typical examples illustrating thepresence or absence of kallikreins in prostatic epithelial cells. A, C, E, and G: Reaction with antibodies lAI and 1A3 directed against hK3 orPSA. B, D, F, and H: Staining with antibody 8A3 developed against hK2. A diffuse staining with either hK3 (A) or hK2 (B) characterizes the exocrine secretory activity, which is typical of benign prostatic hyperplasia and of normal prostatic epithelium. C and D: Both entities from agradeIVtumorare composed ofa tightly packed cluster of small glands the staining of which corresponds to + + for hK3 (C) and +++ for hK2 (D). E and F: Glands from a
.7
.Z,.G -W..'
1-
grade II tumor appear largely single with evidence of stratification of luminal cell layers. hK3 staining is very intense (E); hK2 staining is generally weak (+) or negative (F). G and H: Small glandular-like structures of a grade III tumor do not elaborate hK3 according to the absence of staining illustrated in G, whereas many cells are highly positive for hK2 (H).
trends were observed with metastases to 8 different organs obtained at the autopsies of patients who died of invasive grade IV tumors. Negative results were associated with the dedifferentiation process, which occurs within certain metastases.
Discussion With the exception of one case report illustrating the simultaneous presence of both hK3 and hK2 in the prostatic tissue of a cystic ovarian teratoma,9 this
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Table 1. Each Tumor Showing Discrepancy in hK3 and hK2 Expression or a Lack of Kallikrein Expression Is Correlated with the MDAH Grading System Tumor 1 2 3 4 5 6 7 8 9
hK3
hK2
+
+
+
Grading
III III 11 IV IV III IV IV IV
MDAH, M.D. Anderson Hospital.
work presents the first immunohistochemical study using monoclonal antibodies directed against hK3 and hK2 in cancerous prostatic tissue. Such a comparative work was made possible by the isolation of native hK2 in human seminal fluid and development of highly specific antibodies that recognized hK2 with a minimum of cross-reactivity (
