Correlation between neovascularisation and neuroendocrine differentiation in prostatic carcinoma

PATHOLOGY

"j~rj~i'h~j!~a~rl:i;

RESEARCH AND PRAcnCE

. . ,'

© Urban & Fischer Verlag hnp:llwww.urbanfischer.deJjournalylprp

'"

', . .

. "'<', .

Correlation between Neovascularisation and Neuroendocrine Differentiation in Prostatic Carcinoma* Rainer Grobholzl, Manfred H. Bohrer 2 , Michael Siegsmund 3 , Klaus-Peter Junemann 3 , Uwe Bleyl1 and Matthias Woenckhaus' ' Department of Pathology and 3Department of Urology, Ruprecht-Karls-University Heidelberg, University Hospital Mannheim, Mannheim, Germany; 'Department of Pathology, Klinikum Ludwigshafen gGmbH, Ludwigshafen, Germany

Summary Neuroendocrine (NE) differentiated tumor cells are found in almost all prostatic carcinomas. Prostatic carcinomas with a high NE ditferentiation have a poor prognosis and increased metastatic potential. A relationship between the neovascularisation density in the tumor and the metastatic potential in prostatic carcinoma is well known. NE cells and microvessels were demonstrated immunohistochemically on 102 radical prostatectomy specimens using antibodies against Chromogranin A and C034. Standard areas (7.9 mm' ) of maxi mal Chromogranin A expression and highest vascularisation were determined and topographically related by light microscopy. Area density of microvessels was evaluated morphometric ally. NE tumor cells were present in all prostatic carcinomas studied. High grade prostatic carcinomas expressed significantly more NE tumor cells and exhibited a higher neovasc ularisation than low grade carcinomas. There was significantly higher neovascularisation in high grade tumors with many, ascompared to high grade tumors with few, NE tumor cells. Poorer pathological staging correlated with increased neovascularisation and stronger NE differentiation. A topographical relationship between the area of maximal NE tumor cells and the area of highest neovascularisation was found in 80.4% of all cases. An analysis of variance revealed a large number of NE tumor cells as the only predictor of an increased neovascularisation (p = 0.0006). These observations sup-

* This research was supported by grants from the Faculty of Clinical Medicine Mannheim of the University Heidelberg. Pathol. Res. Pract. 196: 277- 284 (2000)

port the concept that increased neovascularisation is intluenced not only by poor pathological grading but also by a high NE differentiation. Key words: Neuroendocrine differentiation - Neovascularisation - Prostatic carcinoma

Introduction Neuroendocrine (endocrine-paracrine) cells, secreting various peptide hormones and biogenic amines, are components of the normal human prostate epithelium. These cells most likely regulate growth and differentiation, as well as secretory function s of the prostate [23, 39]. Neuroendocrine (NE) differentiation in prostatic carcinoma is a frequent occurrence and manifests itself in several forms. Thc most common form is the focal NE differentiation in adenocarcinoma. There is evidence that all or nearly all prostatic carcinomas show at least some focal NE differentiation 122}. In about 10% of these cases this differentiation is extensive 123]. Increased proliferati ve acti vity in non-NE tumor cells of prostatic carcinoma adjacent to NE tumor cells indicates that neurosecretory products may control proliferative acti vities {l2, 491. NE tumor cells, however, are Address for correspondence: Rainer Grobholz. Pathologisches Insti tut, Ruprecht-Karls-Uni versit.t Heidelberg, UniversiHitsklinikum Mannheim. Thcodor-Kutzer-Ufer 1-3, D-68 167 Mannheim, Germany. Tel.: +49(0)6211383 3505, Fax: +49(0)621/383 2005 . E-mail : rainer.grobholz@ path.ma.uni-heidelberg.de 0344-0338/2000/1 96/5-2 77 $12.0010

278 . R. Grobholz et al.

mostly androgen-insensitive, and do not belong to the proliferating cell compartment [9, II , 39]. Some studies showed a correlation between a high NE differentiation and increased metastatic behavior in prostatic carcinoma [1,3 , 4). Tumor growth and metastasis require neovascularisation. Newly formed vessels provide tumor cells with oxygen and nutrients as well as paracrine mediators [26/. Tumor cells, intratumoral macrophages, mast cells and endothelial cells have been found to secrete several angiogenic and angiostatic factors [26, 31, 38, 41). The balance between these regulators determines whether neovascularisation will occur or not [27). The microvessel density, a measure of tumor angiogenesis, has shown to give prognostic information in breast [46, 47}, lung [301 and prostatic carcinoma [13, 45}. A low vascular density in prostatic carcinomas correlates with a significantly longer survival time than in carcinomas with a high vascular density [36, 44}. Thus, neovascularisation has provcn to be an independent predictor of pathologic state in prostatic carcinoma [14, 36, 45). In this study we therefore investigated the relationship between the neuroendocrine differentiation and the vascularisation in prostatic adenocarcinoma.

Materials and Methods Tissue selection and l:la.\'sification: 102 prostate glands removed by radical prostatectomy between 1993 and 1995 from patients without prior therapy were sampled. The mean age of patients was 65.2 ± 6.6 years. Tissues were formalintixed in 4% buffered formalin for 24 h, followed by paraffinembedding. Sections from each tissue block were stained with hematoxyHn and eosin for tumor c1a. . sific3tion in accordance

with the combined histo- and cytomorphological malignancy grading: Ia-IUb {32J. Tumor staging was performed according to the International Union Against Cancer TNM classification [34J. Four blocks containing the tumor tissue of each carcinoma were selected and immunohi stochemically evalu· ated.

Alltibodie-,: A monoclonal antibody against Chromogranin A (Camon, Wiesbaden, FRG) was used for the demonstration of NE differentiated cells. For the detection of microvessels, a monoclonal antibody against CD34 of endothelial cells (Medae, Hamburg, FRG) was applied. ImmWlOhistochem istry: For the detection of antigenic sites we used abiotin-streptavidin-amplified indirect immunoalkaline phosphatase (A P) method. Serial sections 3 )Jm thick were cut in a HN 40 microtome (Leica, Nussloch, FRG). After deparaffinizing and rehydration, sections for the detection of microvessels were treated

with a 0.1 % trypsine (Sigma, Deisenhofen, FRG) solution (pH 7.2) for 25 min for antigen retrieval. Sections were then rinsed in deioni zed water for 5 min. The primary anti-endothelium antibody was applied and sections were incubated overnight at 4

<'Ie in a dark, humid incubation cha mber.

Pretreatment of the sections for Chromogranin A demon-

stration was unnecessary; after deparaffinizing and rehydration the primary antibody was applied for 60 min. at room temperature. Control sections were incubated with Tris buffered saline (TBS, pH 7.6) instead of the primary antibody. Sections were then rinsed in TBS for 5 min. and incubated with the secondary biotinylated antibody (Zymed, San Francisco, USA) for 10 min. Streptavidin-Iabeled AP (Zy med, San Francisco, USA) was added for 10 min. after washing in TBS for 5 min. The substrate naphtol AS-MX phosphate (Sigma, Deisenhofen, FRG) containing the chromogen Fast Red TR (Sigma, Deisenhofen , FRG) and 0.24 mglml Iveamisol (Sigma, Deisenhofen, FRG) was applied for 30 min. Finally, the sections were washed in tap water, slightly counterstained with hematoxylin and mounted in Kaiser's Glycerin Gelatine (Merck, Darmstadt, FRG). Categorization of tumors: In each case the following standard areas (7.9 mm:!) within serial sections were determined according to the "hot spot" method [36, 45 J: (I) maximal Chromogranin A expression in tumor tissue, (2) highest vascularisation in neoplastic and (3) in surrounding non-neoplastic tissue. Areas (I) and (2) were topographi cally related by conventional light microscopy and projection. Topographical relationship was subdivided into 3 categories: marked standard areas in serial sections (a) had contact, (b) were closely related (i.e., less than I di ameter of the standard area apart from each other) and (c) had no contact at all. Chromugranin A-positive tumor cells in standard areas were counted by conven tional means, NE differentiation was considered as "hi gh" if more than 30 NE tumor cells/standard area, as "low" if fewer than 30 NE tumor cells/standard area were expressed, Tumors were categorized according to the nature of NE differentiation (distribution pattern): a) tumors with indi vidually scattered NE tumor celis (no clusters), b) tumors with small clusters of NE tumor cells ($10 NE tumor cells per cluster), c) tumors with large clusters of NE tumor cells (> 10 NE tumor cells per cluster). Morphometric evaluation: Vascular density was determined with the aid of a Leitz Orthoplan microscope (Leica, Bensheim, FRG) connected to a black-and-white camera (Leica, Bensheim, FRG) following visualization on a high resolution di splay monitor (Leica, Bensheim, FRG), combined with the semiautomatic image-analyzing system Quantimed 520 (Leica, Bensheim, FRG). To measure the vascular area. a threshold level was set so that objects darker than tho selected gray scale were illuminated, distinguishing between objects of positive immunoreactivity and the counterstained hackground. The area (~m ') of all highlighted objects was measured by the computer in the designated field . Within standard areas the area of mkrovcssels in 5 fields of tumorous and the surrounding non-tumorous tissue or the same case was measured at a 200fold magnification (x20 objective and xlO ocular). The mean area of microvessels o f these 5 fields was calculated and thereafter, the area fraction (%) was determined as the mean vascular area per measure field. For the evaluation of the vascular area a computer program was used, supplied with the Quantimed 520 (Leica, Benshcim. FRG).

Neuroendocrine Differentiation and Neovasculari,"tion in Prostatic Carcinoma· 279

Statistical analysis: Arithmetic means and standard devia-

tions were calculated as descriptive parameters. The results

shown are the mean ± standard error of mean (SEM). Data were proven for normal distribution using the KolmogorovSmimov test, proof of significance between arithmetic means was performed using Student's Hest or the Wilcoxon rank sum test. Analysis of variance (ANOYA) was used to study the association between ncovascularisation . the number of

neuroendocrine tumor cells, tumor staging and tumor grading (SAS, Yersion 6.12). P-values <0.05 were considered to be significant.

Results Histological findings Histologic evaluation of primary prostatic tumors confirmed the presence of acinar adenocarcinoma. Small cell and carcinoid tumors were excluded from the study, as well as specimens with a hormonal pretreatment. Sixty-six prostatic adenocarcinomas were poorlydifferentiated (high grade) according to the grading llb!lllallilb (equivalent to Gleason score 7-10), 36 well-differentiated (low grade) according to the grading lallbflIa (equivalent to Gleason score 2-6). The staging revealed one case of pTl, 36 cases of pT2 , 58 cascs of pT3 and 6 cases of pT4.

NE differentiation Chromogranin A-expressing NE tumor cells were present in all carcinomas when 4 blocks of the main tumor bulk were studied. The cytoplasmic staining was intense and similar to that found in NE cells in adjacent benign epithelium. The mean number of NE cells was 70.5 ± 11 .2 for all 102 adenocarcinomas (mcdian 20, range 1-524). High grade carcinomas had significantly more NE tumor cells than low grade carcinomas (p < 0.05), Table I). The distribution of NE cells in tumor tissue showed 3 different patterns: a) individually scattered, b) arrangement in small and c) in large clusters (Figs. lA, 8, C). In 20 cases of low grade and 26

cases of high grade carcinomas, NE cells were scattered in an individual fashion. Small clusters of NE cells were found in 15 low gradc and 23 high grade, large clusters of NE cells only in I low grade butin 16 high grade carcinomas. Tumors expressing NE cells in a solitary patlern showed a mean NE cell number of 14.6 ± 2.2 (median: 9.5; range: 1-75), tumors with NE cells in small clusters a mean number of 82.8 ± 16.3 (median: 52, range: 5-427) and tumors with large clusters 291.3 ± 40.1 (median: 272; range 120-524), respectively (Table 3). This increase in number of NE cells was significant among all 3 groups (p < 0.0 I). Also, with an increasing pT stage, significantly more NE tumor cells per standard area were expressed (p < 0.01, Table 4). One carcinoma classified with a pTt stage was not included in the statistical evaluation . Microvessel density Microvessels staining positive for CD34 were present in variable amounts in all sections studied with an emphasis on the tumor compartment (Fig. 2). The mean area fraction (%5) of microvessels in the surrounding non-neoplastic tissue was constant and never significantly different in any group examined (Tables I, 3, 4). The mean vascular area fraction in neoplastic tissue compared to the surrounding non-neoplastic tissue was I.7 fold higher for all cases studied (p < 0.0001), 2.76% ± 0.12 versus 1.59% ± 0.07, respectively. High grade tumors did not exhibit a significantly increased neovascularisation compared to low grade carcinomas (p = 0.16; Table I). When subdividing all carcinomas in tumors with a significant NE differentiation (more than 30 NE tumor cells per standard area), a significant increase in neovasc ularisation between high grade and low grade carcinomas was detectab le (p = 0.026, Table 2). In contrast, carcinomas with a low NE differentiation (fewer than 30 NE tumor cells per standard area) did not exhibit a difference in neovascularisation between high grade and low grade carcinomas (p = 0.28, Table 2). High grade tumors wilh a high NE differentia-

Table 1. Number of neuroendocrine tumor cells and area fraction of neovascularisation in low (G Ia/lb/lla; equi vale nt to Gleason score 2-ti) and high grade carcinomas (G Ub/lllaf/llb; equivalcnt to Gleason score 7-10) per standard area (7.9 mm') of greatest neuroendocrine differentiation and highest vascular density. P-values <0.05 were considered to be significant. n = number of lumors studied.

Low grade carcinomas

High grade carcinomas

n = 36

n =66

p valuc*

NE tumor cells (N umber)

33.8 ± 6.7 **

90.5 ± l6.4

0.028

Neovascularisation (%J

2.52 ± 0.18

2.88 ± 0.16

0.1575

Vasculari~ation in surrounding

U7 + n. l ?

1/\1 ± 0.08

n.41

nOll-neoplastic tissue (%)

* Wilcoxon rank sum test, ** Mean ± SEM

280 . R. Grobholz ct a!.

Fig. 1. Chromogranin A expression in neuroendocrine tumor cells in prostatic carcinoma, indirect immuno-alkaline phosphatase method. Focal neuroendocrine (NE) differentiation in prostatic carcinoma, categorized according to the nature of NE differentiation (distribution pattern). A: Prostatic carcinoma with individually scattered NE tumor cells (no ciusters). Magnification xiOO. B: Prostatic carcinoma with small clusters of NE tumor cells (:":10 NE tumor cells per cluster). Magnification xl 00. C: Prostatic carcinoma with large clusters of NE tumor cells (> 10 NE tumor cells per cluster). Magnification x 100, Fig. 2. Expression of CD 34 in endothelial cells of microvessels in prostatic carcinoma, indirect immuno-alkaline phosphatase method. Intense neovascularisation in the tumor compartment of prostatic carcinoma. Magnification x60.

tion showed a significantly larger area fraction of microvessels than high grade tumors with a low NE differentiation (p = 0.007, Table 2). Low grade carcinomas, however, had no different neovascularisation irrespective of the amount of NE differentiation (p = 0.30, Table 2).

Looking at the distribution pattern of NE cells, an increase in neovascularisation was noted for all 3 categories: Tumors with small and large clusters had a significantly greater neovascularisation than tumors with an individually scattered distribution pattern of NE cells (p < 0.05, Table 3). A larger microvessel area frac-

Neuroendocrine Differentiation and Neovascularisation in Prostatic Carcinoma tion was found in tumors with a pT4 stage than in those with a pT3 and pT2 stage (p < 0.0 I). Between the pT2 and the pT3 stage a trend towards this increasing neovascularisation was noted which was not significant

281

(p = 0.28, Table 4). Analysis of variance of the parameters staging, NE tumor cells, high grading and low grading revealed that neovascularisation was influenced solely by the number of NE tumor cells (p = 0.0006).

Table 2. Ncovascularisation in high and low grade carcinomas with high NE differentiation (>30 NE tumor cells per standard area) and low NE differentiation «30 NE tumor cells per standard area). P-values <0.05 were considered to be significant, n = number of tumors studied.

Neovascularisation (%)

Ncovascularisation (%)

Low grade carcinomas High NE differentiation n = 14

High grade carcinomas High NE differentiation n = 32

p value*

2040 ± 0.26**

3.29 ± 0.26

0.026

Low grade carcinomas Low NE differentiation n=22

High grade carcinomas Low NE differentiation n=34

p value*

2.61 ± 0.24

2.51 ±0.17

0.28

* Wilcoxon rank sum lest, ** Mean ± SEM Table 3. Area fraction of neovascularisation in carcinomas categorized according to their NE cell distribution pattern per standard area (7.9 mm' ). A small cluster was considered to be an aggregation of ';10 NE tumor cells, a large cluster was considered to be an aggregation of > lONE cells. All NE tumor cells were counted within the standard area. Data are shown as mean ± SEM, n = number of tumors studied, p-values < 0.05 were considered to be significant. Solitary NE tumor cells only n 52

Small clusters of NE tumor cells n = 39

Large clusters of NE tumor cells n = II

14.6 ± 2.2'"

82.8 ± 16.31.3

291.3 ± 40.11.2

=

NE tumor cells (Number) Neovascularisation (%)

2049 ± O.W

2.85 ± 0.17 3

3.68 ± 0.57 1,2

Vascularisation in surrounding non-neoplastic tissue (%)

1.67 ± 0.09

1.51 ±0.12

1.53 ± 0,21

P < 0.05 versus tumors with solitary NE tumor cells only p < 0.05 versuS tumors with small clusters of NE tumor cells ., p <0.05 versus tumors with large clusters of NE tumor cells I

2

Table 4. Neovascularisation and number of NE cells per standard area (7.9 mm') in carcinomas according to the staging. Data are shown as mean ± SEM, n = number of tumors studied. p-values < 0.05 were considered to be significant.

NE tumor cells (Number)

pT2 n = 36

pT3 n =58

pT4 n=6

46.9 ± 10.6'

73.3 ± 15.2'

201.8 ± 86.61.2

Neovascularisation (%)

2.56 ± 0.18'

2.79±O.16'

3.89 ± 00411.2

Vascularisation in surrounding non-neoplastic tissue (%)

1.61 ±O.IO

1.63 ±O.IO

1.33 ± 0.22

I

P < 0.05 versus pT2 lumors, 2 p < 0.05 versus pT3 tumors. 3 p < 0.05 versus pT4 tumors

282 . R. Grobholz et aJ. Topographical relation Within serial sections, standard areas of maximal NE tumor cells and maximal vascularisation density had contact in 59 tumors (57.S%), close relation in 23 cases (22.6%) and no contact in 20 cases (19.6%) indicating a topographical relation between the NE differentiation and the neovascularisation in 80.4% of all tumors examined.

Discussion This study shows a correlation between the NE differentiation and increased neovascularisation in prostatic carcinoma. The NE differentiation correlated well with a poorer differentiation of tumors as well as with the neovascularisation and a progredient tumor staging. In serial sections a close proximity between the area of maximal NE tumor cells and maximal neovascularisaton was present in SO.4% of all cases. The at least partial NE differentiation of prostatic carcinoma is a well known phenomenon [22}. The functional role of these tumor cells is not clear yet, but there is evidence that NE tumor cells may have both a secretory and regulatory function concerning growth and angiogenesis /21, 31, 33}. Bombesin, calcitonin, parathyroid hormone-related protein and interleukin-6 have been shown to be present in tumor cells as potential stimulants, as evidenced by in vitro studies in prostatic cancer cell lines [20, 35, 42, 43]. Also, an increased proliferation within close vicinity of NE tumor cells could be demonstrated /12, 49}. Therefore, NE differentiation, whenever occurring in a considerable amount, seems to portend a poor prognosis in terms of expanding tumor growth [IS, 19,21]. Thi s concept is supported by the fact that the number of NE tumor cells was significantly higher when correlated with the grading, as shown in the present and in previous studies [5, 6}. Also, a significantly higher NE differentiation was present with a progredient pT stage. This is in contrast to the study of Aprikian et al. {3/, who could not show a correlation with the stage of disease. This may be due to the number of specimens studied, namely 31 untreated and 21 hormonally pretreated carcinomas. In 102 untreated cases studied herein a statistically significant increase in the number of NE tumor cells was observed when correlated with the pT stage. The origin of prostatic NE tumor cells is so far unkown. There is evidence that NE cells in benign prostatic glands originate from a local stem cell that differentiates to an intermediate cell type with secretory and endocrine features. This intermediate cell type may differentiate into an androgen-dependent secretory cell type or end up in a te rminal androgen-independent and postmitotic NE differentiation {S-II, 51]. In prostatic carci-

noma it is strongly suggested that NE tumor cells derive from exocrine cell types during tumor progression [S, IOJ as evidenced by the occurrence of amphicrine tumor cells expressing both endocrine and exocrine markers. The stimulus for this specific differentiation of tumor cells, however, still remains unclear. Increasi ng tumor growth and poor grading may accompany a more extensive NE differentiation due to a continuous specific differentiation. So far, no proliferative markers could be demonstrated in NE tumor cells by immunohistochemical means [11 , 12, 49 j . Therefore, this tumor cell type is considered to be "postmitotic" {II, 51}. The reason why proliferaring tumor cells end up in a postmitotic status still remains unclear. The fact that NE tumor cells occur not only individually scattered but also in large clusters, especially in high grade tumors, may indicate that, once a NE differentiation of tumor cells has occurred, a continuous stimulus is given by the altered microenvironment in the immediate vicinity of NE cells, inducing NE phenotype in exocrine tumor cells. Also, with the frequent occurrence of NE cells, an additional stimulus toward more extensive tumor proliferation and progression by paracrine secretion may be given. Studies of the prognostic value of NE cells in prostatic carcinoma have revealed controversial results. A correlation between the numher of NE cells and tumor grade as well as tumor stage andlor prognosis was not found in several studies [3, 15, 17, 40]. On the other hand, several groups have demonstrated a correlation with tumor grade {2, 7, 4S}, tumor stage {2, 37/ and/or patient survival [IS, 37]. Studies on core needle biopsies of previously untreated patients [16} and patients treated by external beam radiotherapy [50} revealed no correlation between the neuroendocrine differentiation, tumor-related survival and the histological grading. According to our results and the results of other studies [3, 21/. NE tumor cells arc focally di stributed throughout the tumor, so that core needle biopsies do not necessarily reflect the extent of neuroendocrine differentiation of the tumor. This study of 102 radical prostatectomy specimens shows acorrelation between the extent ofNE differentiation and the pathological grading as well as the stage of disease, but further follow-up studies of high grade tumors with low NE differentiation and high grade tumors with an extensive NE differentiation are needed to clarify the question of prognostic significance. It is widely accepted that tumor growth must be preceded by an increase in capillaries supplying the neoplasm {25}. In the prevascular phase little or no angiogenic activity is present in solid tumors. Despite the proliferative activity, however, the tumor cells are unable to expand the tumor population beyond a few cubic millimeters in this phase [29]. But once angiogenic factors are released in sutlicient number, the onset of

Neuroendocrine Differentiation and Neovascularisation in Prostatic Carcinoma angiogenic activity permits the rapid expansion of the tumor [25, 47]. Neovascularisaton is mediated by angiogenic growth factors, such as the vascular endothelial growth factor, basic fibroblast growth faclOr or angiogenin [28]. These factors have shown to be present in various tumors such as lung, breast and prostatic carcinoma [24, 31 , 33,38/. In this study we could show a 1.7 fold increase in microvessel density in prostatic carcinoma as compared to the surrounding non-neoplastic tissue. Moreover, a direct correlation between the areas of maximal chromogranin A expression and the area with the highest microvessel density could be found in the majority of all cases. The intensity of neovascularisation seems to be influenced by both NE differentiation and grading. The microvessel density is increased in high grade tumors with a high NE differentiation, compared to high grade tumors with a low NE differentiation. Also, a significantly higher neovascularisation was found in high grade tumors with a high NE differentiation, compared to low grade tumors with a high NE differentiation. High grade tumors also showed a stronger neovascularisation with an increasing number of NE cells. On the other hand. neovascularisation showed no changes in low grade carcinomas irrespective of the NE ditlerentiation, as well as in tumors with a low NE differentiation, irrespective of the grading. These results indicate that the intensity of neovascularisation is neither influenced by the grading alone nor by the NE differentiation alone, but rather by a combination of an extensive NE differentiation combined with a poor pathological grading. Analysis of variance pointed out NE differentiation as a prominent factor in this orchestration. Moreover, former studies could show a correlation between the density of microvessels and increasing metastatic behavior in prostatic carcinoma [14, 36, 45/. High grade tumors with a high NE differenti ation and increased neovascularisation therefore represent a high risk group with an unfavorable outcome. It is thus conceivable that the vicinity of NE tumor cells is characterized by a distinct, paracrine-mediated microenvironment that may also play an important role in the neovascularisation within the tumor. leading to tumor progress combined with a poor prognosis.

References l. Abrahamsson PA. Falkmer S. Fait K, Grimelius L (1989)

The course of neuroendocrine differenliation in prostatic carcinomas. An immunohistochemical study et sting chromogranin A as an "endocrine marker" . Path Res Pract

185:373-380

2. Allen FJ. van Velden DJ, Heyns CF (1995) Are neuroendocnne cells of practical value as an independent prognostic parameter in prostate cancer? Br JUral 75: 75 1-754

283

3. Aprikian AG. Cordon Cardo C, Fair WR, Reuter VE (1993) Characterization of neuroendocrine differentiation in human benign prostate and prostatic adenocarcinoma. Cancer 7/: 3952-3965 4. Aprikian AG, Cordon Cardo C, Fair WR, Zhang ZF, Bazinet M, Hamdy SM, Rcuter VE (1994) Neuroendocrine differentiation in metastatic prostatic adenocarcinoma. J Uro1/5/ : 914-919 5. Bohrer MH, Grubholz R, Woenckhaus M, Riehle HM, Schmoll J. Siegsmund M, Jilnemann K-P, Aiken P (1995) Beziehung zwischen neuroendokriner Differenziemng und Neovaskularisation im Prostatakarzinom. Verh Dtsch Ges Pathol 79: 445 6. Bohrer MH, Riehle HM, Schmoll J, Jilnemann K-P, Aiken P (1995) Relationship between neuroendocrine differentiation and neovascularisaton in prostatic carcinoma. J Urol 153: 485 A (Abstract) 7. Bohrer MH, Schmoll J (1993) Immunohistochemische und morphomclri sche Untersuchungen zur neurocndokrincn Differenzierung in Prostatakarzinornen. Verh Dtsch Ges Pathol 77: 107-110 8. Bonkhoff H (1998) Neuroendocrine cells in benign and malignant prostate tissue: morphogenesis, proliferation, and androgen receptor status. Prostate Suppl 8: 18-22 9. Bonkhoff H, Rcmberger K (1996) Differentiation pathways and histogenetic aspects of normal and abnormal prostatic growth: a stem cell model Prostate 28: 98- 106 10. Bonkhoff H, Stein U, Remberger K (1994) Multidirectional differentiation in the normal, hyperplastic, and neoplastic human prostate: simultaneous demonstration of cell-specific epithclial markers. Hum Pathol25: 42-46 II. Bonkhoff H, Stein U, Remberger K (1995) Endocrineparacrinc cell types in the prostate and prostatic adenocarcinoma are postmitotic cells. Hum Pathol 26: 167- 170 12. Bonkhoff H, Wernert N, Dhom G, Remberger K (1991) Relation of endocrine-paracrine cells to cell proliferation in normal, hyperplastic, and neoplasti c human prostate. Prostate 19: 91-98 13. Brawer MK. Bigler SA, Deering RE (1992) Quantitative morphometric analysis of the microcirculation in prostate carcinoma. J Cell Biochem Suppl17H: 62- 64 14. Brawer MK, Deering RE, Brown M . Preston SD, Bigler SA (1994) Predictors of pathologic stage in prostatic carcinoma. The role of neovascularity. Cancer 73: 678-687 15. Bubendorf L. Sauter G, Moch H. Schmid HP. Gasser TC. Jordan P, Mihatsch MJ (1996) Ki67 labelling index: an independcnt predictor of progression in prostate cancer treated by radical prostatectomy. J Pat hol 178: 437-44 1 16. Casella R, Budendorf L, Sauter G, Moch H. Mihatsch MJ . Gasser TC ( 1998) Fucal neuroendocrine differentiation lacks prognostic significance in prostHte core needle biopsies. J Urol 160: 406-410 17. Cohen MK, Arber DA. Coffield KS, Keegan GT. McClintock J .Speights VO, Jr. ( 1994) Neuroendocrine differentiation in prostatic adenocarcinoma and its relationship to tumor progression. Cancer 74: 1899-1903 18. Cohen RJ, Glezerson G, Haffejee Z (1 99 1) Neuroendocrine cells - A new prognostic parameter in prostate cancer. Br J Uro168: 258-262 19. Cohen RJ, G1ezerson G. Haffcjee Z, Afrika D (1990) Prostatic carcinoma: histological and immunohistological facLors affecting prognosis. Br J Uro166: 405-410

284 . R. Grobholz et al. 20. di Sant' Agnese PA (1986) Calcitonin-like immunoreactive and bombesin-like immunoreactive endocrine-paracrine cells of the human prostate. Arch Pathol Lab Med 110: 412-415. 21. di Sant' Agnese PA (1992) Neuroendocrine differentiation

39.

in carcinoma of the prostate. Diagnostic. prognostic, and thcrapcutic implications. Cancer 70: 254-268 22. di Sant' Agnese PA (1998) Neuroendocrine differentiation in prostatic carcinoma: an update. Prostate Suppl 8: 74-79 23. di Sant' Agnese PA, Cockett AT (1994) The prostatic endocrinc-paracrine (neuroendocrine) regulatory system and neuroendocrine differentiation in prostatic carcinoma: a review and future directions in basic research. J Uro1152: 1927-1931 24. Ferrer FA, Miller LJ, Andrawis RI, Kurtzman SH, Albertsen PC, Laudone VP, Kreutzer DL (1998) Angiogenesis

and prostate cancer: in vivo and in vitro expression of an25. 26. 27. 28. 29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

giogenesis factors by prostate cancer cells. Urology 51: 161-167 Folkman J (1990) What is the evidence that tumors are angiogenesis dependent'! J Natl Cancer Inst 82: 4-6 Folkman J (1992) The role of angiogenesis in tumor growth. Semin Cancer Bioi 3: 65-71 Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med I: 27-31 Folkman J, D'Amore PA (1996) Blood vessel formation: what is its molecular basis'! Cell 87: 1153-1155 Folkman J, Watson K, Ingber D, Hanahan D (1989) Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 339: 58-{i1 Fontanini G, Bigini D, Vignati S, Basolo F, Mussi A, Lucchi M, Chine S, Angeletti CA, Harris AL, Bevilacqua G (1995) Microvessel count predicts metastatic disease and survival in non -small cell lung cancer. J Pathol J77: 57-{i3 Harper ME, Glynne Jones E, Goddard L, Thurston VJ, Orifiths K. (1996) Vascular endothelial growth factor (VEGF) expression in prostatic tumours and its relationship to neuroendocrine cells. Br J Cancer 74:910-916 Helpap B, Biicking A, Dhom G, Faul P, Kastendiek H, Leistenschneider W, Muller HA (1985) Klassifikation, hislologisches und zytologisches Grading sowie Regressionsgrading des Prostatakarzinoms. Pathologe 6: 3-7 Hepburn PJ, Griffiths K, Harper ME (1997) Angiogenic factors expressed by human prostatic cell lines: effect on endothelial cell growth in vitro. Prostate 33: 123- 132 International Union Against Cancer (1993) TNM Klassifikation maligner Tumoren, 4th ed .. 2nd REv. , Springer Verlag, Berlin Iwamura M, Wu G, Abrahamsson PA, di Sant' Agnese PA, Cockett AT, Deftos LJ (1994) Parathyroid hormonerelated protein is expressed by prostatic neuroendocrine cells. Urology 43: 667-{i74 Lissbrant IF. Stattin P, Damber JE, Bcrgh A (1997) Vascular density is a predictor of cancer-specific survival in prostatic carcinoma. Prostate 33:38-45 McWilliam LJ, Manson C, George NJ (1997) Neuroendocrine differentiation and prognosis in prostatic adenocarcinoma. Br J Urol 80: 287-290 Meyer GE, Yu E, Siegal JA, Petteway JC, Blumenstein BA, Brawer MK (1995) Serum basic fibroblast growth

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

factor in men with and without prostate carcinoma. Cancer 76: 2304-2311 Nakada SY, di Sant' Agnese PA, Moynes RA , Hiipakka RA, Liao S, Cockett AT, Abrahamsson PA (1993) The androgen receptor status of neuroendocrine cells in human benign and malignant prostatic tissue. Cancer Res 53: 1967-1970 Noordzij MA, van der Kwast TH, van Steenbrugge GJ, Hop WJ , Schroder FH (1995) The prognostic influence of neuroendocrine cells in prostate cancer: results of a longterm follow-up study with patients treated by radical prostatectomy. 1m J Cancer 62: 252-258 O' Reilly MS, Bochm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277-285 Okamoto M, Lee C, Oyasu R (1997) Interleukin-6 as a paracrinc and autocrine growth factor in human prostatic carcinoma cells in vitro. Cancer Res 57: 141-146 Shah GV, Rayford W, Noble MJ, Austenfeld M, Weigel J, Vamos S. Mebust W K (1994) Calcitonin stimulates growth of human prostate cancer cells through receptormediated increase in cyclic adenosine 3',5'-monophosphates and cytoplasmic Ca" transients. Endocrinology 134: 59ti-{i02 Vartanian RK, Weidner N (1995) Endothelial cell proliferation in prostatic carcinoma and prostatic hyperplasia: correlation with Gleason 's score, microvessel density, and epithelial cell proliferation. Lab Invest 73: 844-850 Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J (1993) Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol143: 401-409 Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, Moore DH, Meli S, Gasparini G (1992) Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 84: 1875-1887 Weidner N, Semple JP, Welch WR. Folkman J (1991) Tumor angiogenesis and metastasis - correlation in invasive breast carcinoma. N Engl J Med 324: 1-8 Weinstein MH, Partin AW, Veltri RW, Epstein JI (1996) Neuroendocrine differentiation in prostate cancer: enhanced prediction of progression after radical prostatectomy. Hum Pathol 27: 683-{i87 Woenckhaus M, Bohrer MH (1997) Beziehung zwischen neuroendokrin differenzierten und proliferationsaktiven Tumorkompartimenten im Prostatakarzinom. Verh Dtsch Ges Pathol81: 751 Wright C. Grignon D, Shum D , Porter A (1992) Neuroendocrine differentiation in prostatic adenocarcinoma is not an independent prognostic indicator. Mod Pathol 5: 61A (Abstract) Xue Y, Smedts, F, Verhofstad A, Debruyne F, de la Rosette J, Schalken J (1998) Cell kinetics of prostate exocrine and neuroendocrine epithelium and their differential interrelationship: new perspectives. Prostate Supp! 8: 62-73

Received: October 9, 1998 Accepted in revised version: November 17, 1999