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Molecular Genetic Evidence for Different Clonal Origins of Epithelial and Stromal Components of Phyllodes Tumor of the Prostate

      Phyllodes tumor of the prostate is a rare neoplasm, composed of epithelium-lined cysts and channels embedded in a variably cellular stroma. The pathogenetic relationship of the epithelium and stroma is unknown and whether each is a clonal neoplastic element is uncertain. We studied the clonality of phyllodes tumors from six patients who underwent either enucleation or transurethral resection as their initial treatment. This was followed by total prostatectomy in three of the patients. Laser-assisted microdissection was performed to extract epithelial and stromal components of phyllodes tumor from formalin-fixed, paraffin-embedded tissue. Polymerase chain reaction was used to amplify genomic DNA at specific loci on chromosome 7q31 (D7S522), 8p21.3-q11.1 (D8S133, D8S137), 8p22 (D8S261), 10q23 (D10S168, D10S571), 17p13 (TP53), 16q23.2 (D16S507), 12q11–12 (D12S264), 17q (D17S855), 18p11.22-p11 (D18S53), and 22q11.2 (D22S264). In each tumor, stroma and epithelium were analyzed separately. Gel electrophoresis with autoradiography was used to detect loss of heterozygosity. All tumors showed allelic loss in one or more loci of both the epithelial and stromal components. The frequency of allelic loss in the epithelial component was 2 of 5 (40%) at D7S522, 2 of 6 (33%) at D8S133, 1 of 5 (20%) at D8S137, 3 of 6 (50%) at D8S261, 4 of 4 (100%) at D10S168, 4 of 6 (67%) at TP53, 2 of 6 (33%) at D10S571, 6 of 6 (100%) at D16S507, 1 of 5 (20%) at D12S264, 1 of 6 (17%) at D17S855, 2 of 6 (33%) at D18S53, and 2 of 5 (40%) at D22S264. The frequency of allelic loss in the stromal component was 2 of 5 (40%) at D7S522, 1 of 6 (17%) at D8S133, 2 of 5 (40%) at D8S137, 3 of 6 (50%) at D8S261, 1 of 4 (25%) at D10S168, 3 of 6 (50%) at TP53, 2 of 6 (33%) at D10S571, 3 of 6 (50%) at D16S507, 1 of 5 (20%) at D12S264, 0 of 6 (0%) at D17S855, 1 of 6 (17%) at D18S53, and 0 of 5 (0%) at D22S264. The pattern of allelic loss is significantly different in both stroma and epithelium statistically; completely concordant allelic loss patterns were not seen in any tumor examined. Our data demonstrate that both epithelial and stromal components of phyllodes tumor of the prostate are clonal, supporting the hypothesis that both elements are neoplastic. While both epithelium and stroma are clonal proliferations, they appear to have different clonal origins.
      Phyllodes tumor of the prostate is a rare neoplasm with poorly understood pathogenesis. Histologically, it resembles phyllodes tumor of the breast with hyperplastic epithelium-lined cysts and channels embedded in a variably cellular stroma.
      • Reese JH
      • Lombard CM
      • Krone K
      • Stamey TA
      Phyllodes type of atypical prostatic hyperplasia: a report of 3 new cases.
      • Attah EB
      • Nkposong EO
      Phyllodes type of atypical prostatic hyperplasia.
      A variety of terms have been used to describe these lesions, including phyllodes type of atypical hyperplasia, cystosarcoma phyllodes, and prostatic cystic epithelial-stromal tumor.
      • Reese JH
      • Lombard CM
      • Krone K
      • Stamey TA
      Phyllodes type of atypical prostatic hyperplasia: a report of 3 new cases.
      • Attah EB
      • Nkposong EO
      Phyllodes type of atypical prostatic hyperplasia.
      • Kevwitch MK
      • Walloch JL
      • Waters WB
      • Flanigan RC
      Prostatic cystic epithelial-stromal tumors: a report of 2 new cases.
      • Yokota T
      • Yamashita Y
      • Okuzono Y
      • Takahashi M
      • Fujihara S
      • Akizuki S
      • Ishihara T
      • Uchino F
      • Iwata T
      Malignant cystosarcoma phyllodes of prostate.
      The malignant potential of this tumor is unclear and has resulted in confusion in terms of prognosis and treatment. Unquestionably, the potential for sarcomatous transformation, recurrence, and infiltrative growth exists, but the frequency of these changes and their prognostic significance is unclear.
      • Kevwitch MK
      • Walloch JL
      • Waters WB
      • Flanigan RC
      Prostatic cystic epithelial-stromal tumors: a report of 2 new cases.
      • Yokota T
      • Yamashita Y
      • Okuzono Y
      • Takahashi M
      • Fujihara S
      • Akizuki S
      • Ishihara T
      • Uchino F
      • Iwata T
      Malignant cystosarcoma phyllodes of prostate.
      • Lopez-Beltran A
      • Gaeta JF
      • Huben R
      • Croghan GA
      Malignant phyllodes tumor of prostate.
      • De Siati M
      • Busolo A
      • Contin F
      • Shah J
      • Visona A
      • Franzolin N
      High-grade phyllodes tumour of the prostate.
      • Watanabe M
      • Yamada Y
      • Kato H
      • Imai H
      • Nakano H
      • Araki T
      • Shiraishi T
      Malignant phyllodes tumor of the prostate: retrospective review of specimens obtained by sequential transurethral resection.
      Little is known about the genetic abnormalities in this tumor. Oncogene activation and tumor suppressor gene inactivation are important mechanisms in the genesis, propagation, and spread of most cancers, but the role of these processes in phyllodes tumor has not been previously explored. It is well known that allelic loss is a common early genetic alteration during tumorigenesis.
      • Bostwick DG
      • Shan A
      • Qian J
      • Darson M
      • Maihle NJ
      • Jenkins RB
      • Cheng L
      Independent origin of multiple foci of prostate intraepithelial neoplasia (PIN): comparison with matched foci of prostate cancer.
      • Cheng L
      • Shan A
      • Cheville JC
      • Qian J
      • Bostwick DG
      Atypical adenomatous hyperplasia of the prostate: a premalignant lesion?.
      • Diaz-Cano SJ
      • Blanes A
      • Wolfe HJ
      PCR techniques for clonality assays.
      • Cheng L
      • Song SY
      • Pretlow TG
      • Abdul-Karim FW
      • Kung HJ
      • Dawson DV
      • Park WS
      • Moon YW
      • Tsai ML
      • Linehan WM
      • Emmert-Buck MR
      • Liotta LA
      • Zhuang Z
      Evidence of independent origin of multiple tumors from patients with prostate cancer.
      Previously, loss of heterozygosity analysis has been used to study the clonal relationship of different components of the same tumor.
      • Cheng L
      • Song SY
      • Pretlow TG
      • Abdul-Karim FW
      • Kung HJ
      • Dawson DV
      • Park WS
      • Moon YW
      • Tsai ML
      • Linehan WM
      • Emmert-Buck MR
      • Liotta LA
      • Zhuang Z
      Evidence of independent origin of multiple tumors from patients with prostate cancer.
      • Brandli DW
      • Ulbright TM
      • Foster RS
      • Cummings OW
      • Zhang S
      • Sweeney CJ
      • Eble JN
      • Cheng L
      Stroma adjacent to metastatic mature teratoma after chemotherapy for testicular germ cell tumor is derived from the same progenitor cells as the teratoma.
      • Cheng L
      • Bostwick DG
      • Li G
      • Wang Q
      • Hu N
      • Vortmeyer AO
      • Zhuang Z
      Allelic imbalance in the clonal evolution of prostate carcinoma.
      • Cheng L
      • Gu J
      • Eble JN
      • Bostwick DG
      • Younger C
      • MacLennan GT
      • Abdul-Karim FW
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      • Koch MO
      • Zhang S
      • Ulbright TM
      Molecular genetic evidence for different clonal origin of components of human renal angiomyolipomas.
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      • Bostwick DG
      • Li G
      • Zhang S
      • Vortmeyer AO
      • Zhuang Z
      Conserved genetic findings in metastatic bladder cancer: a possible utility of allelic loss of chromosome 9p21 and 17p13 in diagnosis.
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      • Gu J
      • Ulbright TM
      • MacLennan GT
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      • Zhang S
      • Sanchez K
      • Koch MO
      • Eble JN
      Precise microdissection of human bladder carcinomas reveals divergent tumor subclones in the same tumor.
      • Kernek KM
      • Ulbright TM
      • Zhang S
      • Billings SD
      • Cummings OW
      • Henley JD
      • Michael H
      • Brunelli M
      • Martignoni G
      • Eble JN
      • Cheng L
      Identical allelic loss in mature teratoma and different histologic components of malignant mixed germ cell tumors of the testis.
      • Emerson RE
      • Ulbright TM
      • Zhang S
      • Foster RS
      • Eble JN
      • Cheng L
      Nephroblastoma arising in a germ cell tumor of testicular origin.
      Since these genetic changes can be identified by allelic typing at polymorphic chromosomal loci, we compared the frequency of loss of heterozygosity and analyzed the pattern of allelic loss between the epithelial and stromal components.

      Materials and Methods

      Patients

      Six men with phyllodes tumor of the prostate were included in our study. None of these cases were reported previously. Patients ranged in age from 25 to 88 years of age, with a mean age of 55, and all presented with urinary obstruction or painless hematuria. Four tumors were diagnosed by transurethral resection and two by retropubic prostatic enucleation. Three of the patients had no recurrence. The other three patients had multiple recurrences, first occurring at 1 and 6 months, and 10 years after diagnosis. Sarcoma emerged in one patient 11 years after the original diagnosis following three recurrences, while metastasis to the abdominal wall occurred in another patient. This research was approved by the Indiana University Institutional Review Board.

      Tissue Samples and Microdissection

      Histological sections were prepared from formalin-fixed, paraffin-embedded tissue and were stained with hematoxylin and eosin for microscopic evaluation. From these slides, the two components of the phyllodes tumors were identified (Figure 1). Laser-assisted microdissection of the two components was performed on the unstained sections using a PixCell II Laser Capture Microdissection (LCM) system (Arcturus Engineering, Mountain View, CA), as previously described.
      • Brandli DW
      • Ulbright TM
      • Foster RS
      • Cummings OW
      • Zhang S
      • Sweeney CJ
      • Eble JN
      • Cheng L
      Stroma adjacent to metastatic mature teratoma after chemotherapy for testicular germ cell tumor is derived from the same progenitor cells as the teratoma.
      • Bonner RF
      • Emmert-Buck M
      • Cole K
      • Pohida T
      • Chuaqui R
      • Goldstein S
      • Liotta LA
      Laser capture microdissection: molecular analysis of tissue.
      • Emmert-Buck MR
      • Vocke CD
      • Pozzatti RO
      • Duray PH
      • Jennings SB
      • Florence CD
      • Zhuang Z
      • Bostwick DG
      • Liotta LA
      • Linehan WM
      Allelic loss on chromosome 8p12–21 in microdissected prostatic intraepithelial neoplasia.
      • Younger C
      • Ulbright TM
      • Zhang S
      • Billings SD
      • Cummings OW
      • Foster RS
      • Eble JN
      • Cheng L
      Molecular evidence supporting the neoplastic nature of some epidermoid cysts of the testis.
      • Cheng L
      • MacLennan GT
      • Zhang S
      • Wang M
      • Pan CX
      • Koch MO
      Laser capture microdissection analysis reveals frequent allelic losses in papillary urothelial neoplasm of low malignant potential (PUNLMP) of the urinary bladder.
      Approximately 400 to 1000 cells of each component were microdissected from the 5-μm histological sections. Normal tissue (lymphoid tissue when present or normal epithelial cells) from each case was microdissected as a control.
      Figure thumbnail gr1
      Figure 1Laser microdissection of a phyllodes tumor of prostate (Case 3). It illustrated epithelial (*) and stromal (♦) components of tumor before microdissection (A) and after microdissection (B).
      We also performed experiments to compare allelic patterns from different parts of the same tumor. Different parts of the same component (epithelial or stromal) were sampled in three cases and left and right sides of the prostate, respectively, in one case (Case 6), in which prostatectomy had been performed.

      Amplification of DNA

      The dissected cells were de-paraffinized with xylene and ethyl alcohol. Polymerase chain reaction (PCR) was used to amplify genomic DNA at various specific loci on chromosome 7q31 (D7S522), 8p21.3-q11.1 (D8S133, D8S137), 8p22 (D8S261), 10q23 (D10S168, D10S571), 17p13 (TP53), 16q23.2 (D16S507), 12q11–12 (D12S264), 17q (D17S855), 18p11.22-p11 (D18S53), and 22q11.2 (D22S264). Previous studies demonstrated frequent loss of heterozygosity (LOH) on these chromosomes in prostatic intraepithielial neoplasia, prostatic carcinoma, and atypical adenomatous hyperplasia.
      • Bostwick DG
      • Shan A
      • Qian J
      • Darson M
      • Maihle NJ
      • Jenkins RB
      • Cheng L
      Independent origin of multiple foci of prostate intraepithelial neoplasia (PIN): comparison with matched foci of prostate cancer.
      • Cheng L
      • Shan A
      • Cheville JC
      • Qian J
      • Bostwick DG
      Atypical adenomatous hyperplasia of the prostate: a premalignant lesion?.
      • Cheng L
      • Song SY
      • Pretlow TG
      • Abdul-Karim FW
      • Kung HJ
      • Dawson DV
      • Park WS
      • Moon YW
      • Tsai ML
      • Linehan WM
      • Emmert-Buck MR
      • Liotta LA
      • Zhuang Z
      Evidence of independent origin of multiple tumors from patients with prostate cancer.
      • Cheng L
      • Bostwick DG
      • Li G
      • Wang Q
      • Hu N
      • Vortmeyer AO
      • Zhuang Z
      Allelic imbalance in the clonal evolution of prostate carcinoma.
      • Emmert-Buck MR
      • Vocke CD
      • Pozzatti RO
      • Duray PH
      • Jennings SB
      • Florence CD
      • Zhuang Z
      • Bostwick DG
      • Liotta LA
      • Linehan WM
      Allelic loss on chromosome 8p12–21 in microdissected prostatic intraepithelial neoplasia.
      • Vocke CD
      • Pozzatti RO
      • Bostwick DG
      • Florence CD
      • Jennings SB
      • Strup SE
      • Duray PH
      • Liotta LA
      • Emmert-Buck MR
      • Linehan WM
      Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12–21.
      PCR amplification and gel electrophoresis were performed as previously described.
      • Cheng L
      • Shan A
      • Cheville JC
      • Qian J
      • Bostwick DG
      Atypical adenomatous hyperplasia of the prostate: a premalignant lesion?.
      • Cheng L
      • Song SY
      • Pretlow TG
      • Abdul-Karim FW
      • Kung HJ
      • Dawson DV
      • Park WS
      • Moon YW
      • Tsai ML
      • Linehan WM
      • Emmert-Buck MR
      • Liotta LA
      • Zhuang Z
      Evidence of independent origin of multiple tumors from patients with prostate cancer.
      • Brandli DW
      • Ulbright TM
      • Foster RS
      • Cummings OW
      • Zhang S
      • Sweeney CJ
      • Eble JN
      • Cheng L
      Stroma adjacent to metastatic mature teratoma after chemotherapy for testicular germ cell tumor is derived from the same progenitor cells as the teratoma.
      • Cheng L
      • Bostwick DG
      • Li G
      • Wang Q
      • Hu N
      • Vortmeyer AO
      • Zhuang Z
      Allelic imbalance in the clonal evolution of prostate carcinoma.
      • Cheng L
      • Gu J
      • Eble JN
      • Bostwick DG
      • Younger C
      • MacLennan GT
      • Abdul-Karim FW
      • Geary WA
      • Koch MO
      • Zhang S
      • Ulbright TM
      Molecular genetic evidence for different clonal origin of components of human renal angiomyolipomas.
      • Cheng L
      • Bostwick DG
      • Li G
      • Zhang S
      • Vortmeyer AO
      • Zhuang Z
      Conserved genetic findings in metastatic bladder cancer: a possible utility of allelic loss of chromosome 9p21 and 17p13 in diagnosis.
      • Cheng L
      • Gu J
      • Ulbright TM
      • MacLennan GT
      • Sweeney CJ
      • Zhang S
      • Sanchez K
      • Koch MO
      • Eble JN
      Precise microdissection of human bladder carcinomas reveals divergent tumor subclones in the same tumor.
      • Gu J
      • Roth LM
      • Younger C
      • Michael H
      • Abdul-Karim FW
      • Zhang S
      • Ulbright TM
      • Eble JN
      • Cheng L
      Molecular evidence for the independent origin of extra-ovarian papillary serous tumors of low malignant potential.
      PCR for each polymorphic microsatellite marker was repeated at least twice from the same DNA preparations and the same results were obtained.

      Analysis of Allelic Loss Pattern

      When the genetic material in a patient was found to be homozygous for the polymorphic markers (ie, showing only one allele in the normal control tissue), the case was considered non-informative. DNA sampled from separate epithelial and stromal cells demonstrating identical allelic loss pattern is compatible with either similar or independent clonal origin, whereas different patterns of allelic deletions are compatible with independent clonal origins of these tumors.
      • Cheng L
      • Song SY
      • Pretlow TG
      • Abdul-Karim FW
      • Kung HJ
      • Dawson DV
      • Park WS
      • Moon YW
      • Tsai ML
      • Linehan WM
      • Emmert-Buck MR
      • Liotta LA
      • Zhuang Z
      Evidence of independent origin of multiple tumors from patients with prostate cancer.
      • Brandli DW
      • Ulbright TM
      • Foster RS
      • Cummings OW
      • Zhang S
      • Sweeney CJ
      • Eble JN
      • Cheng L
      Stroma adjacent to metastatic mature teratoma after chemotherapy for testicular germ cell tumor is derived from the same progenitor cells as the teratoma.
      • Cheng L
      • Bostwick DG
      • Li G
      • Wang Q
      • Hu N
      • Vortmeyer AO
      • Zhuang Z
      Allelic imbalance in the clonal evolution of prostate carcinoma.
      • Cheng L
      • Bostwick DG
      • Li G
      • Zhang S
      • Vortmeyer AO
      • Zhuang Z
      Conserved genetic findings in metastatic bladder cancer: a possible utility of allelic loss of chromosome 9p21 and 17p13 in diagnosis.
      • Gu J
      • Roth LM
      • Younger C
      • Michael H
      • Abdul-Karim FW
      • Zhang S
      • Ulbright TM
      • Eble JN
      • Cheng L
      Molecular evidence for the independent origin of extra-ovarian papillary serous tumors of low malignant potential.
      • Zhuang Z
      • Merino MJ
      • Chuaqui R
      • Liotta LA
      • Emmert-Buck MR
      Identical allelic loss on chromosome 11q13 in microdissected in situ and invasive human breast cancer.

      Single-Stranded Conformation Polymorphism (SSCP) Analysis and p53 Immunostaining

      Since mutations or functional inactivation of the p53 gene are the most common genetic abnormalities in cancer,
      • Levine AJ
      • Momand J
      • Finlay CA
      The p53 tumour suppressor gene.
      p53 analysis by immunohistochemistry as well as single-stranded conformation polymorphism (SSCP) were performed to determine whether mutations were present, and if so, whether the mutations were the same or different in the respective components. Genomic DNA was isolated from tissue using proteinase K digestion and phenol/chloroform extraction and subsequently amplified by PCR. The conditions for PCR were similar to the routine PCR except that 0.2 μl of α-[32P]dATP (Perkin Elmer, Boston, MA) was added per reaction. Eight pair primers were chosen from exons 5–8 of the p53 gene. Twenty-five μl of PCR product was mixed with 3.5 μl of 95% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol and heated at 95°C for 8 minutes. Two μl of this solution was loaded onto a 6% polyacrylamide gel with 10% glycerol. Gel electrophoresis was performed at 7 watts (W) for overnight. The gels were dried on filter paper and exposed to x-ray films at −80°C.
      • Uchida T
      • Wada C
      • Ishida H
      • Wang C
      • Egawa S
      • Yokoyama E
      • Kameya T
      • Koshiba K
      p53 mutations and prognosis in bladder tumors.
      • Bookstein R
      • MacGrogan D
      • Hilsenbeck SG
      • Sharkey F
      • Allred DC
      p53 is mutated in a subset of advanced-stage prostate cancers.
      P53 immunostaining was performed on formalin-fixed, paraffin-embedded sections using the avidin-biotin complex technique, as described previously.
      • Cheng L
      • Leibovich BC
      • Bergstralh EJ
      • Scherer BG
      • Pacelli A
      • Ramnani DM
      • Zincke H
      • Bostwick DG
      p53 alteration in regional lymph node metastases from prostate carcinoma: a marker for progression?.
      • Cheng L
      • Sebo TJ
      • Cheville JC
      • Slezak J
      • Bergstralh EJ
      • Paceli A
      • Neumann RM
      • Zincke H
      • Bostwick DG
      P53 protein overexpression is associated with increased cell proliferation in patients with locally recurrent prostate carcinoma after radiation therapy.
      • Cheng L
      • Pisansky TM
      • Sebo TJ
      • Leibovich BC
      • Ramnani DM
      • Weaver Al
      • Schere BG
      • Blute ML
      • Zincke H
      • Bostwick DG
      Cell proliferation in prostate cancer patients with lymph node metastasis: a marker for progression.
      Primary monoclonal antibodies were used for evaluation of p53 overexpression (DO-7, DAKO, Carpinteria, CA; dilution 1:100).

      Statistical Analysis

      Lack of association of allelic loss in the tumors between the epithelial and stromal sites would suggest independent origins of phyllodes tumor. Statistical analysis of the association was performed to confirm or disprove the hypothesis of independent origin.
      • Skrondal A
      • Rabe-Hesketh S
      Multilevel logistic regression for polytomous data and rankings.
      In the analysis, response variable is allele loss in epithelial component and is a three-level categorical response, with 0 coded as no allele loss, 1 coded as loss of upper allele, and 2 coded as loss of lower allele. Homozygous controls (non-informative) response is not included in the analysis. The fixed predictor is allele loss in stromal component and is coded similarly as that of the response variable. Correlated multinomial logistic regression was performed; a random intercept for each case was used to account for correlation within a same case. Additionally, fixed or random effects of the twelve loci on the chromosomes within each case were included to account for the effect of the loci.

      Results

      The frequency of allelic loss is summarized in Figure 2. All patients with phyllodes tumor of the prostate showed allelic loss in both epithelial and stromal components (Table 1, Figure 3). All cases showed allelic loss in at least five loci when epithelial and stromal components are combined. The number of specific loci lost ranged from four to seven in the epithelial component and two to four in the stromal component.
      Figure thumbnail gr2
      Figure 2Comparison of frequency of allelic loss between epithelial and stromal components of phyllodes tumor.
      Figure thumbnail gr3
      Figure 3Representative results of loss of heterozygosity analysis (Case 3). DNA was prepared from normal tissue (N), epithelial (E), and stromal cells (S) of the phyllodes tumor, amplified by polymerase chain reaction using polymorphic markers D7S522, D8S133, D8S137, D8S261, D10S168, D10S571, D12S264, D16S507, TP53, D17S855 D18S53, D22S264, and separated by gel electrophoresis. Arrows, allelic bands.
      Completely concordant allelic loss patterns between epithelium and stroma were not present in any of the tumors examined (Table 1). Case 2 showed allelic loss of four loci of both epithelial and stromal components, but only two of these loci (D8S137, D16S507) showed identical loss patterns. Similar results were seen in other cases, including case 3 with loss of seven epithelial and three stromal alleles, with complete concordance at three loci (TP53, D16S507, D12S264). Case 4 showed loss of four epithelial and two stromal alleles, with concordance at one locus (D16S507). Case 5 showed loss of five epithelial and three stromal alleles, with concordance at one locus (TP53). Case 6 showed loss of six epithelial and four stromal alleles, with complete concordance at two loci (D8S261, D18S53) and loss of opposite alleles at one locus (D10S168).
      There were no completely identical allelic loss patterns when comparing epithelial and stromal components separately. High frequency of allelic loss of the epithelial component was seen at TP53, D10S168, and D16S507. In the epithelial component, 4 of 6 cases demonstrated identical allelic loss at TP53, 5 of 6 cases demonstrated identical allelic loss at D16S507 with the other having loss of the opposite allele, and 2 of 4 cases demonstrated identical allelic loss at D10S168 with the other two having loss of the opposite allele. The stromal component did not show as much similarity in allelic loss patterns and appeared more random, with only 3 of 6 cases showing identical allelic loss at TP53 and D16S507.
      We further compared LOH patterns from different parts of the same tumor. These LOH findings were identical on multiple parts of specimens obtained from both left and right sides of a radical prostatectomy specimen (Case 6) (Figure 4). In three other cases where tumor was sampled from multiple sites, identical allelic loss patterns were also observed. Different parts of the same component (epithelial or stromal) yield the same LOH pattern (Figure 4).
      Figure thumbnail gr4
      Figure 4Different parts of the same component (epithelial or stromal) showed the same LOH pattern. DNA was prepared from normal tissue (N), epithelial (E), and stromal cells (S) of the phyllodes tumor and amplified by polymerase chain reaction. E1 and E2 represent epithelial components sampled from different tumor parts; S1 and S2 represent stromal components sampled from different tumor parts. The same LOH pattern was seen in tumor components of left and right sides of a radical prostatectomy specimen (Case 6) and multiple areas of different case (Case 2). Arrows, allelic bands.
      P53 immunohistochemistry failed to reveal p53 protein overexpression in either epithelial or stromal components. Additionally, SSCP analysis did not demonstrate any evidence of p53 mutations in either case where both alleles were present, or when one was lost.
      From the statistical analysis using correlated multinomial logistic model, the upper allele loss in stromal component is not associated with the upper allele loss in epithelial component (P value = 0.48); the lower allele loss in stromal component is also not associated with the lower allele loss in epithelial component (P value = 0.28). This result indicates independent origin of epithelial and stromal components of the phyllodes tumors.

      Discussion

      In this study, we found a high frequency of allelic loss on chromosome 10q23, 16q23.2, and 17p13 in phyllodes tumor of the prostate. High frequency of allelic loss was found in the epithelial and stromal components, suggesting both components are clonal and neoplastic. The pattern of allelic loss is statistically different between the stroma and epithelium, supporting different clonal origins of the epithelial and stromal components of phyllodes tumor of the prostate.
      Phyllodes tumor is a rare neoplasm of the prostate which may undergo sarcomatous transformation, is prone to early recurrences, infiltrative growth, and has potential for extraprostatic spread.
      • Kevwitch MK
      • Walloch JL
      • Waters WB
      • Flanigan RC
      Prostatic cystic epithelial-stromal tumors: a report of 2 new cases.
      • Yokota T
      • Yamashita Y
      • Okuzono Y
      • Takahashi M
      • Fujihara S
      • Akizuki S
      • Ishihara T
      • Uchino F
      • Iwata T
      Malignant cystosarcoma phyllodes of prostate.
      • Lopez-Beltran A
      • Gaeta JF
      • Huben R
      • Croghan GA
      Malignant phyllodes tumor of prostate.
      • De Siati M
      • Busolo A
      • Contin F
      • Shah J
      • Visona A
      • Franzolin N
      High-grade phyllodes tumour of the prostate.
      • Watanabe M
      • Yamada Y
      • Kato H
      • Imai H
      • Nakano H
      • Araki T
      • Shiraishi T
      Malignant phyllodes tumor of the prostate: retrospective review of specimens obtained by sequential transurethral resection.
      Benign clinical courses have been cited, but the follow-up time has been limited.
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      Cystosarcoma phyllodes of the prostate.
      In regards to the stroma, Gaudin et al
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      • Rosai J
      • Epstein JI
      Sarcomas and related proliferative lesions of specialized prostatic stroma: a clinicopathologic study of 22 cases.
      proposed the clinicopathologic categories of prostatic stromal proliferation of uncertain malignant potential and prostatic stromal sarcoma to differentiate these entities from other mesenchymal lesions of the prostate, using immunohistochemical staining data in conjunction with morphological criteria.
      Clonal evolution of transformed cell populations requires one or more genetic changes to obtain a growth advantage over adjacent cells. This results in the formation of a clinically detectable tumor. The specific types of genetic alterations associated with tumorigenesis are variable. They may range from DNA point mutations to major chromosomal structural aberrations or changes in chromosome numbers. Chromosomal analyses of phyllodes tumor of the breast have shown contradictory results. Noguchi et al
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      • Koyama H
      Clonal analysis of fibroadenoma and phyllodes tumor of the breast.
      demonstrated the epithelial component to be polyclonal and the stromal component to be monoclonal. This led to the conclusion that phyllodes tumor is a neoplasm of stromal cells. However, Sawyer et al
      • Sawyer EJ
      • Hanby AM
      • Ellis P
      • Lakhani SR
      • Ellis IO
      • Boyle S
      • Tomlinson IP
      Molecular analysis of phyllodes tumors reveals distinct changes in the epithelial and stromal components.
      demonstrated that allelic instability occurs in both the stroma and epithelia, indicating that both components are neoplastic.
      The pathogenesis of phyllodes tumor of the prostate is unknown. This is the first study to assess loss of heterozygosity of this tumor. The high frequency of allelic loss of the epithelial component at 16q23.2 (D16S507) is striking. This locus encodes the gene HSD17B2, which is involved in steroid biosynthesis in the prostate. It has been suggested that the regulation of intraprostatic concentrations of active androgens is involved in the maintenance of organ homeostasis by modulating the balance between proliferation and apoptotic death of prostatic epithelial cells.
      • Isaacs JT
      • Lundmo PI
      • Berges R
      • Martikainen P
      • Kyprianou N
      • English HF
      Androgen regulation of programmed death of normal and malignant prostatic cells.
      Recently, Harkonen et al
      • Harkonen P
      • Torn S
      • Kurkela R
      • Porvari K
      • Pulkka A
      • Lindfors A
      • Isomaa V
      • Vihko P
      Sex hormone metabolism in prostate cancer cells during transition to an androgen-independent state.
      observed remarkable changes of 17HSD enzyme activities in prostate carcinoma, suggesting that this enzyme influences steroid hormone bioavailability locally and may contribute to the progression of prostate cancer.
      The frequent epithelial allelic loss of p53 (TP53) and PTEN/MMAC1 (D10S168) suggests phyllodes tumor development may occur through inactivation of tumor suppressor genes, as has been shown in prostate carcinoma.
      • Cheng L
      • Leibovich BC
      • Bergstralh EJ
      • Scherer BG
      • Pacelli A
      • Ramnani DM
      • Zincke H
      • Bostwick DG
      p53 alteration in regional lymph node metastases from prostate carcinoma: a marker for progression?.
      • Cheng L
      • Sebo TJ
      • Cheville JC
      • Slezak J
      • Bergstralh EJ
      • Paceli A
      • Neumann RM
      • Zincke H
      • Bostwick DG
      P53 protein overexpression is associated with increased cell proliferation in patients with locally recurrent prostate carcinoma after radiation therapy.
      • Cheng L
      • Pisansky TM
      • Sebo TJ
      • Leibovich BC
      • Ramnani DM
      • Weaver Al
      • Schere BG
      • Blute ML
      • Zincke H
      • Bostwick DG
      Cell proliferation in prostate cancer patients with lymph node metastasis: a marker for progression.
      • Brooks JD
      • Bova GS
      • Ewing CM
      • Piantadosi S
      • Carter BS
      • Robinson JC
      • Epstein JI
      • Isaacs WB
      An uncertain role for p53 gene alterations in human prostate cancers.
      • Sharrard RM
      • Maitland NJ
      Phenotypic effects of overexpression of the MMAC1 gene in prostate epithelial cells.
      Our data showed detection of allelic loss as a poor indicator of mutation of the remaining copy of the p53 gene. This is not an uncommon finding as Brooks et al
      • Brooks JD
      • Bova GS
      • Ewing CM
      • Piantadosi S
      • Carter BS
      • Robinson JC
      • Epstein JI
      • Isaacs WB
      An uncertain role for p53 gene alterations in human prostate cancers.
      had one of 10 tumors in their series of prostatic carcinoma with a mutation of its remaining allele documented using SSCP, and none of the tumors with allelic loss were immunohistochemically positive. This lack of concordance between 17p allelic loss and p53 mutation has been occasionally observed in bladder and head and neck tumors and more commonly in breast cancers and astrocytomas.
      • Davidoff AM
      • Humphrey PA
      • Iglehart JD
      • Marks JR
      Genetic basis for p53 overexpression in human breast cancer.
      • Ahomadegbe JC
      • Barrois M
      • Fogel S
      • Le Bihan ML
      • Douc-Rasy S
      • Duvillard P
      • Armand JP
      • Riou G
      High incidence of p53 alterations (mutation, deletion, overexpression) in head and neck primary tumors and metastases; absence of correlation with clinical outcome. Frequent protein overexpression in normal epithelium and in early non-invasive lesions.
      • Chang F
      • Syrjanen S
      • Syrjanen K
      Implications of the p53 tumor-suppressor gene in clinical oncology.
      • Saxena A
      • Clark WC
      • Robertson JT
      • Ikejiri B
      • Oldfield EH
      • Ali IU
      Evidence for the involvement of a potential second tumor suppressor gene on chromosome 17 distinct from p53 in malignant astrocytomas.
      In summary, our study demonstrated that phyllodes tumors of the prostate are clonal proliferations. Epithelial and stromal components may have different clonal origins and appear to be true neoplastic conditions. Our data support that the epithelial and stromal components may arise independently and provide support for a pathogenetic model of phyllodes tumor of the prostate where both epithelial and stromal components are neoplastic. However, given that there are occasional similarities in LOH patterns, although statistically insignificant, one must also consider common clonality with loss of additional alleles separately after divergence of the two components.

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