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Future Directions in Research of Prostate Carcinoma
Path. Res. Pract. 189,497-509 (1993)
Diagnostic Seminar
Future Directions in Research of Prostate Carcinoma~:B. Helpap Institute of Pathology, Singen and Academical Hospital of the University of FreiburglBrsg, FRG
SUMMARY Histological, cytological and growth characteristics of prostate carcinoma are presented in this review, as well as the importance of various cell kinetic methods such as autoradiography, DNA- and AgNOR-analysis. By combining these methods with nucleolar grading, carcinoma of the prostate can be divided into two different groups of malignancy regarding therapy and prognosis: I. Carcinoma patients with well differentiated carcinomas and low risk. II. Carcinoma patients with poorly differentiated carcinomas and high risk. Cytogenetic and oncogenetic findings are discussed according to these groups. Special features of neuroendocrinically differentiated carcinomas are also presented. This review contains our own data in addition to information from the literature, and it describes future directions in the research of prostate carcinoma. Prostatic carcinoma is at present the second most common tumor in males. This has led to extensive research and numerous clinical studies on the prostate gland in the recent years.
Morphology Knowledge of growth pattern of prostatic carcinoma cells, their proliferative ability and their stage of expansion (both within the prostate gland as well as into surrounding structures) have influenced the indication of radical prostatectomy. McNea125,26 has contributed a great deal of research in this area (Figs. 1, 2, 3, 4). Infiltration of the neurovascular bundle and perineural invasion in the vicinity of the so-called prostatic capsule can be demonstrated by modern punch biopsy techniques. Several biopsies are taken from different areas of both lobes under sonographic control. The results influence the urologist's decision as to the extent of the operation, ie with or without resection of neural tissue 10,27,39 (Fig. Sa,b).
* Paper presented at "Urological Pathology" conference of the International Society of Urological Pathology, lAP meeting in Madrid on Oct. 23, 1992 ©
1993 by Gustav Fischer Verlag, Stuttgart
The prostate gland can be subdivided into peripheral, transitional and central zones. Clinical carcinoma is generally found in the peripheral zone, though central/transitional zone carcinoma can be found in 10-20 % of cases. This has increased the importance of early diagnosis and therapy24,25,26 (Figs. 1,2,3,4). In transitional zone carcinoma, tumor volume is of major importance, and this can be estimated by transurethral resection. The classification into peripheral or central carcinoma is necessary, since the prognosis for central carcinoma of small volume and low grade of malignancy is much more favorable than for peripheral carcino-· ma 11 ,30. In studying the development of carcinoma, the question of premalignant lesions arises. Through detailed investigations of central or transitional zones, atypical adenomatous hyperplasia is recognized as a precursor of highly differentiated microglandular prostate carcinoma. A precursor or concomitant lesion of peripheral carcinoma is the peripherally located prostatic intraepithelial neoplasia (PIN) of moderated to marked grade of cellular atypia 2 (Tab. 1) (Figs. 6, 7). 0344-0338/93/0189-0497$3.50/0
498 . B. Helpap
Fig. 1. Carcinoma of the central or tansitional zone of the prostate gland (in most cases "incidental carcinoma"). H.E.
Fig. 2. Typical prostatic carcinoma of the peripheral zone, so-called "clinical carcinoma", with invasion of seminal vesicles. H.E.
Fig. 3. Combination of carcinoma of the center and periphery. H.E. (slide from Prof. Dr. H. Kastendieck, Hamburg).
Prostate Carcinoma . 499 Table 1. Atypical prostatic hyperplasia/PIN: prostatic carcinoma 8ENIGN
Localization
HYPERPLASIA
Central
: : ~:' ~ ~Jf/ ~ ( V (J. D 1;\ CLiNICAL!7n CARCINOMA
Peripheral
Grading Slight (1) Moderate (2) Severe (3) Well or moderately differentiated (GIa-IIa) incidental carcinoma mostly with uniform pattern Clinical carcinoma (GlIb, III) mostly pluriform pattern
~
Fig. 4. Location and distribution of benign hyperplasia (BPH), central (incidental) and peripheral (clinical) carcinomas of the prostate gland (Kastendieck H in the prostate vol. edts. B. Helpap, Th Senge, W. Vahlensieck 1984, PMI-Verlag, p 159).
Fig. 5. Infiltration of the neurovascular bundle and perineural invasion in the vicinity of the so-called capsule of the prostate gland. a: perineural carcinomatous invasion. - b: infiltration of fat and connective tissue of the so-called capsule. H.E. 80 x.
500 . B. Helpap
Fig. 6. Prostatic intraepithelial neoplasia of the prostate gland. Intraductal cell proliferations with marked cellular atypias (PIN III). Precursor lesion or concomicant lesion of peripheral carcinoma. H.E.250 x.
Grading - Prognosis Exact knowledge of proliferative activity is necessary in order to judge the malignant potential during the development of prostatic carcinoma. It is important to emphasize that the tumor's development and volume are dependent on the proliferative tendency of individual tumor cells, and whether they all arise from one clone or from several. The assumption that prostatic carcinoma with a volume of less than 0.5 ml corresponds to a slower tumor growth rate should be examined more closely. Here, particularly, it is necessary to have knowledge about grading with its array of tests. If a diploid, highly differentiated tumor is present without cytological atypias or prominent nucleoli status, then it is controversial in the literature regarding clonal selection. We know that prostatic carcinoma is a heterogenic tumor which can contain a mixture of cells of varying proliferative tendencies, or of a single clone. At first glance, this differentiation may not be evident using routine histological and cytological grading methods 18 . (Figs. 8-11).
Fig. 7. Atypical (microglandular) adenomatous hyperplasia (AAH) in central parts of the prostate gland. Precursor of centraUincidental carcinomas. H.E. 250' x.
I do not wish to discuss the numerous attempts at grading prostatic carcinoma in this paper. I would just like to point out the more common grading systems used worldwide. This is first of all Gleason's grading system 17. It is based on the degree of histological dedifferentiation. Since the cytological parameters are less important in the Gleason grading system, it should be used primarily for radical prostatectomy specimens instead of punch biopsies. Cytological grading is not possible with this method (Tab. 2). The grading system according to Mostofi or WHO is based on histological structural analysis of carcinoma including grade of cellular and nuclear anaplasia. Three grades of malignancy are described 14 ,29,3o (Tab. 2). In Germany, we have worked extensively on possibilities of grading. I would like to present a grading system of the "Pathological Urological Working Group of Prostatic Carcinoma", which is based on Mostofi's WHO grading system. Prostate carcinoma is differentiated on the basis of uniform or pluriform structure, with particular attention paid to the degree of nuclear atypia. To this, we added a
Prostate Carcinoma . 501
Fig. 8. Highly differentiated glandular carcinoma of the prostate gland with only few singular nucleoli. Low grade of malignancy lb. H.E. 318 x.
Fig. 9. Moderately differentiated glandular carcinoma of the prostate gland with prominent nucleoli in eccentric location. Grade of malignancy IIa. H.E. 318 x .
nucleolar grading and divided the original grades of malignancy I, II and III into subgroups a and b. This can be correlated with the WHO grading system as well as Gleason'slR (Tab. 2).
Patients with prostate carcinoma can be divided into two groups based on survival and mortality rates. The first group, which includes grades of malignancy la to I1a, is of low risk, whereas patients with grades lIb, IlIa and IIIb are of high risk. The death rates in our own statistics show mortality within 10 years for 2.8 % in Group 1 and 38.2 % in Group 2. The low-risk group is compatible with WHO Grade I and partly Grade II, as well as Gleason groups I-III. The Gleason pattern III with a cribriform structure, pattern IV and V and WHO Grade III correspond to our high-risk group. Since survival rates do not seem to be affected by different forms of therapy, we can conclude that it is essential to apply methods of differentiation between the high and low risk groups19 (Tab. 2, Fig. 12) . Possibilities for future directions of research:
Table 2. Comparison between different grading systems - Gleason - Pathol. Urol. Working Group: "Prostatic Carcinoma" WHO/MOSTOFI Gleason
Pathol. Urol. WK.
MostofilWHO Prognosis
Pattern 1 2 3 (Glandular)
Grade
IA Is IIA
Grade 1 2
3 (Cribriform) 4 5
lIB
3
lIlA IIIB
Favorable Low Risk
Cell kinetics Unfavorable High Risk
I. Combination of nucleolar pattern of prostatic carcinoma and cell kinetic auto radiographic proliferative behavior.
502 . B. Helpap
Fig. 10. Prostate carcinoma with cribriform pattern. Grade of malignancy lIb. H.E. 310 x. 1,0 _Ib/lia DoD
0,9 --0
:tJ 0
co co r
:tJ
0,8 0,7
n = 118 n
= 45
!
0,6
L..........
!
0,5
'LL . . . . ..
0,4 logranktesl p = 0,01
0,3 0,2
n=108 n= 3
~··---····1
---1
-c
lib/III DoD
..........,
i
I·
2
3
4
5
6
7
8
Fig. 11. Poorly differentiated carcinoma of the prostate gland with solid pattern and marked cellular atypias with multiple large nucleoli in eccentric location. Grade of malignancy IlIa. H.E. 318 x .
3
10
VEARS Fig. 12. High survival rates of over ten years of patients with prostatic carcinoma grade of malignany Ib-IIa (---) with low rates of death due to disease. Shorter survival rates and higher rates for death due to disease of patients with prostatic cancer grades of malignancy lIb-III (---).
We studied proliferation indices and DNA synthesis rates in prostatic carcinoma of varying degrees of differentiation. This was done by labeling DNA synthesizing cell elements in vitro in a proliferating pool of prostatic carcinoma with tritiated or 14C thymidine in autologous patient serum under increased carbogen pressure (Tab. 3). In other cell kinetic studies we used immunohistochemical analysis with Ki67 or PCNA (proliferating cell nuclear antigen) (Tab. 3; Fig. 13). We also determined the percentage of nuclei with nucleoli, the number of nucleoli per nucleus, and the central or peripheral locations of nucleoli (Fig. 14, 15). We observed an increase in labeling indices corresponding to increased dedifferentiation. This correlates to the increased frequency of nuclei containing nucleoli, the number of nucleoli per nucleus, and peripheral location of nucleoli. The behavior of nucleoli corresponds to measurable proliferation parameters such as a shortening of the DNA synthesis phase with increased malignancy of prostate carcinoma. This emphasizes the value of nucleolar grading. Other researchers have
Prostate Carcinoma . 503
Fig. 14. Distribution of number and size of nucleoli within nuclei (solitary of multiple nucleoli).
~
IIi~'\ ,. ,'.-:.'
1\ '. ':.<" ':::' /
~\ :. ",-,:'
-:.:: -'.'
.
r--,--
f@1= ~.
)
:
.. ..... : ..
--
Fig. 15, Location of nucleoli within nuclei (central or eccentric) .
./j
Fig. 13. Immunohistochemicalla beling of proliferating cells with proliferating cell nuclear antigen (PCNA) of carcinoma of the prostate gland. ABC-method, 318 x .
t t 11 :1 r----------~-- ~:~~:--- ------ ,/
Table 3. Grading of malignant tumors
::IHH
100
.-.'.~\
90
Table 4. Nuclear and nucleolar grading Nucleus
Nucleolus
Frequency of Mitosis Frequency of Apoptosis Size Shape Degree of Chromasia
Frequency of Nuclei with Nucleoli Size Shape Location
. _ - -•......."
80
j
a
Routine Histology and Cytology AgNOR Analysis DNA Cytometry DNA Autoradiography Immunohistochemistry Ki 67 Frozen Section PCNA Paraffin Section
.---.
i
J,
0,1
0,5
=lS: :
I
./.
60
b
1,0
1,5
2,0
2,5 '
3,0
I
3,5
4,0
4,5
5,6 loU''''
Fig. 16. a: Correlation of nuclei with one, two or more nucleoli to the labeling index of carcinomas of the prostate gland. N+n nucleolar frequency. - b: Correlation of the location of nucleoli within nuclei to the labeling index of carcinomas of the prostate gland c = central, i = intermediate, p = peripheral (from Helpap B, Pathologie der ableitenden Harowege und der Prostata. Springer Heidelberg, Berlin 1989 p 260).
504 . B. Helpap
Fig. 17. Low number of silver-stained nucleolar orgallizlllg regions (AgNORs) in prostate carcinoma of low malignancy. 390 x.
Fig. 18. Increased number of AgNORs in prostatic carcinoma with high grade of malignancy. 390 x.
achieved similar results using the same methods . An increase in nucleolar size has also been observed9,28 (Tab. 4, 5; Fig. 16). Similar results have been found by determining the number of silver-stained nucleolar organizer regions (AgNORs per unit area of nucleus). In the succession from benign hyperplasia to atypical hyperplasia, PIN, highly differentiated carcinoma and poorly differentiated carci-
noma, there is a distinct increase in the number of AgNORs per unit area of nucleus. Here, too, similar results were obtained by different researchers using varying procedures. However, this method is not adequate to determine regressive changes in proliferative potential under therapy. It also cannot measure differences between carcinomas with similar proliferation activity 1, 5, 9,13,35; (Tab. 6) (Figs. 17, 18).
Table 5. Mean diameter, frequency of nuclei with nucleoli and number of nucleoli per nucleus of prostatic tissue: modified to Deschenes and Weidner (1990), Montironi et al (1991) Histology
Mean diameter of Frequency (%) of nuclei with nucleoli nucleoli (UM)
Number of nucleoli per nucleus N1
N2
N3
BPH* PAH"* PIN*""
0.6-1.9 0.8-2.7 1.1-2.9
16.1 ± 5.1 57.7 ± 8.9 58.7 ± 7.6
96.0 ± 4.6 85.5 ± 7.5 83.4 ± 4.6
3.9 ±4.6 13.7 ± 7.4 15.1 ± 4.0
0.8 ± 1.1 1.8 ± 1.4
Carcinoma G IB -IIA G II B-I1IA,B
1.2-2.1 3.3
78.6 ± 7.5
80.4 ± 7.9
16.4 ± 5.9
3.2 ±2.6
* Benign prostatic hyperplasia; * * prostatic atypical hyperplasia; * * * prostatic intraepithelial neoplasia
Prostate Carcinoma . 505 Table 6. Nucleolar and AgNOR pattern of prostatic tissue Histology
N
Nucleoli Frequency/ nucleus
Number/ nucleus
Location in nucleus
N
AgNOR per unit area of nucleus
Typical Hyperplasia (BPH)
10
0.5 ± 1.2
solitary
central
10
1.7 ± 0.5
Atypical Hyperplasia (Transition Zone) mild moderate-marked
55 24
1.6± 0.4 5.3 ± 1.3
solitary solitary
central central
10
2.1 ± O.S
Prostatic intra epithelial Neoplasia moderate-marked
27
20.0± 6.4
solitary multiple
central eccentric
10
4.3 ± 1.S
Carcinomas Grading Ib-IIa
113
41.4 ± 12.5
solitary multiple
central eccentric
34
3.5 ± 0.5
Grading lIb-III
72
SO.l ± 15.6
multiple
eccentric
37
5.1 ±0.7
Another important cell kinetic method is the utilization of flow cytometry or single cell DNA cytophotometry. If we recognize the presently accepted subdivision of DNA values into nearly diploid, hypertetraploid and aneuploid, the highest percentage of diploid DNA values are found in the highly differentiated prostatic carcinomas. 20 to 25 % of highly differentiated prostatic carcinomas have aneuploid DNA values. Poorly differentiated prostatic carcinomas in the high risk group are solely aneuploid. The GIl group, which is difficult to interpret, shows varying fractions of diploid, hypertetraploid and aneuploid values. It is particularly in this group that an increase in grade of aneuploidy with transition to progression can be seen after two to three years of observation. Because of the heterogeneous tumor structure, single cell cytophotometry should be utilized on several different sectors of the tumor for its evaluation 8, 13. The classification of prostatic carcinoma should only be carried out in conjunction with the usual histological and cytological findings. It is best to utilize both flow cytometry and single cell cytometry. In addition, DNA analysis is important for evaluating response to the therapy. DNA ploidy values offer prognostic information 12 • By applying all these described methods, we believe that there is obvious value in the subclassification and subgrading of prostatic carcinoma, especially the division into high and low-risk groups18. Prostatic carcinoma with higher aneuploid values in the group of highly differentiated tumors can belong to those that progress earlier, and can be the small percentage of the low risk tumors that do lead to death. More detailed studies are necessary in order to obtain more precise values. Cytogenetics
Cytogenetic studies will surely play an important role in future research on prostatic carcinoma. By utilizing DNA hybridization techniques, more than 50 % of karyotyped prostatic carcinomas show numerical
aberrations, and to a lesser extent, structural changes (Sesterhenn et al1992). The loss of chromosomes 1,2,5 and Y; addition of chromosomes 7, 14,20 and 22; and translocation of 7p, 7q and 10q have been observed in prostatic carcinoma. Other researchers have discovered deletion, duplication, inversion and translocation mainly in chromosomes 2,7,10 and 16. Mutation of the gene p53 on chromosome 7p can be seen more often in metastasizing prostatic carcinoma13,31. Research up to now has shown that no single chromosomal change can be made responsible for prostatic carcinoma. There is no karyotypemarker for carcinoma of the prostate gland. Lundgren et aF3 found that patients with only one clonal karyotypical abnormality live on average 1.3 years after diagnosis, whereas carcinoma patients with a combination of normal and abnormal karyotypes generally survive another 3 years. Oncogenetics
Studies on oncogenes in prostatic tissue are just beginning34 . Some oncogenes are expressed in both benign and malignant prostatic tissues, as for instance c-erbB24o. DNA in situ hybridization techniques have found differing results on prot%ncogenes. Oncogenes are genes which have the ability to transform cells neoplastically. Nuclear oncogenes modulate DNA transcription and influence the phases of the cell cycle. Cytoplasmic oncogenes activate tumorgenesis. Both together can induce neoplasms. For example, c-myc and c-fos are nuclear oncogenes and vH-ras is a cytoplasmic gene. V-erb-B-oncogene codes a part of the receptor for epidermal growth factor. Cellular oncogenes are of major influence in the regulation of cellular growth 13 (Tab. 7). In one case studied, a highly differentiated (GI) prostatic carcinoma was found to release a large amount of c-myc mRNA and c-sis m RNA, with small amounts of c-fos mRNA. Interestingly, an increased expression of c-sis and c-fos mRNA in hyperplastic prostatic glands was found in the vicinity of prostatic carcinoma. This appears to show
506 . B. Helpap Table 7. Expression of oncogenes in prostatic carcinoma (Pc) Grading
V-ErbBC-Fos C-Myc C-sis
Low Grade PC + +-(+) High Grade PC +++ + ++
VHras Kras
++
+
++
(+)
that local production of growth factor for prostatic cells plays an important role in the proliferative activity of prostatic carcinoma16. Activation of K-ras-oncogenes in conjunction with an increased ras-P-21 expression has been observed in latent pro~tatic c~rcinoma 'Yith low grade of malignancy as well as m carcmomas wIth both higher staging and grading3,4,22,37. Deletion of genetic material from a chromosome can lead to a loss of impeding gene sequences in the vicinity of ?ncogenes. The loss of tumor suppression genes is an Important event, since it can trigger tumor production. Tumor suppressor antigens are also called antioncogenes. Suppressor genes code a protein that is essential for normal c~llul~r growth behavior. Its loss induces tumor progresSIOn, l.e. progression of prostatic tumor tissue 13 ,21. A crucial step in tumor formation is the uncontrolled cell div~sion. This. res?l~s in aberration of chromosomal replicatIon and mItosIS m the neoplastic cells. A decrease in tumor suppressor genes has been found in car~inoma. This shows similar behavior to blood group antIgens. In BPH, blood group antigens Lewis a and bare released in great amounts. This expression is decreased in the vicinity of carcinoma, weak in highly differentiated prostatic carcinoma, and absent in poorly differentiated prostatic carcinoma l ,20 (Tab. 8). Proto-oncogene-protein R-erb-B related to epidermal growth factor receptor shows the opposite behavior. Its expression is weak in benign tumors and strong in undifferentiated carcinomas. No differences were seen between highly differentiated carcinoma and regions of BPI! near carcinoma. The behavior of blood group antigens supports the division into low and high risk groups20,38. The expression pattern of androgen receptors also play an important role in postsurgical hormone therapy or ~hemotherapy. Cases with positive receptors correlate to responders and negative receptors to non-responders. The importance of other receptors such as prolactine receptors or estramustine binding protein receptor regarding malignancy and response to therapy must still be evaluated 1,13.
Histology, Immunohistochemistry, Prognosis Th~ pre?ictiv.e v~lue ~f th~ discussed investigative
~echm9ues IS mamly m conjUnctIon with ordinary prostatIC carCl~oma. ~he rare squamous cell or urothelial type of
prostatic carcmoma are not included in these evulations. One. tU!ll0r type should still be mentioned. Recently more fllldlllgS have been made on prostatic carcinoma with neuroendocrine cells 32 . As in other organs, eosinophilic g~anulated Grimelius-Silver-stained cells have been recogmzed be~een s.ecretory cells in benign prostate changes as wel~ ~s m carcmoma. T~ese are immunohistochemically posltlve for chromogranm A and neuron specific enolase (Figs. 19,20). These cells can also secrete a number of other neuroendocrine proteins. These neuroendocrine cells a.re present in high percentages in carcinomas. A coexpresSlon of PS~ and neuroendocrine markers, especially chr?I?ogranlll~, has. been found. Interestingly, no nuclei posltlve for prohferation marker Ki 67 or PCNA have been recognized. On the other hand, positivity has been found in
Table 8. Expression of blood group antigens in prostatic carcinoma Grading
Blood group antigens A
Low grade PC + High grade PC 0/( +)
B
+
0/(+)
H
+
0/(+)
Lewis A Lewis B
+
0/(+)
+
0/(+)
Fig. 19. Glandular carcinoma of the prostate gland with neuroendocrine differentiation. Grimelius-silver-staining method. 200 x.
Prostate Carcinoma . 507
non-neuroendocrinally differentiated typical prostate tumor cells near the neuroendocrine tumor cells. It is suspected that the neuroendocrine cells secrete substances that stimulate proliferation kinetics. Since the neuroendocrinologically differentiated tumor cells do not respond to hormonal therapy, these types of carcinoma should be classified into the group of non-responders. Cohen and coworkers 6, 7 could observe a poorer prognosis for patients with neuroendocrinally differentiated prostatic carcinomas in contrast to prostatic carcinomas which were negative for neuroendocrine cells. Finding a therapeutic method to treat these endocrinally differentiated prostatic carcinomas is a task for the future. Conclusion
Let us review what is now possible and what goals future research has. I believe that primary diagnosis of prostatic carcinoma with specification of grade and volume is of great importance. Analyses have shown that the grading methods used now can be made more precise through a number of additional tests, yet methods such as DNA ploidy measurement or cytogenetic studies cannot be used as a replacement. Only the combination of all the methods
Fig. 20. Glandular carcinoma of the prostate gland with neuroendocrine differentiation. Immunohistochemical analysis with chromogranin A. ABC-method.250 x.
can improve our knowledge about the nature of carcinoma of the prostate gland. The significance of GI prostatic carcinoma with an aneuploid DNA pattern is not yet clear. We can only suspect that this group has a greater tendency for progression than diploid GI-tumors. On the other hand, the question arises whether diploid GIll tumors exist and whether the diploid fraction is significant or negligable. I believe that the cases of high and low malignancy prostatic carcinoma should be studied in order to structure a clinically orientated therapy plan. The many questions concerning the pathogenesis of prostatic carcinoma and molecular pathological diagnostic steps including genetic studies should be continued, especially for their clinically orientated value. I believe that this provides an adequate stimulus for future research directions 14,18 (Tab. 9).
References 1 Allsbrook WC, Simms WW (1992) Histochemistry of the prostate. Hum Pathol 23: 297-305 2 Brawer MK (1992) Prostatic intraepithelial neoplasia. A premalignant lesion. Hum Pathol 23 : 242-248 3 Bushmann EW, Bushmann WA, Kingston TE, Lippitt Rg, Chiapella CD, Stein BS (1989) Ras (p 21) expression in proliferative lesions of the prostate. J Urol 141: 205 A 4 Carter BS, Epstein JI, Isaacs WB (1990) Ras gene mutations in human prostate cancer. Cancer Res 50: 6830-6832 5 ChevilleJC, Calmon GH, Robinson RA (1990) Silver stained nucleolar organizer regions in the differentiation of prostatic hyperplasia, intraepithelial neoplasia and adenocarcinoma. Mod Pathol3: 596-598 6 Cohen RJ, Glezerson G, Haffejee Z, Afrika D (1990) Prostatic carcinoma. Histological and immunohistological factors affecting prognosis. Br J Urol 66: 405-410 7 Cohen RJ, Glezerson G, Haffejee Z (1991) Neuroendocrine cells - a new prognostic parameter in prostatic cancer. Br J Urol 68: 258-262 8 Deitch AD, Vere White de RW (1992) Flow cytometry as a predictive modality in prostate cancer. Hum Pathol 23: 352-359 9 Deschenes J, Weidner N (1990) Nucleolar organizer regions (NOR) in hyperplastic and neoplastic prostate disease. Am J Surg Pathol14: 1148-1155 10 Epstein JI (1990) Evaluation of radical prostatectomy capsular margins of resection - the significance of margins designated as negative, closely approaching, and positive. Am J Surg Pathol14: 626-632 11 Epstein JI, Steinberg GD (1990) The significance of lowgrade prostate cancer on needle biopsy. A radical prostatectomy study of tumor grade, volume, and stage of the biopsied and multifocal tumor. Cancer 66: 1927-1932 12 Falkmer UG (1992) Methodologic sources of errors in image and flow cytometric DNA assessments of the malignancy potential of prostatic carcinoma. Hum Pathol 23: 360-367 13 Foster CS, Abel PD (1992) Clinical and molecular techniques for diagnosis and monitoring of prostatic cancer. Hum Pathol 23: 395-401 14 Foster CS, Mostofi FK (1992) Prostate cancer - Quo vadis? Hum Pathol 23: 402-406 15 Foster CS,McLoughlin J, Bashir I, Abel PD (1992) Markers of the metastatic phenotype in prostate cancer. Hum Pathol 23: 381-394
508 . B. Helpap Table 9. Morphologic, cell kinetic and immunohistochemical differential diagnosis criteria of low and high malignant carcinomas [18] Low malignant carcinomas
High malignant carcinomas
IA,IB,II A High to moderate differentiation Slight atypias nucleus uniform nuclei mild nuclear chromasia nuclear-cytoplasmic ratio 0.25 nucleolus low frequency central localization one nucleolus per nucleus
IIB,I1IA,I1IB (IV) Low to no differentiation Marked atypias Nucleus marked variability of nuclear shape increased chromasia nuclear-cytoplasmic ratio more than 0.75 nucleolus increased frequency eccentric localization multiple nucleoli per nucleus
Low cell proliferation DNA eu(an-)ploidy low frequency of mitoses low 3H-thymidine labelling index low Ki 67 growth fraction long duration of S phase low number of AgNORs
Increased cell proliferation DNA aneuploidy increased frequency of mitoses increased 3H-thymidine labelling index increased Ki 67 growth fraction short duration of S-phase increased number of AgNORs
Cell loss
Low index of apoptosis
Increased index of apoptosis
Immunohistochemistry
Homogeneous expression of antigens Diminished ABO blood group antigens Diminished suppressor antigens
Heterogeneous expression of antigens Lack of ABO blood group antigens Lack of supressor antigens
Histochemistry
Lectin binding sites
Lack of lectin binding sites
Prognosis
Favorable rare recurrences or metastases low DoD rates
Unfavorable frequent recurrences or metastases high DoD rates
Sub grading Histology Cytology
Cell kinetics
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Prostate Carcinoma . 509 organizing regions in prostatic hyperplasia, intraepithelial neoplasia, and prostatic carcinoma. J Urol Patholl: 61-74 36 Sesterhenn lA, Mostofi FK, Davis Ch jr, van Dekken H (1991) Numerical chromosomal aberrations in interphase nuclei of genitourinary tumors by in situ hybridochemistry (abstract). Lab Invest 64: 298 37 Sumiya H, Masai M, Akimoto S, Yatnai R, Shimazaki J (1990) Histochemical examination of expression of ras p21 protein and R 1881-binding protein in human prostatic cancers. Eur J Cancer 26: 786-789
38 Thompson TC (1990) Growth factors and oncogenes in prostate cancer. Cancer Cells 2: 345-354 39 Voges GE, McNealJE, Redwine EA, Freiha FS, Stamey TH (1992) Morphologic analysis of surgical margins with positive findings in prostatectomy for adenocarcinoma of the prostate. Cancer 69: 520-526 40 Ware JL, Maygarden SJ, Koontz WW, Strom SC (1991) Immunohistochemical detection of c-erbB-2 protein in human benign and neoplastic prostate. Hum Pathol 22: 254-258
Received November 4, 1992 . Accepted January 19, 1993
Key words: Prostate carcinoma - DNA analysis - AgNOR - nucleolar grading. Prof. Dr. B. Helpap, PO Box 720, W-7700 Singen, FRG
Diagnostic Seminar
Future Directions in Research of Prostate Carcinoma~:B. Helpap Institute of Pathology, Singen and Academical Hospital of the University of FreiburglBrsg, FRG
SUMMARY Histological, cytological and growth characteristics of prostate carcinoma are presented in this review, as well as the importance of various cell kinetic methods such as autoradiography, DNA- and AgNOR-analysis. By combining these methods with nucleolar grading, carcinoma of the prostate can be divided into two different groups of malignancy regarding therapy and prognosis: I. Carcinoma patients with well differentiated carcinomas and low risk. II. Carcinoma patients with poorly differentiated carcinomas and high risk. Cytogenetic and oncogenetic findings are discussed according to these groups. Special features of neuroendocrinically differentiated carcinomas are also presented. This review contains our own data in addition to information from the literature, and it describes future directions in the research of prostate carcinoma. Prostatic carcinoma is at present the second most common tumor in males. This has led to extensive research and numerous clinical studies on the prostate gland in the recent years.
Morphology Knowledge of growth pattern of prostatic carcinoma cells, their proliferative ability and their stage of expansion (both within the prostate gland as well as into surrounding structures) have influenced the indication of radical prostatectomy. McNea125,26 has contributed a great deal of research in this area (Figs. 1, 2, 3, 4). Infiltration of the neurovascular bundle and perineural invasion in the vicinity of the so-called prostatic capsule can be demonstrated by modern punch biopsy techniques. Several biopsies are taken from different areas of both lobes under sonographic control. The results influence the urologist's decision as to the extent of the operation, ie with or without resection of neural tissue 10,27,39 (Fig. Sa,b).
* Paper presented at "Urological Pathology" conference of the International Society of Urological Pathology, lAP meeting in Madrid on Oct. 23, 1992 ©
1993 by Gustav Fischer Verlag, Stuttgart
The prostate gland can be subdivided into peripheral, transitional and central zones. Clinical carcinoma is generally found in the peripheral zone, though central/transitional zone carcinoma can be found in 10-20 % of cases. This has increased the importance of early diagnosis and therapy24,25,26 (Figs. 1,2,3,4). In transitional zone carcinoma, tumor volume is of major importance, and this can be estimated by transurethral resection. The classification into peripheral or central carcinoma is necessary, since the prognosis for central carcinoma of small volume and low grade of malignancy is much more favorable than for peripheral carcino-· ma 11 ,30. In studying the development of carcinoma, the question of premalignant lesions arises. Through detailed investigations of central or transitional zones, atypical adenomatous hyperplasia is recognized as a precursor of highly differentiated microglandular prostate carcinoma. A precursor or concomitant lesion of peripheral carcinoma is the peripherally located prostatic intraepithelial neoplasia (PIN) of moderated to marked grade of cellular atypia 2 (Tab. 1) (Figs. 6, 7). 0344-0338/93/0189-0497$3.50/0
498 . B. Helpap
Fig. 1. Carcinoma of the central or tansitional zone of the prostate gland (in most cases "incidental carcinoma"). H.E.
Fig. 2. Typical prostatic carcinoma of the peripheral zone, so-called "clinical carcinoma", with invasion of seminal vesicles. H.E.
Fig. 3. Combination of carcinoma of the center and periphery. H.E. (slide from Prof. Dr. H. Kastendieck, Hamburg).
Prostate Carcinoma . 499 Table 1. Atypical prostatic hyperplasia/PIN: prostatic carcinoma 8ENIGN
Localization
HYPERPLASIA
Central
: : ~:' ~ ~Jf/ ~ ( V (J. D 1;\ CLiNICAL!7n CARCINOMA
Peripheral
Grading Slight (1) Moderate (2) Severe (3) Well or moderately differentiated (GIa-IIa) incidental carcinoma mostly with uniform pattern Clinical carcinoma (GlIb, III) mostly pluriform pattern
~
Fig. 4. Location and distribution of benign hyperplasia (BPH), central (incidental) and peripheral (clinical) carcinomas of the prostate gland (Kastendieck H in the prostate vol. edts. B. Helpap, Th Senge, W. Vahlensieck 1984, PMI-Verlag, p 159).
Fig. 5. Infiltration of the neurovascular bundle and perineural invasion in the vicinity of the so-called capsule of the prostate gland. a: perineural carcinomatous invasion. - b: infiltration of fat and connective tissue of the so-called capsule. H.E. 80 x.
500 . B. Helpap
Fig. 6. Prostatic intraepithelial neoplasia of the prostate gland. Intraductal cell proliferations with marked cellular atypias (PIN III). Precursor lesion or concomicant lesion of peripheral carcinoma. H.E.250 x.
Grading - Prognosis Exact knowledge of proliferative activity is necessary in order to judge the malignant potential during the development of prostatic carcinoma. It is important to emphasize that the tumor's development and volume are dependent on the proliferative tendency of individual tumor cells, and whether they all arise from one clone or from several. The assumption that prostatic carcinoma with a volume of less than 0.5 ml corresponds to a slower tumor growth rate should be examined more closely. Here, particularly, it is necessary to have knowledge about grading with its array of tests. If a diploid, highly differentiated tumor is present without cytological atypias or prominent nucleoli status, then it is controversial in the literature regarding clonal selection. We know that prostatic carcinoma is a heterogenic tumor which can contain a mixture of cells of varying proliferative tendencies, or of a single clone. At first glance, this differentiation may not be evident using routine histological and cytological grading methods 18 . (Figs. 8-11).
Fig. 7. Atypical (microglandular) adenomatous hyperplasia (AAH) in central parts of the prostate gland. Precursor of centraUincidental carcinomas. H.E. 250' x.
I do not wish to discuss the numerous attempts at grading prostatic carcinoma in this paper. I would just like to point out the more common grading systems used worldwide. This is first of all Gleason's grading system 17. It is based on the degree of histological dedifferentiation. Since the cytological parameters are less important in the Gleason grading system, it should be used primarily for radical prostatectomy specimens instead of punch biopsies. Cytological grading is not possible with this method (Tab. 2). The grading system according to Mostofi or WHO is based on histological structural analysis of carcinoma including grade of cellular and nuclear anaplasia. Three grades of malignancy are described 14 ,29,3o (Tab. 2). In Germany, we have worked extensively on possibilities of grading. I would like to present a grading system of the "Pathological Urological Working Group of Prostatic Carcinoma", which is based on Mostofi's WHO grading system. Prostate carcinoma is differentiated on the basis of uniform or pluriform structure, with particular attention paid to the degree of nuclear atypia. To this, we added a
Prostate Carcinoma . 501
Fig. 8. Highly differentiated glandular carcinoma of the prostate gland with only few singular nucleoli. Low grade of malignancy lb. H.E. 318 x.
Fig. 9. Moderately differentiated glandular carcinoma of the prostate gland with prominent nucleoli in eccentric location. Grade of malignancy IIa. H.E. 318 x .
nucleolar grading and divided the original grades of malignancy I, II and III into subgroups a and b. This can be correlated with the WHO grading system as well as Gleason'slR (Tab. 2).
Patients with prostate carcinoma can be divided into two groups based on survival and mortality rates. The first group, which includes grades of malignancy la to I1a, is of low risk, whereas patients with grades lIb, IlIa and IIIb are of high risk. The death rates in our own statistics show mortality within 10 years for 2.8 % in Group 1 and 38.2 % in Group 2. The low-risk group is compatible with WHO Grade I and partly Grade II, as well as Gleason groups I-III. The Gleason pattern III with a cribriform structure, pattern IV and V and WHO Grade III correspond to our high-risk group. Since survival rates do not seem to be affected by different forms of therapy, we can conclude that it is essential to apply methods of differentiation between the high and low risk groups19 (Tab. 2, Fig. 12) . Possibilities for future directions of research:
Table 2. Comparison between different grading systems - Gleason - Pathol. Urol. Working Group: "Prostatic Carcinoma" WHO/MOSTOFI Gleason
Pathol. Urol. WK.
MostofilWHO Prognosis
Pattern 1 2 3 (Glandular)
Grade
IA Is IIA
Grade 1 2
3 (Cribriform) 4 5
lIB
3
lIlA IIIB
Favorable Low Risk
Cell kinetics Unfavorable High Risk
I. Combination of nucleolar pattern of prostatic carcinoma and cell kinetic auto radiographic proliferative behavior.
502 . B. Helpap
Fig. 10. Prostate carcinoma with cribriform pattern. Grade of malignancy lIb. H.E. 310 x. 1,0 _Ib/lia DoD
0,9 --0
:tJ 0
co co r
:tJ
0,8 0,7
n = 118 n
= 45
!
0,6
L..........
!
0,5
'LL . . . . ..
0,4 logranktesl p = 0,01
0,3 0,2
n=108 n= 3
~··---····1
---1
-c
lib/III DoD
..........,
i
I·
2
3
4
5
6
7
8
Fig. 11. Poorly differentiated carcinoma of the prostate gland with solid pattern and marked cellular atypias with multiple large nucleoli in eccentric location. Grade of malignancy IlIa. H.E. 318 x .
3
10
VEARS Fig. 12. High survival rates of over ten years of patients with prostatic carcinoma grade of malignany Ib-IIa (---) with low rates of death due to disease. Shorter survival rates and higher rates for death due to disease of patients with prostatic cancer grades of malignancy lIb-III (---).
We studied proliferation indices and DNA synthesis rates in prostatic carcinoma of varying degrees of differentiation. This was done by labeling DNA synthesizing cell elements in vitro in a proliferating pool of prostatic carcinoma with tritiated or 14C thymidine in autologous patient serum under increased carbogen pressure (Tab. 3). In other cell kinetic studies we used immunohistochemical analysis with Ki67 or PCNA (proliferating cell nuclear antigen) (Tab. 3; Fig. 13). We also determined the percentage of nuclei with nucleoli, the number of nucleoli per nucleus, and the central or peripheral locations of nucleoli (Fig. 14, 15). We observed an increase in labeling indices corresponding to increased dedifferentiation. This correlates to the increased frequency of nuclei containing nucleoli, the number of nucleoli per nucleus, and peripheral location of nucleoli. The behavior of nucleoli corresponds to measurable proliferation parameters such as a shortening of the DNA synthesis phase with increased malignancy of prostate carcinoma. This emphasizes the value of nucleolar grading. Other researchers have
Prostate Carcinoma . 503
Fig. 14. Distribution of number and size of nucleoli within nuclei (solitary of multiple nucleoli).
~
IIi~'\ ,. ,'.-:.'
1\ '. ':.<" ':::' /
~\ :. ",-,:'
-:.:: -'.'
.
r--,--
f@1= ~.
)
:
.. ..... : ..
--
Fig. 15, Location of nucleoli within nuclei (central or eccentric) .
./j
Fig. 13. Immunohistochemicalla beling of proliferating cells with proliferating cell nuclear antigen (PCNA) of carcinoma of the prostate gland. ABC-method, 318 x .
t t 11 :1 r----------~-- ~:~~:--- ------ ,/
Table 3. Grading of malignant tumors
::IHH
100
.-.'.~\
90
Table 4. Nuclear and nucleolar grading Nucleus
Nucleolus
Frequency of Mitosis Frequency of Apoptosis Size Shape Degree of Chromasia
Frequency of Nuclei with Nucleoli Size Shape Location
. _ - -•......."
80
j
a
Routine Histology and Cytology AgNOR Analysis DNA Cytometry DNA Autoradiography Immunohistochemistry Ki 67 Frozen Section PCNA Paraffin Section
.---.
i
J,
0,1
0,5
=lS: :
I
./.
60
b
1,0
1,5
2,0
2,5 '
3,0
I
3,5
4,0
4,5
5,6 loU''''
Fig. 16. a: Correlation of nuclei with one, two or more nucleoli to the labeling index of carcinomas of the prostate gland. N+n nucleolar frequency. - b: Correlation of the location of nucleoli within nuclei to the labeling index of carcinomas of the prostate gland c = central, i = intermediate, p = peripheral (from Helpap B, Pathologie der ableitenden Harowege und der Prostata. Springer Heidelberg, Berlin 1989 p 260).
504 . B. Helpap
Fig. 17. Low number of silver-stained nucleolar orgallizlllg regions (AgNORs) in prostate carcinoma of low malignancy. 390 x.
Fig. 18. Increased number of AgNORs in prostatic carcinoma with high grade of malignancy. 390 x.
achieved similar results using the same methods . An increase in nucleolar size has also been observed9,28 (Tab. 4, 5; Fig. 16). Similar results have been found by determining the number of silver-stained nucleolar organizer regions (AgNORs per unit area of nucleus). In the succession from benign hyperplasia to atypical hyperplasia, PIN, highly differentiated carcinoma and poorly differentiated carci-
noma, there is a distinct increase in the number of AgNORs per unit area of nucleus. Here, too, similar results were obtained by different researchers using varying procedures. However, this method is not adequate to determine regressive changes in proliferative potential under therapy. It also cannot measure differences between carcinomas with similar proliferation activity 1, 5, 9,13,35; (Tab. 6) (Figs. 17, 18).
Table 5. Mean diameter, frequency of nuclei with nucleoli and number of nucleoli per nucleus of prostatic tissue: modified to Deschenes and Weidner (1990), Montironi et al (1991) Histology
Mean diameter of Frequency (%) of nuclei with nucleoli nucleoli (UM)
Number of nucleoli per nucleus N1
N2
N3
BPH* PAH"* PIN*""
0.6-1.9 0.8-2.7 1.1-2.9
16.1 ± 5.1 57.7 ± 8.9 58.7 ± 7.6
96.0 ± 4.6 85.5 ± 7.5 83.4 ± 4.6
3.9 ±4.6 13.7 ± 7.4 15.1 ± 4.0
0.8 ± 1.1 1.8 ± 1.4
Carcinoma G IB -IIA G II B-I1IA,B
1.2-2.1 3.3
78.6 ± 7.5
80.4 ± 7.9
16.4 ± 5.9
3.2 ±2.6
* Benign prostatic hyperplasia; * * prostatic atypical hyperplasia; * * * prostatic intraepithelial neoplasia
Prostate Carcinoma . 505 Table 6. Nucleolar and AgNOR pattern of prostatic tissue Histology
N
Nucleoli Frequency/ nucleus
Number/ nucleus
Location in nucleus
N
AgNOR per unit area of nucleus
Typical Hyperplasia (BPH)
10
0.5 ± 1.2
solitary
central
10
1.7 ± 0.5
Atypical Hyperplasia (Transition Zone) mild moderate-marked
55 24
1.6± 0.4 5.3 ± 1.3
solitary solitary
central central
10
2.1 ± O.S
Prostatic intra epithelial Neoplasia moderate-marked
27
20.0± 6.4
solitary multiple
central eccentric
10
4.3 ± 1.S
Carcinomas Grading Ib-IIa
113
41.4 ± 12.5
solitary multiple
central eccentric
34
3.5 ± 0.5
Grading lIb-III
72
SO.l ± 15.6
multiple
eccentric
37
5.1 ±0.7
Another important cell kinetic method is the utilization of flow cytometry or single cell DNA cytophotometry. If we recognize the presently accepted subdivision of DNA values into nearly diploid, hypertetraploid and aneuploid, the highest percentage of diploid DNA values are found in the highly differentiated prostatic carcinomas. 20 to 25 % of highly differentiated prostatic carcinomas have aneuploid DNA values. Poorly differentiated prostatic carcinomas in the high risk group are solely aneuploid. The GIl group, which is difficult to interpret, shows varying fractions of diploid, hypertetraploid and aneuploid values. It is particularly in this group that an increase in grade of aneuploidy with transition to progression can be seen after two to three years of observation. Because of the heterogeneous tumor structure, single cell cytophotometry should be utilized on several different sectors of the tumor for its evaluation 8, 13. The classification of prostatic carcinoma should only be carried out in conjunction with the usual histological and cytological findings. It is best to utilize both flow cytometry and single cell cytometry. In addition, DNA analysis is important for evaluating response to the therapy. DNA ploidy values offer prognostic information 12 • By applying all these described methods, we believe that there is obvious value in the subclassification and subgrading of prostatic carcinoma, especially the division into high and low-risk groups18. Prostatic carcinoma with higher aneuploid values in the group of highly differentiated tumors can belong to those that progress earlier, and can be the small percentage of the low risk tumors that do lead to death. More detailed studies are necessary in order to obtain more precise values. Cytogenetics
Cytogenetic studies will surely play an important role in future research on prostatic carcinoma. By utilizing DNA hybridization techniques, more than 50 % of karyotyped prostatic carcinomas show numerical
aberrations, and to a lesser extent, structural changes (Sesterhenn et al1992). The loss of chromosomes 1,2,5 and Y; addition of chromosomes 7, 14,20 and 22; and translocation of 7p, 7q and 10q have been observed in prostatic carcinoma. Other researchers have discovered deletion, duplication, inversion and translocation mainly in chromosomes 2,7,10 and 16. Mutation of the gene p53 on chromosome 7p can be seen more often in metastasizing prostatic carcinoma13,31. Research up to now has shown that no single chromosomal change can be made responsible for prostatic carcinoma. There is no karyotypemarker for carcinoma of the prostate gland. Lundgren et aF3 found that patients with only one clonal karyotypical abnormality live on average 1.3 years after diagnosis, whereas carcinoma patients with a combination of normal and abnormal karyotypes generally survive another 3 years. Oncogenetics
Studies on oncogenes in prostatic tissue are just beginning34 . Some oncogenes are expressed in both benign and malignant prostatic tissues, as for instance c-erbB24o. DNA in situ hybridization techniques have found differing results on prot%ncogenes. Oncogenes are genes which have the ability to transform cells neoplastically. Nuclear oncogenes modulate DNA transcription and influence the phases of the cell cycle. Cytoplasmic oncogenes activate tumorgenesis. Both together can induce neoplasms. For example, c-myc and c-fos are nuclear oncogenes and vH-ras is a cytoplasmic gene. V-erb-B-oncogene codes a part of the receptor for epidermal growth factor. Cellular oncogenes are of major influence in the regulation of cellular growth 13 (Tab. 7). In one case studied, a highly differentiated (GI) prostatic carcinoma was found to release a large amount of c-myc mRNA and c-sis m RNA, with small amounts of c-fos mRNA. Interestingly, an increased expression of c-sis and c-fos mRNA in hyperplastic prostatic glands was found in the vicinity of prostatic carcinoma. This appears to show
506 . B. Helpap Table 7. Expression of oncogenes in prostatic carcinoma (Pc) Grading
V-ErbBC-Fos C-Myc C-sis
Low Grade PC + +-(+) High Grade PC +++ + ++
VHras Kras
++
+
++
(+)
that local production of growth factor for prostatic cells plays an important role in the proliferative activity of prostatic carcinoma16. Activation of K-ras-oncogenes in conjunction with an increased ras-P-21 expression has been observed in latent pro~tatic c~rcinoma 'Yith low grade of malignancy as well as m carcmomas wIth both higher staging and grading3,4,22,37. Deletion of genetic material from a chromosome can lead to a loss of impeding gene sequences in the vicinity of ?ncogenes. The loss of tumor suppression genes is an Important event, since it can trigger tumor production. Tumor suppressor antigens are also called antioncogenes. Suppressor genes code a protein that is essential for normal c~llul~r growth behavior. Its loss induces tumor progresSIOn, l.e. progression of prostatic tumor tissue 13 ,21. A crucial step in tumor formation is the uncontrolled cell div~sion. This. res?l~s in aberration of chromosomal replicatIon and mItosIS m the neoplastic cells. A decrease in tumor suppressor genes has been found in car~inoma. This shows similar behavior to blood group antIgens. In BPH, blood group antigens Lewis a and bare released in great amounts. This expression is decreased in the vicinity of carcinoma, weak in highly differentiated prostatic carcinoma, and absent in poorly differentiated prostatic carcinoma l ,20 (Tab. 8). Proto-oncogene-protein R-erb-B related to epidermal growth factor receptor shows the opposite behavior. Its expression is weak in benign tumors and strong in undifferentiated carcinomas. No differences were seen between highly differentiated carcinoma and regions of BPI! near carcinoma. The behavior of blood group antigens supports the division into low and high risk groups20,38. The expression pattern of androgen receptors also play an important role in postsurgical hormone therapy or ~hemotherapy. Cases with positive receptors correlate to responders and negative receptors to non-responders. The importance of other receptors such as prolactine receptors or estramustine binding protein receptor regarding malignancy and response to therapy must still be evaluated 1,13.
Histology, Immunohistochemistry, Prognosis Th~ pre?ictiv.e v~lue ~f th~ discussed investigative
~echm9ues IS mamly m conjUnctIon with ordinary prostatIC carCl~oma. ~he rare squamous cell or urothelial type of
prostatic carcmoma are not included in these evulations. One. tU!ll0r type should still be mentioned. Recently more fllldlllgS have been made on prostatic carcinoma with neuroendocrine cells 32 . As in other organs, eosinophilic g~anulated Grimelius-Silver-stained cells have been recogmzed be~een s.ecretory cells in benign prostate changes as wel~ ~s m carcmoma. T~ese are immunohistochemically posltlve for chromogranm A and neuron specific enolase (Figs. 19,20). These cells can also secrete a number of other neuroendocrine proteins. These neuroendocrine cells a.re present in high percentages in carcinomas. A coexpresSlon of PS~ and neuroendocrine markers, especially chr?I?ogranlll~, has. been found. Interestingly, no nuclei posltlve for prohferation marker Ki 67 or PCNA have been recognized. On the other hand, positivity has been found in
Table 8. Expression of blood group antigens in prostatic carcinoma Grading
Blood group antigens A
Low grade PC + High grade PC 0/( +)
B
+
0/(+)
H
+
0/(+)
Lewis A Lewis B
+
0/(+)
+
0/(+)
Fig. 19. Glandular carcinoma of the prostate gland with neuroendocrine differentiation. Grimelius-silver-staining method. 200 x.
Prostate Carcinoma . 507
non-neuroendocrinally differentiated typical prostate tumor cells near the neuroendocrine tumor cells. It is suspected that the neuroendocrine cells secrete substances that stimulate proliferation kinetics. Since the neuroendocrinologically differentiated tumor cells do not respond to hormonal therapy, these types of carcinoma should be classified into the group of non-responders. Cohen and coworkers 6, 7 could observe a poorer prognosis for patients with neuroendocrinally differentiated prostatic carcinomas in contrast to prostatic carcinomas which were negative for neuroendocrine cells. Finding a therapeutic method to treat these endocrinally differentiated prostatic carcinomas is a task for the future. Conclusion
Let us review what is now possible and what goals future research has. I believe that primary diagnosis of prostatic carcinoma with specification of grade and volume is of great importance. Analyses have shown that the grading methods used now can be made more precise through a number of additional tests, yet methods such as DNA ploidy measurement or cytogenetic studies cannot be used as a replacement. Only the combination of all the methods
Fig. 20. Glandular carcinoma of the prostate gland with neuroendocrine differentiation. Immunohistochemical analysis with chromogranin A. ABC-method.250 x.
can improve our knowledge about the nature of carcinoma of the prostate gland. The significance of GI prostatic carcinoma with an aneuploid DNA pattern is not yet clear. We can only suspect that this group has a greater tendency for progression than diploid GI-tumors. On the other hand, the question arises whether diploid GIll tumors exist and whether the diploid fraction is significant or negligable. I believe that the cases of high and low malignancy prostatic carcinoma should be studied in order to structure a clinically orientated therapy plan. The many questions concerning the pathogenesis of prostatic carcinoma and molecular pathological diagnostic steps including genetic studies should be continued, especially for their clinically orientated value. I believe that this provides an adequate stimulus for future research directions 14,18 (Tab. 9).
References 1 Allsbrook WC, Simms WW (1992) Histochemistry of the prostate. Hum Pathol 23: 297-305 2 Brawer MK (1992) Prostatic intraepithelial neoplasia. A premalignant lesion. Hum Pathol 23 : 242-248 3 Bushmann EW, Bushmann WA, Kingston TE, Lippitt Rg, Chiapella CD, Stein BS (1989) Ras (p 21) expression in proliferative lesions of the prostate. J Urol 141: 205 A 4 Carter BS, Epstein JI, Isaacs WB (1990) Ras gene mutations in human prostate cancer. Cancer Res 50: 6830-6832 5 ChevilleJC, Calmon GH, Robinson RA (1990) Silver stained nucleolar organizer regions in the differentiation of prostatic hyperplasia, intraepithelial neoplasia and adenocarcinoma. Mod Pathol3: 596-598 6 Cohen RJ, Glezerson G, Haffejee Z, Afrika D (1990) Prostatic carcinoma. Histological and immunohistological factors affecting prognosis. Br J Urol 66: 405-410 7 Cohen RJ, Glezerson G, Haffejee Z (1991) Neuroendocrine cells - a new prognostic parameter in prostatic cancer. Br J Urol 68: 258-262 8 Deitch AD, Vere White de RW (1992) Flow cytometry as a predictive modality in prostate cancer. Hum Pathol 23: 352-359 9 Deschenes J, Weidner N (1990) Nucleolar organizer regions (NOR) in hyperplastic and neoplastic prostate disease. Am J Surg Pathol14: 1148-1155 10 Epstein JI (1990) Evaluation of radical prostatectomy capsular margins of resection - the significance of margins designated as negative, closely approaching, and positive. Am J Surg Pathol14: 626-632 11 Epstein JI, Steinberg GD (1990) The significance of lowgrade prostate cancer on needle biopsy. A radical prostatectomy study of tumor grade, volume, and stage of the biopsied and multifocal tumor. Cancer 66: 1927-1932 12 Falkmer UG (1992) Methodologic sources of errors in image and flow cytometric DNA assessments of the malignancy potential of prostatic carcinoma. Hum Pathol 23: 360-367 13 Foster CS, Abel PD (1992) Clinical and molecular techniques for diagnosis and monitoring of prostatic cancer. Hum Pathol 23: 395-401 14 Foster CS, Mostofi FK (1992) Prostate cancer - Quo vadis? Hum Pathol 23: 402-406 15 Foster CS,McLoughlin J, Bashir I, Abel PD (1992) Markers of the metastatic phenotype in prostate cancer. Hum Pathol 23: 381-394
508 . B. Helpap Table 9. Morphologic, cell kinetic and immunohistochemical differential diagnosis criteria of low and high malignant carcinomas [18] Low malignant carcinomas
High malignant carcinomas
IA,IB,II A High to moderate differentiation Slight atypias nucleus uniform nuclei mild nuclear chromasia nuclear-cytoplasmic ratio 0.25 nucleolus low frequency central localization one nucleolus per nucleus
IIB,I1IA,I1IB (IV) Low to no differentiation Marked atypias Nucleus marked variability of nuclear shape increased chromasia nuclear-cytoplasmic ratio more than 0.75 nucleolus increased frequency eccentric localization multiple nucleoli per nucleus
Low cell proliferation DNA eu(an-)ploidy low frequency of mitoses low 3H-thymidine labelling index low Ki 67 growth fraction long duration of S phase low number of AgNORs
Increased cell proliferation DNA aneuploidy increased frequency of mitoses increased 3H-thymidine labelling index increased Ki 67 growth fraction short duration of S-phase increased number of AgNORs
Cell loss
Low index of apoptosis
Increased index of apoptosis
Immunohistochemistry
Homogeneous expression of antigens Diminished ABO blood group antigens Diminished suppressor antigens
Heterogeneous expression of antigens Lack of ABO blood group antigens Lack of supressor antigens
Histochemistry
Lectin binding sites
Lack of lectin binding sites
Prognosis
Favorable rare recurrences or metastases low DoD rates
Unfavorable frequent recurrences or metastases high DoD rates
Sub grading Histology Cytology
Cell kinetics
16 Funa K, Nordgren H, Nilsson S (1991) In situ expression of mRNA for protooncogenes in benign prostatic hyperplasia and in prostatic carcinoma. Scand J Urol Nephrol25: 95-100 17 Gleason DF (1992) Histologic grading of prostate cancer. A perspective. Hum Pathol 23: 273-279 18 Helpap B (1992) Grading and prognostic significance of urologic carcinomas. Urol Int 48: 245-257 19 Helpap B, Koch V (1991) Histological and immunohistochemical findings of prostatic carcinoma after external or interstitial radiotherapy. J Cancer Res Clin Onco1117: 608-614 20 Idikio HA, Manickavel V (1991) Correlation of blood group antigen expression and oncogene-related proteins in malignant prostatic tissues. Path Res Pract 187: 189-197 21 Isaacs WE, Carter BS, Ewing CM (1991) Wild-type p53 suppresses growth of human prostate cells containing mutant p53 alleles. Cancer Res 51: 4716-4720 22 Konishi N, Enomoto T, Buzard G, Ohshima M, Ward JM, Rice JM (1992) K-ras activation and ras p21 expression in latent prostatic carcinoma in Japanese men. Cancer 69: 2293-2299 23 Lundgren R, Kristofferson U, Heim S, Mendahl N, Mitelman F (1988) Multiple structured chromosome rearrangements including del (7q) and del (lOp) in an adenocarcinoma of the prostate. Cancer Genet Cytogenet 35: 103-109 24 McNeal JE, Redwine EA, Freiha FS, Stamey TA (1988) Zonal distribution of prostatic adenocarcinoma. Correlation with histologic pattern and direction of spread. Am J Surg Pathol 12 (12): 897-906 25 McNeal JE (1988) Normal histology of the prostate. Am J Surg Pathol 12: 619-633
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Received November 4, 1992 . Accepted January 19, 1993
Key words: Prostate carcinoma - DNA analysis - AgNOR - nucleolar grading. Prof. Dr. B. Helpap, PO Box 720, W-7700 Singen, FRG