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Clinical Cancer Research Vol. 10, 1064-1073, February 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Genetic Pathways and New Progression Markers for Prostate Cancer Suggested by Microsatellite Allelotyping

Rolf von Knobloch1, Lutz Konrad1, Peter J. Barth2, Heidrun Brandt1, Sebastian Wille1, Axel Heidenreich1, Roland Moll2 and Rainer Hofmann1

1 Departments of Urology and 2 Pathology, Philipps-University Marburg Medical School, Marburg, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: At diagnosis, the biological behavior of prostate cancer is uncertain, making the choice of an adequate therapy option difficult. Performing microsatellite allelotyping on a large series of consecutive prostate cancers procured during radical prostatectomy at our institution, we sought to identify molecular markers associated with disease progression.

Experimental Design: A total of 156 consecutive fresh tumor samples was prospectively collected and macroscopically dissected from the whole prostatectomy specimen immediately after operation. Histologically 100 samples contained >75% tumor cells and were therefore enrolled in the microsatellite allelotyping, using a total of 24 polymorphic markers for the chromosomal regions 5p, 5q, 7q, 8p, 9p, 9q, 13q, 17p, 17q, and 18q. Fresh paired normal and tumor DNA was investigated in fluorescent microsatellite analysis with automated laser product detection.

Results: The incidence of tumor–DNA alterations [loss of heterozygosity or allelic imbalance (AI)] was highest for chromosomal regions 13q and 8p with 72 and 71%, respectively, followed by chromosomes 7q, 18q, 5q, and 17p with 57, 53, 41, and 39%, respectively. Alterations at chromosomes 8p, 9p, 13q, and 17p were significantly (P < 0.05) associated with advanced tumor stage, whereas AI at 8p and 17p was also associated with high Gleason score (P < 0.05). AI at 5q and 9p was associated with regional lymph node metastasis (P < 0.05). The combination of AI at 8p and 13q was strongly associated with advanced tumor stage (P < 0.0001).

Conclusions: With the obtained results, we are able to postulate three distinct pathways in prostate carcinogenesis, and we identified microsatellite markers of prognostic value.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostate cancer (CaP) is one of the leading causes of male cancer death in western countries. In the era of prostate-specific antigen screening, more patients are being diagnosed with early stage disease. Because of the uncertain biological behavior of this tumor entity, estimating prognosis at time of diagnosis is difficult, rendering decision making on various therapy options a challenging task. Discriminating aggressive and potentially progressive tumors from tumors with a low threat of progression is crucial when planning nerve-sparing radical prostatectomy procedures to preserve potency in younger men harboring cancer of the prostate. The broad application of cytogenetic and molecular genetic methods has led to the identification of tumor-associated chromosomal regions substantial for the tumorigenesis of CaP (1, 2, 3, 4, 5) . Comparative genomic hybridization predominantly showed losses at chromosomes 5q, 6q, 8p, 10q, 13q, 16q, and 18q to be of importance in CaP initiation and progression (4, 5, 6) . Allelotyping studies applying microsatellite analysis were able to confirm the obtained results and further delineated the tumor-associated regions (2 , 7, 8, 9) . Nevertheless, the genetics of CaP are not completely understood, therefore demanding additional investigations on large cohorts of CaP specimens of various pathological stages. The heterogeneity of the tumor and a large proportion of accompanying stromal and glandular cells harboring normal unaltered DNA have an adverse influence on procuring pure DNA from CaP specimens after radical prostatectomy. These CaP specimen characteristics warrant the application of microdissection techniques. As a result, most of the studies investigating genomic DNA in CaP were performed on archival paraffin-embedded material. In contrast to most previous studies, we undertook a prospective investigation sampling "fresh" DNA during surgery for clinically localized CaP on a large cohort of 156 consecutive men receiving radical retropubic prostatectomy at our institution. By this strategy, we were able to collect large amounts of CaP DNA from untreated and unfixed tumor tissue and cancers of all pathological stages. This also implies that the cohort comprised more early stage disease rather than progressive disease stages. To further improve the method of microsatellite analysis (MSA), we applied computer-assisted laser detection of the PCR products, allowing a highly sensitive identification of allelic imbalance (AI). With the underlying comprehensive allelotyping study of chromosomal regions implicated in the tumorigenesis of CaP, our goal was to identify marker regions significantly associated with adverse pathological features at the time of surgery. Besides identifying tumor-associated molecular marker sites, we are also able to propose possible genetic pathways for prostate carcinogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor and Blood Sampling.
Between 1999 and 2002, preoperative peripheral blood (10 ml of EDTA blood) and fresh tumor samples were prospectively collected from 156 consecutive men undergoing radical retropubic ascending prostatectomy at our institution for sporadic adenocarcinoma of the prostate. An extended regional lymphadenectomy was performed in every case. The fresh tumor samples were macroscopically dissected from the prostatectomy specimen immediately after operation by a pathologist (P. J. B.). Without further delay, all samples were then shock frozen in liquid nitrogen and stored at -80°C before further processing. A representative piece of the CaP sample was H&E stained after cryosectioning. Histologically, only 100 of the initial 156 samples contained >75% tumor cells and were therefore enrolled in the MSA. The pathohistological work up of the main tumor specimen was performed by the Department of Pathology at the Philipps-University Marburg Medical School according to the Unio Internationale Contra Cancrum classification of 1997. Informed consent was obtained from the patients, and the tissue sampling was approved by the ethics committee of the Philipps-University Marburg Medical School (No. 39/03).

DNA Isolation.
The DNA isolation was performed as reported previously for bladder and kidney tumors (10 , 11) . Briefly, a small piece (~5 x 5 x 2 mm) of frozen tumor tissue was allowed to thaw in TE9 buffer in a Petri dish. The tumor cells then were carefully scraped and pressed from the stromal tissue. Afterward, the tumor cells were resuspended in TE9 buffer and incubated in 2% SDS plus 0.5 mg/ml proteinase-K for >=3 h at 56°C. After digestion, the DNA was isolated by the phenol–chloroform method with final ethanol precipitation. Extracted DNA was resuspended in TE1 buffer. By this technique, the contamination of the tumor DNA with unaltered normal stromal DNA was reduced to a minimum. To obtain the corresponding normal DNA, the same method was applied to peripheral blood lymphocytes from the EDTA blood samples.

MSA and PCR Conditions.
For the identification of CaP-DNA AI or loss of heterozygosity (LOH), 24 highly polymorphic markers for the chromosomal regions 5p, 5q, 8p, 9p, 9q, 13q, 17p, 17q, and 18q (D5S1460, D5S1720, D5S818, D5S476, D7S1797, D7S1796, D7S1837, D7S1807, D8S264, D8S261, D8S1477, D9S925, D9S171, D9S15, D9S1826, D13S153, D13S317, D13S796, D17S1298, D17S799, D17S1306, D18S851, D18S846, and D18S858) were used in the PCR-based MSA (Table 1)Citation . If possible, tetranucleotide rather than dinucleotide markers (tandem repeats) were chosen. DNA sequences for the microsatellite markers were obtained from the genome database.3 Markers D5S1460 for chromosome 5p and marker D17S1306 for 17q were used as background markers to identify rates of incidental genetic events at loci not known to be over-represented in CaP. Fifty to 100 ng of normal and tumor DNA were used as templates in 10 µl of PCR reactions as reported previously (10 , 11) . PCR amplifications were performed using the same conditions for all microsatellites in a PTC100 thermocycler (MJ Research, Watertown, MA). Four microliters of the PCR products were then separated on 8% polyacrylamide gels (ReproGel "long read"; Amersham Pharmacia Biotech, Freiburg, Germany) at constant 1500 V, 60 mA, 30 W, and 55°C gel temperature on 200-mm gel cassettes in 0.5x Tris-borate EDTA buffer for 5 h. Fragment analysis was performed on an automated DNA laser sequencer (ALFexpressII; Amersham Pharmacia Biotech). Results were computed using the Fragment Manager (FM 1.02; Amersham Pharmacia Biotech) software. AI in heterozygous PCR products was described as LOH or deletion, when loss of genetic information was known to occur for this chromosomal region, but AI could also describe differences in allele intensity caused by genetic gain from, e.g., duplication, when known for the specific site. AI caused by either loss or gain was summarized as alteration of the genetic locus.


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  		Table 1 Microsatellite markers used for PCRs

 
The association of each microsatellite marker with the pathological features Gleason score, pT stage, and pN status was evaluated independently and in combination with the other markers for the chromosome arm.

Statistical Analysis.
To identify possible associations between detected chromosomal and molecular alterations with tumor stage, Gleason score, and lymph node metastasis, the Mann-Whitney and Kruskal-Wallis tests were used as applicable. A P < 0.05 was regarded as significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pathological Distribution of Cancer Specimens.
The distribution of the analyzed CaP specimens according to the Unio Internationale Contra Cancrum classification of 1997 is shown in Table 2Citation . The high percentage of tumors of higher grade and pathological stage does not represent the true distribution of cancer stages within our operative series. It is a result of advanced stage tumors being more suitable for the procurement of tumor samples attributable to higher tumor volumes. Initially, 156 tumor samples were harvested, but histologically, only 100 samples proved to contain >75% tumor cells. The high rate of locally advanced and high-grade tumors is of value for defining markers associated with disease progression.


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  		Table 2 Distribution of 100 prostate cancers analyzed in microsatellite allelotyping according to the TNM system (UICC 1997)a

 
AI.
Incidences of AI at the 24 loci investigated together with the heterozygosity rates of the microsatellite markers, as well as the incidences of alterations for the 10 investigated chromosome arms in 100 different CaP specimens, are given in Table 3Citation . All but three cancer DNA specimens exhibited alterations at least at one examined locus. Interpreting our results cytogenetically, the highest incidences of AI were observed for chromosomes 8p and 13q with 71 and 72%, respectively. Alterations at chromosomes 5q, 7q, 17p, and 18q were seen in 37% (5q) to 57% (7q). The lowest incidences of AI were observed for chromosomes 9p, 9q, and 17q in 17% (9q) to 25% (17q) of cases. (Table 3Citation and Figs. 2Citation and 3Citation ). The background marker D5S1460 at 5p led to the identification of AI in 31% but only in conjunction with a heterozygosity rate of 62% (Table 3Citation and Figs. 2Citation and 3Citation ). The highest incidence of AI for a single microsatellite marker was found for D13S153 at chromosome 13q14.1 located in the second intron of the RB tumor suppressor gene (Table 3Citation ; Figs. 1Citation , 2Citation , and 3Citation ). Markers D8S264 (8p23), D8S261 (8p22), and D13S317 (13q22) also displayed AI in >50%, accompanied by heterozygosity rates > 80% (Table 3)Citation . Curves obtained from the computer-assisted evaluation of the MSA PCR products after gel electrophoresis in a laser sequencer (ALFexpressII; Amersham-Pharmacia Biotech) at four marker sites for three paired normal and tumor specimens are shown in Fig. 1Citation . The clear loss of one allele in the MSA demonstrates the amplification of pure ("clean") tumor DNA without contamination from normal DNA. There was no observation of widespread microsatellite instability.


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  		Table 3 Statistical association of alteration at chromosome arm and microsatellite locus with Gleason score, pathological stage, and lymph node metastasis (Mann-Whitney test)

 


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  		Fig. 2. A, incidence of loss of heterozygosity and allelic imbalance for chromosomal arms investigated with a total of 24 microsatellite markers (as shown in Table 3Citation ). B, incidence of loss of heterozygosity and allelic imbalance stratified for organ-confined (pT2) versus nonorgan-confined local tumor stage (>pT2). Incidence of loss of heterozygosity and allelic imbalance stratified for Gleason score < 7 versus >=7.

 


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  		Fig. 3. A, incidence of loss of heterozygosity and allelic imbalance (AI) for chromosome arms and microsatellite markers significantly associated with local tumor stage stratified for organ-confined (pT2) and nonorgan-confined (>pT2) disease. B, incidence of loss of heterozygosity and AI for chromosome arms and microsatellite markers significantly associated with Gleason score (<7 versus >=7). C, incidence of loss of heterozygosity and AI for chromosome arms and microsatellite markers significantly associated with lymph node metastasis (pN0 versus pN1).

 


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  		Fig. 1. Results of fluorescent microsatellite analysis for prostate cancer (CaP)-271 (pT2apN0, Gl 7), CaP-272 (pT2bpN0, Gl 5), and CaP-274 (pT3bpN1, Gl 6). Curves for normal-DNA (N) from circulating blood lymphocytes and tumor DNA (T) are shown. CaP-271 demonstrates clear loss (loss of heterozygosity) of allele-2 in tumor for marker D8S1477 and loss of allele-1 for markers D13S153 and D13S317. Retention of constitutional heterozygosity was observed for locus D17S799. CaP-272 demonstrates clear loss of allele-1 in tumor for marker D8S1477 and loss of allele-2 for marker D13S153. Retention of constitutional heterozygosity was observed for locus D17S799, whereas marker D13S317 was not informative. CaP-274 demonstrates allelic imbalance for markers D8S1477 and D17S799 with retention of heterozygosity at loci D13S153 and D13S317. Clear loss of one allele in tumors CaP-271 and CaP-272 documents pure tumor DNA without contamination from normal DNA.

 
Correlation of AI and LOH with Clinicopathological Features of the Prostatectomy Specimens.
Statistical evaluation rendered AI or LOH at chromosomes 5p, 8p, 9p, 13q, and 17p to be significantly associated with advanced local tumor stages (P < 0.05; Table 3Citation ; Figs. 2Citation and 3Citation ). Looking at the different microsatellite markers independently showed markers D5S1460 (5p13.1), D5S476 (5q31.1), D7S1807 (7q36), D9S171 (9p13), D13S153 (13q14.1), D17S799 (17p12–13.1), and D18S858 (18q21) to each be significantly associated with local tumor stage (P < 0.05; Table 3Citation ; Fig. 3Citation ). Only AI at chromosomes 8p and 17p was significantly associated with the Gleason score of the prostatectomy specimen, and here, only the marker D17S1298 (17p13.3) displayed an association independently (P < 0.05; Table 3Citation ). Regional lymph node metastasis was over-represented in tumors exhibiting AI at chromosomes 5q and 9p (P < 0.05; Table 3Citation ; Figs. 2Citation and 3Citation ). Markers demonstrating a significant association with regional lymph node metastasis were D5S476 (5q31.1), D9S171 (9p13), and D17S799 (17p12–13.1). With a P of 0.05, marker D13S796 (13q33) was just slightly above statistical significance (Table 3Citation ; Fig. 3Citation ). Not shown in Table 3Citation is a highly significant association of a combination of LOH at 8p and 13q with advanced tumor stage (P < 0.0001) and with tumor Gleason score (P = 0.015). Besides the correlation of AI at distinct chromosomal loci with adverse pathological tumor features, the total number of genetic events per tumor investigated was significantly associated with advanced tumor stage but not with Gleason score or regional lymph node metastasis. When looking at Gleason score as a predictor of advanced local tumor stage, the level of statistical significance (association with advanced local tumor stage) is lower than for the prognostic markers delineated above (P < 0.038).

Association with Disease Progression.
To date, we can only provide data on disease courses with a mean follow-up of 32.8 months (11–54 months), which is yet insufficient for CaP. Still of interest, during this period, a disease progression (either rise in serum prostate-specific antigen or morphological progress) was observed in a total of nine patients. All but one patient with cancer progression had loss of chromosome 8p, and of the eight patients with 8p AI, the combination with loss of chromosome 13q was present in seven. Of four patients with recognized distant progress, three had loss of chromosome 9p at time of surgery.

Genetic Pathways in CaP.
With incidences of AI at 8p and 13q being >70%, we assume alterations at these chromosomes to represent early events in the tumorigenesis of CaP. Furthermore, losses of chromosomes 8p and 13q were identified in combination in >80% of cases (significant association with advanced tumor stage, P < 0.0001). The genetic events observed in the analysis of this large cohort of prostatectomy specimens give us the opportunity to postulate three distinct genetic pathways in the carcinogenesis of CaP, which are summarized in Fig. 4Citation . We suggest carcinogenesis to be initiated either by loss of chromosome 8p or 13q or alternatively via alterations of chromosome 7q, 17p, or 18q. In the main pathways with loss at either 8p or 13q and especially the combination of both alterations, AIs at 7q, 17p, or 18q represent genetic events leading to local tumor progression. Additional losses of chromosome 5q, 9p, or 17p may cause distant tumor spread (Fig. 4)Citation .



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  		Fig. 4. Genetic pathways suggested for the development and progression of prostate cancer limited to the chromosomal regions investigated in this study. With incidences of >70%, losses of chromosomes 8p and 13q are postulated as early genetic events (two major pathways). In ~80% of cases, 8p and 13q loss of heterozygosity are identified in combination. The combination of these two genetic events is significantly associated with advanced local tumor stage (P < 0.0001). The synergistic effect of these two chromosomal alterations may therefore be important for local tumor progress. Local tumor progress may also be caused by either loss of chromosome 7q, 17p, or 18q within the two major pathways, when 8p and 13q are not combined. Losses of chromosome 7q, 17p, or 18q may also be genetic events initiating carcinogenesis in the alternative genetic pathway in ~13% of prostate cancers (middle). With alterations of chromosomes 5q, 9p, and 17p being significantly associated with lymph node metastasis, these genetic events are postulated to lead to distant tumor spread in all three pathways. Note, that 87% of prostate cancers in this study either had loss of chromosome 8p or 13q, and in 80%, chromosomal loss of 8p and 13q was observed in combination. The highly significant association of allelic imbalance at chromosome 17p and the combination of 8p and 13q loss of heterozygosity with Gleason score (P < 0.02) is pointed out at the bottom of the figure by "loss of tumor differentiation."

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the underlying study, we performed a microsatellite allelotyping of chromosomal regions known to be involved in the carcinogenesis of the prostate by analyzing 100 paired normal and tumor DNA samples with a total of 24 markers for chromosomes 5p, 5q, 7q, 8p, 9p, 9q, 13q, 17p, 17q, and 18q. In contrast to most previous reports applying the same molecular genetic method for investigating the genetics of CaP, we procured fresh tumor specimens during operation and performed the MSA with the highly sensitive laser detection of fluorescently labeled PCR products. By this prospective methodological approach, we were able to collect large amounts of tumor DNA not subjected to potentially deteriorating processes during the formalin fixation and paraffin embedding for histological work up. With our analysis of 100 paired CaP tumor and normal DNA specimens, we are able to present one of the largest cohorts of primary sporadic CaP prospectively investigated.

Early chromosomal investigations on the genetics of CaP implicated losses of chromosomes 7q and 10q to play a substantial role in the development of this tumor entity (12 , 13) . The understanding of prostate carcinogenesis became more profound with the introduction of comparative genomic hybridization, allowing the analysis of the whole karyotype from archival CaP specimens. Large comparative genomic hybridization studies observed mainly losses of chromosomes 2q, 5q, 6q, 8p, 10q, 13q, 16q, and 18q to be important in CaP development (4 , 5) . Chromosomes exhibiting the highest incidence of allelic loss were 8p and 13q in >50% of the investigated cases (for review, see Ref. 1 ). Comprehensive allelotyping studies and the simultaneous application of molecular techniques confirmed the results obtained with comparative genomic hybridization (2 , 3 , 5) .

In accordance to rates given in literature, we identified LOH at chromosomes 8p and 13q to have the highest incidence throughout all stages of CaP with 71 and 72%, respectively (2 , 3 , 7 , 8 , 14, 15, 16) . In an earlier comprehensive allelotyping study, the incidence of 8p LOH with 69% in 55 primary CaPs almost matched the incidence observed in this study (3) . In two more recent studies investigating primary CaPs, the incidences for chromosome 8p loss varied roughly between 30 and 50% (2 , 16) . For chromosome 13q, the reported incidences of LOH differed between 20 and 70% (2 , 3 , 8 , 15 , 17) . To the best of our knowledge, none of the previous investigations identified similarly high incidences for the two genetic events in the same cohort, although the combination of allelic loss at 8p and 13q has been pointed out previously (18) . With incidences of AI at 8p and 13q in excess of 70% and the observed combination of both alterations in >80%, we suggest these two genetic events to represent early initiating events, although both aberrations are found predominantly in advanced tumor stages (P < 0.05; Table 3Citation ; Figs. 1Citation 2Citation 3Citation 4Citation ). If both alterations are present in combination, a highly significant association with advanced tumor stage is identified (P < 0.0001; Figs. 2Citation and 3Citation ), suggesting a synergistic mechanism of both genetic events or synergistic tumor genes residing at these two loci responsible for the progression of CaP. To our knowledge, an interaction of these two chromosomal loci to date has not been postulated for CaP.

Although extensive research has gone into the identification of potential tumor genes for CaP located on 8p and 13q, an association of specific gene mutations with the narrowed chromosomal loci delineated in mapping studies has not been found. Refined chromosomal loci on 8p postulated to harbor putative tumor genes are 8p12–21 and 8p22 (14 , 19, 20, 21, 22) . The three microsatellite markers used in the underlying study span the two implicated tumor loci on 8p, and with incidences of 46–56% for AI at these loci, we are able to confirm the previous results (Table 3Citation ; Figs. 2Citation and 3Citation ). Despite the fact that loss of chromosome 8p was discovered to have the highest incidence within the tested chromosomal loci throughout all tumor stages and differentiation grades, statistical analysis identified a significant association of this alteration with these pathological features (P < 0.05; Table 3Citation ; Figs. 2Citation and 3Citation ). Of interest, none of the three microsatellite markers alone showed a significant association. In some previous publications, an association of LOH at 8p with advanced local tumor stage or disease progression was also identified (7 , 23) . Some investigators on the other hand did not discover an association and interpreted the high incidence of this genetic event to represent an early alteration in the carcinogenesis of the prostate, which also is our notion (14 , 20) .

Common regions of allelic deletion for chromosome 13q are 13q14.3 just telomeric to the retinoblastoma locus, 13q21–22, and 13q33 (8 , 15 , 17 , 24, 25, 26, 27) . Again, a specific tumor-associated gene residing at these loci has not been revealed. However, it has been assumed that loss at 13q14 unmasks recessive tumor suppressor gene function, resulting in uncontrolled cell growth and proliferation. Again, the three microsatellite markers applied for the allelotype at 13q in this study closely mapped to the three tumor-associated regions implicated in CaP (Table 1)Citation . With incidences of 39–61% for AI at the three sites, our results are in the top ranking of values published previously. We elucidated a significant association of chromosome 13q loss with advanced tumor stage, and here it predominantly was the marker D13S153 located at 13q14.1 within the second intron of the retinoblastoma gene with an incidence of 61% AI, which showed an independent association with advanced local tumor stage (P < 0.034; Table 3Citation ; Fig. 3Citation ). Allelic loss at 13q demonstrating an adverse effect on CaP progression has also been recognized previously (8 , 25) . Dong et al. (8 , 25) could show the chromosomal regions 13q14 and 13q21 but not 13q33 to be associated with aggressiveness of CaP. These data further support our findings where mainly the 13q14 locus was correlated with advanced tumor stage.

Besides alterations at the chromosomal regions 8p and 13q, which had the highest incidence in our cohort of primary CaPs, high incidences of AI were also discovered for chromosomes 5q, 7q, 17p, and 18q. The incidences of 37–57% AI at these chromosomes observed in our study are mostly higher than the incidences reported in previous investigations (Ref. 3 and for summary, see Ref. 1 ). This may be a result of the highly sensitive method of detection applied in this study, as well as a result of processing prospectively procured fresh and not potentially deteriorated normal and tumor DNA. Of interest, out of these four chromosomal regions, loss of chromosome 5q was found to be significantly associated with lymph node metastasis, which was also an observation made by Saric et al. (2) , who had collected DNA in part from autopsy-derived metastasis. Loss of chromosome 5q was also found to be associated with adverse pathological features in bladder cancer (28) . Within the above four chromosomal regions displaying a high incidence of AI in CaP, 17p LOH rendered a significant association with advanced tumor stage and Gleason score, leaving us to implicate this alteration as a progression marker for the investigated tumor entity as it has been by others previously (Table 3Citation ; Figs. 2Citation and 3Citation ; Refs. 29 and 30 ). Although loss of chromosome 18q was associated with adverse tumor characteristics in previous reports, we only identified the marker D18S858 to be correlated with advanced tumor stage (P = 0.038; Table 3Citation ; Fig. 3Citation ). The statistical association is attenuated though by the low heterozygosity rate of the marker (Table 3Citation ; Refs. 31, 32, 33 ). Because of the results of previous investigations and the association of marker D18S858 with tumor stage, we do interpret 18q AI as a putative progression marker in CaP for a subgroup of CaP similar to alterations at 7q (34, 35, 36) . For an alternative genetic pathway of CaP, these two alterations may represent initiating events in prostate carcinogenesis (Fig. 4)Citation .

In the underlying allelotyping study, we elucidated another chromosomal region of possible prognostic value in CaP. Of interest, LOH at 9p was calculated to have a significant association with advanced tumor stage and lymph node metastasis, although the overall incidence in the total cohort was rather low with 23% (P = 0.021 and P = 0.002, respectively; Table 3Citation ; Figs. 2Citation 3Citation 4Citation ). Within chromosome 9p, it seems that locus 9p13 is crucial for disease progression (marker D9S171, P = 0.003 and <0.0001, respectively; Table 3Citation ; Fig. 3Citation ). Our finding is in accordance to earlier reports, although a predominant role of the tumor suppressor gene CDKN2 (p16/MTS1) at 9p22 was implicated in the progression of CaP (2 , 37 , 38) .

With the results of our comprehensive allelotyping study in 100 primary CaPs procured during radical prostatectomy, we are able to suggest an algorithm for the genetic pathway in prostate carcinogenesis (Fig. 4)Citation . Here, we believe losses of chromosomes 8p and 13q to represent the major initiating events and the combination of both alterations to be important for disease progression. AI at chromosomes 7q, 17p, and 18q may represent alterations leading to an alternative genetic pathway in ~15% of cases. Finally, losses at chromosomes 5q and 9p appear to lead to the capability of distant metastatic deposit.

In conclusion, the MSA of chromosomes 5q, 8p, 9p, 13q, and 17p with the nine markers D5S476, D8S264, D8S261, D8S1477, D9S171, D13S153, D13S796, D17S1298, and D17S799 together with the markers D7S1807 and D18S858 for chromosomes 7q and 18q, respectively, may be helpful in discriminating lesser from more aggressive CaPs. If the applied fluorescent MSA, which to date is still time consuming, costly, and limited to research laboratories, were to be technically optimized for routine clinical use, observer independent prognostic genetic information may even be available at the time of diagnosis, investigating the prostate biopsy specimen, and would possibly give the clinician a better chance of planning the optimal therapy for each individual patient. Nevertheless, the above data are the result of a prospective analysis, and warranted further follow-up of the patients having had their cancer analyzed will therefore prove the true value of the results presented. We are confident that ongoing intensive research into gene expression profiles and proteomics may elucidate genes associated with the chromosomal regions implicated in prostate carcinogenesis by a molecular genetic approach in this study.


   FOOTNOTES
 
Grant support: Grant QLRT-2000-00602 from the 5th Framework Program of the European Union.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Rolf von Knobloch, Department of Urology, Philipps-University Marburg Medical School, Baldingerstrasse, D-35043 Marburg/Lahn, Germany. Phone: 49-6421-286-2560; Fax: 49-6421-286-5590; E-mail: Rolf.von-Knobloch{at}med.uni-marburg.de

3 Internet addresses: http://gai.nci.nih.gov/html-chlc/ChlcMarkers.html, http://www-genome.wi.mit.edu/cgi-bin/contig/sts_info?sts, or http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = unists. Back

Received 8/21/03; revised 10/13/03; accepted 10/16/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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