The Prostate 74:217^224 (2014)

Early Over-Expression of GRP Receptors in Prostatic Carcinogenesis Meike Körner, Beatrice Waser, Ruth Rehmann, and Jean Claude Reubi* Division of Cell Biologyand Experimental Cancer Research, Institute of Pathology ofthe University of Berne, Berne, Switzerland

BACKGROUND. The GRP receptor shows high over-expression in prostatic adenocarcinoma and high grade PIN, but low expression in normal prostate glands. This represents the molecular basis for GRP receptor imaging of prostate cancer with radioactive compounds. However, a focal, high density GRP receptor expression can be observed in hitherto uncharacterized prostate glands. METHODS. GRP receptors were quantitatively measured with in vitro receptor autoradiography using 125I-Tyr4-bombesin in samples from 115 prostates. On successive tissue sections, 125 I-Tyr4-bombesin autoradiography was compared with H&E staining and MIB-1 and 34bE12 immunohistochemistry. RESULTS. On one hand, it was confirmed that GRP receptors were expressed in adenocarcinoma and high grade PIN in high density and high incidence (77% and 73%, respectively), but in normal prostate glands in low density and low frequency (18%). On the other hand, a novel and intriguing observation was the existence of focal non-invasive prostate glands with high GRP receptor density, characterized by low grade nuclear atypia and increased proliferation, compatible with lower grade PIN. There was a significant GRP receptor density gradient (P
0.005), increasing from normal prostate glands (mean relative optical density, ROD, of 125 I-Tyr4-bombesin binding: 0.17) over atypical glands without increased MIB-1 labeling (0.28) and atypical glands with increased MIB-1 expression (0.44) to high grade PIN and adenocarcinoma (0.64 and 0.58, respectively). CONCLUSIONS. GRP receptor over-expression may be a novel, specific marker of early prostatic neoplastic transformation, arising in low grade PIN, and progressively increasing during malignant progression. This should be considered when interpreting in vivo GRP receptor imaging in males. Prostate 74:217–224, 2014. # 2013 Wiley Periodicals, Inc. KEY WORDS: bombesin receptor; GRP receptor; intraepithelial neoplasia; in vitro receptor autoradiography

INTRODUCTION Many peptide hormone receptors are highly overexpressed in human cancer. They represent molecular tumor targets of increasing clinical importance [1]. Indeed, they either allow pharmacological interference with effects of peptide hormones on tumor cells using receptor agonists or antagonists. Or they can serve as targets for radiolabelled peptide analogs for nuclear imaging and radiotherapy of tumors. The prime example for such clinical applications is somatostatin receptor targeting of neuroendocrine tumors of the gastroenteropancreatic tract [2]. Stable somatostatin analogs like octreotide effectively inhibit hormone secretion from ß 2013 Wiley Periodicals, Inc.

prostate

cancer;

prostatic

neuroendocrine tumor cells and improve related symptoms. Moreover, nuclear imaging with 111In-labeled octreotide (Octreoscan) is highly effective in the radiologic localization of these tumors and currently represents a routine imaging method for this purpose [3]. PET 

Correspondence to: Jean Claude Reubi, MD, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology of the University of Berne, Murtenstrasse 31, CH—3010, Berne, Switzerland. E-mail: [email protected] Received 15 July 2013; Accepted 18 September 2013 DOI 10.1002/pros.22743 Published online 22 October 2013 in Wiley Online Library (wileyonlinelibrary.com).

218

Ko¨rner et al.

scanning with 68Ga-labelled somatostatin analogs emerges to be even superior to Octreoscan with respect to sensitivity and specificity [4]. Finally, radionuclide therapy with 90Y- or 177Lu-labeled octreotide derivatives extents survival of patients affected by an advanced neuroendocrine tumor [5,6]. Another important peptide hormone receptor in the field of receptor-targeted tumor imaging and radiotherapy is the bombesin type 2 (BB2) receptor, also called gastrin-releasing peptide (GRP) receptor [7]. It is a member of the bombesin receptor family with high affinity for its natural ligands GRP, a mammalian peptide, and bombesin, a toad peptide. The GRP receptor is highly over-expressed in various human tumors, including prostate and breast cancer, gliomas and gastrointestinal stromal tumors [8–11], where it may stimulate tumor cell proliferation via autocrine pathways [7,12]. Irrespective of its biologic role, the high tumoral GRP receptor over-expression is the basis for promising clinical applications with radiolabeled GRP or bombesin analogs, similar to somatostatin receptor targeting of gut neuroendocrine tumors. Analogs suitable for such purposes are currently developed and have already been tested in patients in preliminary clinical trials [13]. For instance, using 99m Tc-labeled bombesin, it was possible to visualize primaries and lymph node metastases of prostate cancers that were not identified with routine radiologic techniques [14]. Similarly, breast cancer primaries and metastases could be detected with another 99mTclabeled bombesin analog [15]. 64Cu- or 68Ga-labeled bombesin derivatives for PET scanning are also promising, having been shown to be superior to 18F-FDG PET in patients with GRP receptor-positive recurrent gliomas [16,17]. Furthermore, an increasing number of GRP receptor agonists coupled with 177Lu are available, like 177Lu-AMBA or 177Lu-DOTA-PESIN [18,19]. Although a 177Lu-labeled bombesin derivative showed significant tumor effects in an in vivo mouse model of prostate cancer [20], similar positive results could not yet be achieved in cancer patients [21]. Of note, GRP receptor antagonists may be better suited for tumor targeting than agonists. Indeed, several different radiolabeled GRP receptor antagonists, such as demobesin 1, RM1 and RM2, were found to exhibit in vivo tumor targeting characteristics clearly superior to those of current agonists [22–24]. While the clinical exploration of GRP receptor-targeting of tumors advances, basic studies on the GRP receptor expression in organs of interest, which represent the foundation of clinical development, have made only little progress since the first publications more than a decade ago [8,9]. With the increased application of GRP receptor tumor imaging, it becomes now important to more thoroughly know the GRP receptor expression in The Prostate

target organs. This is essential for the interpretation of imaging results. As for the prostate, it was reported that GRP receptors were highly expressed not only in invasive cancer, but also in prostatic intraepithelial neoplasia (PIN) [8]. PIN encompasses a spectrum of preinvasive proliferative and atypical epithelial changes of increasing severity, subdivided initially into grades 1 to 3 and then into low and high grade, where high grade PIN represents an established precursor of invasive prostate cancer [25,26]. Initially, a high GRP receptor overexpression was reported in PIN lesions of high degree [8]. In the meantime, we have observed a prominent GRP receptor over-expression, sufficient to potentially yield a positive GRP receptor imaging result, in additional prostatic glandular structures distinct from the high grade PIN lesions described in the original publication. These glands have not been characterized so far. Therefore, the aim of the present study was to assess the different glandular compartments of the human prostate exhibiting a GRP over-expression with in vitro receptor autoradiography, light microscopy, and immunohistochemistry. MATERIALS AND METHODS Prostate Specimens Frozen prostate tissue samples were obtained from 115 prostatectomy specimens removed for prostate cancer, prostatic hyperplasia, or urinary bladder cancer. The samples were collected at the Institute of Pathology of the University of Berne in accordance with international ethical guidelines, including informed consent and approval by the institutional review board. The samples were stored at 80°C. Consecutive frozen tissue sections were cut from the prostate samples, mounted on glass slides and alternately subjected to in vitro receptor autoradiography, hematoxylin and eosin (H&E) staining and immunohistochemistry. This allowed a direct comparison of the results of the three procedures in the same histologic structures of the prostate. This required, however, that the serial tissue sections for all three procedures were cut at a constant thickness, to avoid loss of intervening tissue during sectioning. A thickness of 10 mm was chosen, as this permitted both sufficient radioligand binding in the autoradiography experiments and adequate morphology in the H&E and immunohistochemical stainings. InVitro GRP Receptor Autoradiography In vitro GRP receptor autoradiography was carried out as described before using the radioligand 125I-[Tyr4]bombesin [8]. Briefly, tissue sections were incubated first

GRP Receptor in Prostate Carcinogenesis in 10 mM HEPES buffer (pH 7.4) for 5 min at room temperature and then in the incubation solution containing 10 mM HEPES buffer, 130 mM NaCl, 4.7 mM KCl, 5 mM MgCl2, 1 mM ethyleneglycol-bis (b-aminoethylether)-N-N0 -tetraacetic acid, 0.1% BSA, 100 mg/ml bacitracin (pH 7.4), and 100 pM 125I-[Tyr4]-bombesin-14 (2,000 Ci/mmol; Anawa, Wangen, Switzerland) in the presence or absence of 1 mM cold bombesin for 1 hr at room temperature. For the pharmacological characterization of GRP receptors, additional serial tissue sections were incubated with the incubation solution containing increasing concentrations of cold GRP, neuromedin B (NMB) or [Tyr3]-octreotide. Afterwards, the slides were washed in ice-cold HEPES buffer containing 0.1% BSA and rinsed in ice-cold water. They were then exposed to Kodak films Biomax MR® for 7 days at 4°C. 125I-[Tyr4]bombesin binding to the tissues was analyzed in correlation with morphology using corresponding serial tissue sections stained either with H&E or immunohistochemically for MIB-1 or 34bE12. Specific 125I-[Tyr4]-bombesin binding to the tissues was quantitatively measured as relative optical density (ROD) using a computer-assisted imaging system (Interfocus, Mering, Germany). ROD levels were not converted into the measure dpm/mg tissue, as no calibration system is available for the conversion of ROD levels obtained for 125I-binding to 10 mm-thick tissue sections into dpm/mg tissue. MIB-1and 34bE12 Immunohistochemistry Immunohistochemistry for MIB-1 and 34bE12 was performed on tissue sections immediately adjacent to those subjected to autoradiography. These tissue sections were fixed in acetone and postfixed in 4% formalin. The primary antibody was either a monoclonal MIB-1 antibody (1:50; Dako, Glostrup, Denmark) or a monoclonal 34bE12 antibody (1:50; Dako). The secondary antibody was a biotinylated goat antimouse immunoglobulin (1:300; Dako). Antibody binding was visualized using the ABComplex Elite (Vector Laboratories, Burlingame, CA). Staining was carried out with 3,30 diaminobenzidine, and counterstaining with hemalum.

for their GRP receptor expression using [125I]-Tyr4bombesin autoradiography in conjunction with immunohistochemistry for the proliferation marker MIB-1 and the basal cell marker 34bE12. Samples containing adenocarcinoma as well as samples taken at a distance from invasive cancer were studied. Thus, GRP receptors were found to be expressed in diverse prostatic glandular epithelial structures exhibiting varying degrees of proliferation, atypia and malignancy. This is summarized in Table I and illustrated with typical examples in Figures 1 and 2. A high GRP receptor expression was present in prostatic invasive adenocarcinoma of acinar type (Fig. 1A–C), which was characterized histologically by glandular epithelial structures with invasive architecture, high grade nuclear atypia, high proliferation by MIB-1 immunohistochemistry, and absence of a 34bE12-positive basal cell layer (Fig. 2A–D). As expected [8,9], invasive adenocarcinoma expressed GRP receptors in high incidence, namely in 46 of 60 cases (76.7%), and usually in very high density (mean ROD of [125I]-Tyr4-bombesin binding: 0.58). Of note, GRP receptors often showed a heterogeneous distribution within invasive adenocarcinoma. They were either strongly expressed at the tumor periphery, while present in only small amounts or even absent in the central tumor portion, or vice versa exhibited high expression in the tumor center but were absent at the invasive tumor front. High grade PIN showed a similarly strong GRP receptor expression (Fig. 1D–F). High grade PIN was defined morphologically by non-invasive glands surrounded by 34bE12-positive basal cells and lined by epithelial cells with evidence of prominently increased proliferation and significant atypia [25] (Fig. 2E–H). Excessive proliferation was reflected by a conspicuous

TABLE I. GRP Receptor Expression in Different Prostatic Glandular Epithelial Structures

Na

Statistics For the statistical analysis, the Student’s t-test was used. P < 0.05 was considered statistically significant. RESULTS GRP Receptor Expression in Diverse Prostatic Glandular Structures Tissue samples of 115 prostate glands, 110 of which harbored proven invasive cancer, were investigated

219

Prostatic invasive 46 adenocarcinoma High grade PIN 40 Atypical prostate glands: MIB-1 expression 32 increased MIB-1 expression 28 not increased Normal glands 20 a

GRP receptor density (relative optical density ROD, mean  SEM) 0.58  0.04 0.64  0.04 0.44  0.04 0.28  0.03 0.17  0.02

Only receptor positive cases. The Prostate

220

Ko¨rner et al.

Fig. 1. In vitro GRP receptor autoradiography on serial tissue sections of an invasive prostate adenocarcinoma (A^C), high grade PIN (D ^F ), atypical prostate glands with increased MIB-1 expression (G ^I), atypical prostate glands without increased MIB-1 expression (K^M) and normal prostate glands (N ^P). The rectangles indicate the areas which are displayed at higher magnification in Figure 2. A, D,G, K, N: H&E-stained tissue sections showing invasive adenocarcinoma (A; asterisks), high grade PIN (D; asterisk), a large area with atypical prostate glands (G; asterisks), a small focus of atypical prostate glands (K; arrow), and normal prostate glands (N; asterisks). Bars indicate1mm. B, E, H, L,O: Autoradiograms showing total binding of 125I-[Tyr4 ]-bombesin. There is strong binding to invasive adenocarcinoma (B) and high grade PIN (E) as well as moderate binding to atypical prostate glands (H, L), but no binding to normal prostate glands (O).C, F, I, M, P: Autoradiograms showing non-specific binding of 125I-[Tyr4 ]-bombesin in the presence of1 mm cold bombesin.Complete displacement of 125I[Tyr4 ]-bombesin by excess cold bombesin in all instances provides proof of specificity of 125I-[Tyr4 ]-bombesin for bombesin receptors.

increase in cellularity with prominent intraluminal cell proliferates as well as a marked fraction of MIB-1 positive cells, similar to that in invasive adenocarcinoma. The estimated fraction of MIB-1 positive glandular epithelial cells usually exceeded 10%. However, an exact percentage of MIB-1 positive cells could not be obtained as the nuclear overlap in the thick frozen tissue sections did not allow a reliable enumeration. Nuclear atypia included prominent nuclear enlargeThe Prostate

Fig. 2. Serial tissue sections showing at higher magnification the morphological and immunohistochemical characteristics of the areas marked with rectangles in Figure1. A, E, I,N, R: H&E tissue sections showing invasive adenocarcinoma (A), high grade PIN (E), atypical prostate glands with increased cellularity (I, N) and a normal prostate gland with low cellularity (R). Bars indicate 0.1mm. The rectanglesindicate the areas shown at high magnification in the lastcolumn of Figure 2.B,F,K,O, S: MIB-1immunohistochemistry. Cells with nuclear staining (brown) are numerous in invasive carcinoma (B) and high grade PIN (F), infrequent in the atypical gland in K and virtually absent in the atypical gland in O and in the normal prostate gland (S).C,G, L, P,T: 34bE12 immunohistochemistry. A basal cell layer (brown staining) is absent in invasive adenocarcinoma (C) and present in high grade PIN (G), atypical prostate glands (L, P) and the normal prostate gland (T). D, H, M, Q, U: H&Estained sections showing the areas marked with rectangles in the first column of Figure 2 at high magnification under oil immersion. The nuclei of invasive adenocarcinoma (D), high grade PIN (H), and atypical prostate glands (M, Q) show varying degrees of atypia with increase in size and irregularity. Conversely, the nuclei of normalprostate glands (U) devoid of atypia are small andround.

GRP Receptor in Prostate Carcinogenesis ment, hyperchromasia, pleomorphism, and irregular contours, while nucleoli were barely visible in the frozen tissue sections. Thus defined high grade PIN lesions expressed GRP receptors in high incidence (40 of 55 cases; 72.7%) and very high density (mean ROD of [125I]-Tyr4-bombesin binding: 0.64). The GRP receptor expression was sometimes heterogeneous, with receptor positive and negative areas next to each other. In contrast, GRP receptors were absent or only weakly expressed in normal prostate glands which exhibited low cellularity, no or only single MIB-1 positive cells per glandular cross-section and no atypia (Fig. 1N–P, Fig. 2R–U). Receptors were identified in these areas in 20 of 111 samples (18.0%) in only low density (mean ROD of [125I]-Tyr4-bombesin binding: 0.17). Novel data of particular interest were, in 53 prostates, a focal high density GRP receptor expression observed in non-invasive glandular epithelial structures which showed histomorphologic characteristics distinct from those of the high grade PIN lesions and normal prostate glands. Specifically, these glands exhibited proliferative and nuclear atypical changes of mild to moderate degree not reaching the extent of the qualitatively similar alterations in the high grade PIN lesions (Fig. 2I–Q). Excessive proliferation was reflected by increased cellularity with nuclear overlap and little tufting. The fraction of MIB-1-positive cells was in comparison with the surrounding normal prostate glands either equally low, comprising no or only isolated cells per glandular cross-section (n ¼ 28), or slightly increased (n ¼ 32). In the latter case, MIB-1 positivity affected more than only single cells but less than 10% of glandular cross-sections, thus not reaching the same extent as in high grade PIN lesions. Atypical nuclei showed enlargement and hyperchromasia. Through increased cellularity and nuclear atypia, the affected glands exhibited a characteristic blue appearance contrasting with the surrounding normal prostate glands. A 34bE12-positive basal cell layer was always present, excluding invasion. The GRP receptor density in these atypical glands was moderate to high (Fig. 1G–M): The mean ROD of [125I]-Tyr4-bombesin binding amounted to 0.28 in the absence of increased MIB1 expression and to 0.44 when MIB-1 labeling was increased. Of importance, the GRP receptor overexpressing atypical glands often showed a focal distribution. They usually comprised a small collection of less than ten glands and only rarely covered a larger area. Moreover, the GRP receptor expression in these atypical glands appeared to be heterogeneous: atypical glands with and without GRP receptor expression were often found next to each other. Of interest, these glands were also identified in the few cases without evidence of invasive prostate cancer.

221

Increasing GRP Receptor Expression Across a Morphologic Spectrum of Prostatic Glandular Epithelial Atypia and Proliferation The GRP receptor density levels differed significantly between the various prostatic glandular epithelial structures. This is demonstrated graphically in Figure 3, which shows the 125I-[Tyr4]-bombesin binding levels for each individual case. Figure 3 reveals three main findings. First, the mean GRP receptor density levels were increased in all types of abnormal glands, including the atypical blue glands, high grade PIN lesions, and invasive carcinomas, in comparison with morphologically normal prostate glands. Second, mean GRP receptor density levels gradually and highly significantly increased from normal prostate glands over atypical glands without increased MIB-1 expression and atypical glands with increased MIB-1 labeling to high grade PIN lesions (P
0.005). Conversely, the mean GRP receptor density was slightly lower in invasive adenocarcinoma than in high grade PIN, which was, however, statistically not significant (P ¼ 0.256). Third, as for the individual cases, the vast majority of atypical glands with increased MIB-1

Fig. 3. 125I-[Tyr4 ]-bombesinbinding densitylevels in theindividual cases of normal prostate glands, atypical prostate glands without and with increased MIB-1 expression, high grade PIN and invasive adenocarcinoma.The bars indicate the mean values. ROD values of 125 I-[Tyr4 ]-bombesinbindingdensity above 0.3 are considered to reflect GRP receptor amounts potentially high enough for a positive GRP receptor imaging.The 125I-[Tyr4 ]-bombesin binding density significantly increases from normal prostate glands over atypical prostate glands to high grade PINandinvasive carcinoma. The Prostate

222

Ko¨rner et al.

expression, high grade PIN lesions, and invasive carcinomas as well as a smaller fraction of atypical glands without increased MIB-1 expression exhibited ROD values of 125I-[Tyr4]-bombesin binding above 0.3. On the contrary, ROD levels in normal prostate glands were almost always below 0.3. A ROD of 0.3 or more is considered to correspond to a high GRP receptor density, expected to be potentially sufficient for a positive in vivo GRP receptor imaging, based on extensive experience with somatostatin receptor imaging [27]. Pharmacological Characterization of GRP Receptors in the Atypical Prostate Glands In order to provide evidence that the radioligand I-[Tyr4]-bombesin, which is known to bind GRP (BB2) as well as NMB (BB1) receptors, specifically detected GRP receptors in the atypical prostate glands, pharmacological displacement experiments were performed using various cold receptor ligands in competition with 125I-[Tyr4]-bombesin. In three different cases, 125 I-[Tyr4]-bombesin was displaced by BB2 receptorpreferring GRP in the nanomolar concentration range (mean IC50 1.367 nM), but by BB1 receptor-preferring NMB in the micromolar concentration range (mean IC50 1.659 mM), that is, with 1,000 times lower affinity (Fig. 4). Moreover, [Tyr3]-octreotide, a ligand for somatostatin receptors which are unrelated to the bombesin receptor family, did virtually not displace 125 I-[Tyr4]-bombesin. These results provide strong pharmacological proof that the receptors identified with 125I-[Tyr4]-bombesin in the atypical glands largely correspond to high affinity GRP receptors [7,9]. 125

Fig. 4. Pharmacological competition experiments in the atypical prostate glands. Values represent means  SEM of three different cases.High affinitydisplacementof 125I-[Tyr4 ]-bombesinby GRP (*), butlow affinityor no displacementby NMB (&) or [Tyr3]-octreotide (~). The Prostate

DISCUSSION The GRP receptor is an established marker of prostate cancer. It is well-known to be highly overexpressed in invasive adenocarcinoma as well as in high grade PIN, that is, in the most atypical end of the PIN spectrum, while it shows only little expression in the normal prostate [8,9]. In the present study, using in vitro GRP receptor autoradiography, additional prostatic glandular epithelial structures were identified which over-express GRP receptors at intermediate levels. These glandular structures were characterized morphologically and immunohistochemically by increased proliferation and nuclear atypia of low to moderate degree as well as absence of invasion. Of particular interest is now the emergence of a significant progressive increase of GRP receptor expression levels across a morphologic spectrum of prostatic epithelial atypia, proliferation, and malignancy, ranging from normal prostate glands with low GRP receptor expression over slightly to moderately atypical glands with intermediate GRP receptor levels to severe PIN and invasive adenocarcinoma with high GRP receptor over-expression. 125 I-[Tyr4]-bombesin autoradiography has previously been demonstrated to specifically identify the GRP receptor in invasive prostate cancer and high grade PIN [8,9]. In the present study, evidence had to be provided that it recognized the same receptor type also in the glands with atypia of lower degree. In fact, 125I[Tyr4]-bombesin is known to bind preferentially to GRP (BB2) receptors, but also to a smaller degree to NMB (BB1) receptors, while it exhibits no affinity for BB3 receptors [9]. Therefore, pharmacological competition experiments were performed in the atypical prostate glands with the GRP receptor preferring ligand GRP and the BB1 receptor preferring ligand NMB [7]. The receptors identified with 125I-[Tyr4]-bombesin showed very high affinity for GRP, but only low affinity for NMB. This rank order of potencies of different bombesin receptor ligands at the receptors provides strong pharmacological proof of a predominant GRP receptor expression in the atypical prostate glands. In vitro receptor autoradiography using 125I-[Tyr4]bombesin is thus an excellent method to study GRP receptors in prostate tissue samples. It is well-characterized, highly specific, and sensitive as well as quantifiable, in contrast to immunohistochemistry for bombesin receptors. Moreover, receptor autoradiography is morphology-based. In particular, it can be combined with H&E and immunohistochemical stainings performed on consecutive tissue sections in order to characterize the autoradiographically positive structures. This kind of combination of receptor autoradiography with tissue stainings has already been

GRP Receptor in Prostate Carcinogenesis

223

successfully performed before for other peptide receptors in other organs [28]. It requires, however, that the histologic stainings are carried out on frozen tissue, since autoradiography cannot be performed on formalin-fixed, paraffin-embedded samples, and on relatively thick tissue sections of 10 mm thickness. Morphologic details in H&E-stained frozen tissue sections are limited. In particular, nucleoli, an important distinguishing feature in PIN [25], are barely visible. Furthermore, the use of frozen tissue restricts the use of immunohistochemical antibodies. In the present case, MIB-1 and 34bE12 worked well, while for instance CK5/6, p63, and AMACR did not yield satisfactory results and could therefore not be used in adjunct tests. Finally, the fraction of MIB-1 positive glandular epithelial cells can only be estimated, while an exact percentage cannot be obtained by counting positive and negative cells due to substantial nuclear overlap and difficulties in distinguishing single cells in the thick frozen tissue sections. The combination of GRP receptor autoradiography with H&E staining and immunohistochemistry revealed three distinctive morphologic features of the GRP receptor expressing atypical prostate glands, namely (1) signs of increased proliferation, consisting in increased cellularity and variable increase in MIB-1 expression, (2) nuclear atypia, and (3) normal glandular architecture without evidence of invasion. The atypical glands thus fulfill the diagnostic criteria of PIN, which is defined morphologically by architecturally benign glands lined by cytologically atypical epithelia [25]. In the spectrum of PIN, which exhibits proliferative and atypical changes of increasing severity, the atypical blue glands correspond to low to intermediate grade lesions, whereas the glands designated high grade PIN reflect the most severe end. In fact, according to the original PIN classification, the atypical glands would best be categorized as PIN 1 and 2 and the lesions termed high grade PIN as PIN 3. This division is particularly supported by the differential MIB-1 expression, which is low in the atypical glands and highly increased in high grade PIN lesions [29,30]. Conversely, the presence of nucleoli cannot be used as a criterion distinguishing between low and high grade PIN in the present study due to the use of frozen tissue [25].

ty [26], may be facilitated by an adequate immunohistochemical anti-GRP receptor antibody, once such an antibody becomes available. Moreover, the progressive rise of GRP receptor expression levels in PIN of increasing severity, in parallel with increasing MIB-1 levels, suggests that the GRP receptor may be not only a marker, but potentially also a driver of prostatic carcinogenesis, where its over-expression would represent an early event. In support of this hypothesis, it has been shown experimentally that activation of the GRP receptor is mitogenic in normal and tumor cells. In particular, bombesin exhibits autocrine growth stimulating effects in various cancer types [7]. With regard to the prostate, bombesin has been shown in vitro to specifically stimulate growth of human prostate cancer cell lines [31,32]. Moreover, bombesin/GRP antagonists were found to inhibit growth of invasive prostate cancer as well as of benign prostatic hyperplasia in in vivo animal models [33–35]. It will now be of interest to elucidate the potential role of bombesin and its receptor also in PIN. The GRP receptor over-expression in the prostate glands with atypia of lower degree is also of immediate practical importance, namely for the interpretation of in vivo GRP receptor imaging. Although of focal distribution, the GRP receptor levels in these glands may be sufficient to yield a positive scan result. Therefore, in the prostate, GRP receptor imaging may not be able to reliably distinguish between invasive cancer, high grade PIN or PIN lesions of lower degree and of unknown clinical significance. This is particularly important to be kept in mind when GRP receptor imaging is performed in males for an indication other than prostate cancer.

CONCLUSIONS

4. Gabriel M, Decristoforo C, Kendler D, Dobrozemsky G, Heute D, Uprimny C, Kovacs P, Von Guggenberg E, Bale R, Virgolini IJ. 68Ga-DOTA-Tyr3-octreotide PET in neuroendocrine tumors: Comparison with somatostatin receptor scintigraphy and CT. J Nucl Med 2007;48:508–518.

The present study identifies with the GRP receptor for the first time a putative reliable marker of PIN of lower degree. The GRP receptor thus emerges as a new tool to identify and study PIN of lower grade which so far has only been poorly investigated. For instance, the recognition of low grade PIN in prostate samples, which is presently afflicted with a high interobserver variabili-

REFERENCES 1. Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev 2003;24:389–427. 2. Oberg KE, Reubi JC, Kwekkeboom DJ, Krenning EP. Role of somatostatins in gastroenteropancreatic neuroendocrine tumor development and therapy. Gastroenterology 2010;139: 742–753. 3. Kwekkeboom DJ, Krenning EP, Scheidhauer K, Lewington V, Lebtahi R, Grossman A, Vitek P, Sundin A, Plöckinger U. ENETS Consensus Guidelines for the Standards of Care in Neuroendocrine Tumors: Somatostatin receptor imaging with (111)Inpentetreotide. Neuroendocrinology 2009;90:184–189.

5. Kwekkeboom DJ, de Herder WW, Kam BL, van Eijck CH, van Essen M, Kooij PP, Feelders RA, van Aken MO, Krenning EP. Treatment with the radiolabeled somatostatin analog [177 LuDOTA0, Tyr3]octreotate: Toxicity, efficacy, and survival. J Clin Oncol 2008;26:2124–2130. The Prostate

224

Ko¨rner et al.

6. Imhof A, Brunner P, Marincek N, Briel M, Schindler C, Rasch H, Mäcke HR, Rochlitz C, Müller-Brand J, Walter MA. Response, survival, and long-term toxicity after therapy with the radiolabeled somatostatin analogue [90Y-DOTA]-TOC in metastasized neuroendocrine cancers. J Clin Oncol 2011;29:2416–2423. 7. Jensen RT, Battey JF, Spindel ER, Benya RV. International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: Nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol Rev 2008;60:1–42. 8. Markwalder R, Reubi JC. Gastrin-releasing peptide receptors in the human prostate: Relation to neoplastic transformation. Cancer Res 1999;59:1152–1159. 9. Reubi JC, Wenger S, Schmuckli-Maurer J, Schaer JC, Gugger M. Bombesin receptor subtypes in human cancers: Detection with the universal radioligand (125)I-[D-TYR(6), beta-ALA(11), PHE(13), NLE(14)] bombesin (6-14). Clin Cancer Res 2002;8:1139–1146. 10. Sharif TR, Luo W, Sharif M. Functional expression of bombesin receptor in most adult and pediatric human glioblastoma cell lines; role in mitogenesis and in stimulating the mitogen-activated protein kinase pathway. Mol Cell Endocrinol 1997;130:119–130. 11. Reubi JC, Körner M, Waser B, Mazzucchelli L, Guillou L. High expression of peptide receptors as a novel target in gastrointestinal stromal tumours. Eur J Nucl Med Mol Imaging 2004;31:803–810. 12. Moody TW, Chan D, Fahrenkrug J, Jensen RT. Neuropeptides as autocrine growth factors in cancer cells. Curr Pharm Des 2003;9:495–509. 13. Ambrosini V, Fani M, Fanti S, Forrer F, Maecke HR. Radiopeptide imaging and therapy in Europe. J Nucl Med 2011;52:42S–55S. 14. De Vincentis G, Remediani S, Varvarigou AD, Di Santo G, Iori F, Laurenti C, Scopinaro F. Role of 99mTc-bombesin scan in diagnosis and staging of prostate cancer. Cancer Biother Radiopharm 2004;19:81–84.

21. Bodei L, Ferrari M, Nunn A, Llull J, Cremonesi M, Martano L, Laurora G, Scardino E, Tiberini S, Bufi G, Eaton S, de Cobelli O, Paganelli G. 177Lu-AMBA Bombesin analogue in hormone refractory prostate cancer patients: A phase I escalation study with single-cycle administrations. Eur J Nucl Med Mol Imaging 2007;34:S221. 22. Cescato R, Maina T, Nock B, Nikolopoulou A, Charalambidis D, Piccand V, Reubi JC. Bombesin receptor antagonists may be preferable to agonists for tumor targeting. J Nucl Med 2008;49:318–326. 23. Mansi R, Wang X, Forrer F, Kneifel S, Tamma ML, Waser B, Cescato R, Reubi JC, Maecke HR. Evaluation of a 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated bombesin-based radioantagonist for the labeling with singlephoton emission computed tomography, positron emission tomography, and therapeutic radionuclides. Clin Cancer Res 2009;15:5240–5249. 24. Mansi R, Wang X, Forrer F, Waser B, Cescato R, Graham K, Borkowski S, Reubi JC, Maecke HR. Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPrpositive tumours. Eur J Nucl Med Mol Imaging 2011;38:97–107. 25. Epstein JI. Precursor lesions to prostatic adenocarcinoma. Virchows Arch 2009;454(1):1–16. 26. Bostwick DG, Cheng L. Precursors of prostate cancer. Histopathology 2012;60:4–27. 27. Körner M, Waser B, Schonbrunn A, Perren A, Reubi JC. Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo Somatostatin receptor targeting. Am J Surg Pathol 2012;36:242–252. 28. Körner M, Waser B, Thalmann GN, Reubi JC. High expression of NPY receptors in the human testis. Mol Cell Endocrinol 2011;337:62–70.

15. Van de Wiele C, Phonteyne P, Pauwels P, Goethals I, Van den Broecke R, Cocquyt V, Dierckx RA. Gastrin-releasing peptide receptor imaging in human breast carcinoma versus immunohistochemistry. J Nucl Med 2008;49:260–264.

29. Häussler O, Epstein JI, Amin MB, Heitz PU, Hailemariam S. Cell proliferation, apoptosis, oncogene, and tumor suppressor gene status in adenosis with comparison to benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and cancer. Hum Pathol 1999;30:1077–1086.

16. Gornik G, Mansi R, Abiraj K, Knippen S, Grosu A, Maecke H, Weber W. Evaluation of the GRPR radioantagonist Cu-64-CBTE2A-AR-06 in mice and men. J Nucl Med 2011;52:22.

30. Helpap B. Cell kinetic studies on prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia (AAH) of the prostate. Pathol Res Pract 1995;191:904–907.

17. Dimitrakopoulou-Strauss A, Seiz M, Tuettenberg J, Schmieder K, Eisenhut M, Haberkorn U, Strauss LG. Pharmacokinetic studies of 68Ga-labeled Bombesin (68Ga-BZH3) and F-18 FDG PET in patients with recurrent gliomas and comparison to grading: Preliminary results. Clin Nucl Med 2011;36:101–108.

31. Bologna M, Festuccia C, Muzi P, Biordi L, Ciomei M. Bombesin stimulates growth of human prostatic cancer cells in vitro. Cancer 1989;63:1714–1720.

18. Lantry LE, Cappelletti E, Maddalena ME, Fox JS, Feng W, Chen J, Thomas R, Eaton SM, Bogdan NJ, Arunachalam T, Reubi JC, Raju N, Metcalfe EC, Lattuada L, Linder KE, Swenson RE, Tweedle MF, Nunn AD. 177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer. J Nucl Med 2006;47:1144–1152. 19. Zhang H, Schuhmacher J, Waser B, Wild D, Eisenhut M, Reubi JC, Maecke HR. DOTA-PESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours. Eur J Nucl Med Mol Imaging 2007;34:1198–1208. 20. Johnson CV, Shelton T, Smith CJ, Ma L, Perry MC, Volkert WA, Hoffman TJ. Evaluation of combined (177)Lu-DOTA-8-AOCBBN (7-14)NH(2) GRP receptor-targeted radiotherapy and chemotherapy in PC-3 human prostate tumor cell xenografted SCID mice. Cancer Biother Radiopharm 2006;21:155–166.

The Prostate

32. Xiao D, Qu X, Weber HC. GRP receptor-mediated immediate early gene expression and transcription factor Elk-1 activation in prostate cancer cells. Regul Pept 2002;109:141–148. 33. Rick FG, Abi-Chaker Am, Szalontay L, Perez R, Jaszberenyi M, Jayakumar AR, Shamaladevi N, Szepeshazi K, Vidaurre I, Halmos G, Krishan A, Block NL, Schally AV. Shrinkage of experimental benign prostatic hyperplasia and reduction of prostatic cell volume by a gastrin-releasing peptide antagonist. Proc Natl Acad Sci USA 2013;110:2617–2622. 34. Plonowski A, Nagy A, Schally AV, Sun B, Groot K, Halmos G. In vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue, AN-215. Int J Cancer 2000;88:652–657. 35. Stangelberger A, Schally AV, Varga JL, Zarandi M, Szepeshazi K, Armatis P, Halmos G. Inhibitory effect of antagonists of bombesin and growth hormone-releasing hormone on orthotopic and intraosseous growth and invasiveness of PC-3 human prostate cancer in nude mice. Clin Cancer Res 2005;11:49–57.