Peyronie's disease fibroblasts demonstrate tumorigenicity in the severe combined immunodeficient (SCID) mouse model.

Peyronie's disease is a localized connective tissue disorder, caused by trauma to the erect penis, which results in cellular proliferation and excess extracellular matrix production within the tunica albuginea of the penis. We have previously demonstrated that cells derived from Peyronie's disease plaque tissue demonstrate increased cell growth, increased S-phase on flow cytometry, stabilization and inactivation of p53, and consistent morphologic transformation, all suggesting that these cells are biologically transformed. Severe combined immunodeficient (SCID) mice have been used extensively to study the pathobiology of malignant and benign tissue and cells. This study was undertaken to determine if Peyronie's derived fibroblasts had the potential to demonstrate tumorigenicity in the SCID mouse model, thus confirming their biologically transformed nature. Cultured fibroblasts were derived from three sources, namely, plaque tissue excised from men with Peyronie's disease, tunical tissue excised from young men with congenital penile curvature and neonatal foreskins. BALB/C SCID mice were divided into three groups and each group was inoculated with cultured fibroblasts from each of the three different sources. All animals were evaluated regularly and maintained in isolation for a period of 3 months following inoculation. All SCID mice inoculated with cells derived from Peyronie's disease plaque tissue (n=10) developed subcutaneous nodules at a mean time period of 2.5+/-0.5 months following injection. The mean maximum dimension and weight of the nodules at the time of killing the animal was 1.1+/-0.2 cms and 0.6+/-0.2 g, respectively. Histologically, the nodules were composed of large pleomorphic epithelioid cells with a high mitotic activity, which were negative for cytokeratin but positive for vimentin. None of the SCID mice inoculated with cells cultured from either normal tunica (n=5) or foreskin (n=5) developed subcutaneous nodules. In conclusion, the tumorigenic nature of Peyronie's disease plaque-derived fibroblasts sheds further light on the pathobiologic characteristics of these cells. Specifically, these data confirm that cells cultured from Peyronie's disease plaque are biologically transformed. Future refinement and study of this animal model may permit a more complete understanding of the pathophysiology of Peyronie's disease and fibromatoses in general. Furthermore, such an animal model may, in the future, allow a more ready evaluation of the therapeutic interventions for Peyronie's disease.

Chromosomal instability is demonstrated by fibroblasts derived from the tunica of men with Peyronie's disease.

Peyronie's disease is a fibrotic disorder, a condition characterized by cellular proliferation and excess extracellular matrix production. Previous work in related conditions has demonstrated chromosomal instability. This investigation was undertaken to analyze fibroblasts derived from Peyronie's disease tunical tissue for abnormalities of chromosome number and progression of cytogenetic aberrations during cell culture. Tunical tissue was excised from men with Peyronie's disease from both plaque and nonplaque tissue and cells were explanted in culture. Control cells were derived from both neonatal foreskins and normal tunica from men with congenital penile curvature. Fluorescent in situ hybridization was used to probe for chromosomes 7, 8, 17, 18, X and Y. Control cells demonstrated normal copy number for all chromosomes analyzed. In contrast, Peyronie's disease plaque-derived fibroblasts demonstrated frequent aneusomies in chromosomes 7, 8, 17, 18 and X and recurrent deletions of chromosome Y. Peyronie's disease nonplaque tunica-derived fibroblasts demonstrated infrequent chromosomal changes early in culture; however, with repeated passaging the majority of cell cultures demonstrated aneusomies in at least one chromosome. These data indicate that Peyronie's disease plaque-derived fibroblasts have consistent aneusomies even at early passage and that nonplaque tunica-derived cells from men with Peyronie's disease also demonstrate chromosomal instability. This suggests that the tunica albuginea of men with Peyronie's disease may be predisposed to undergoing unregulated fibrosis. These findings confirm the transformed nature of the Peyronie's disease tunical fibroblasts studied in this analysis. While the etiology of these findings is not clear, it is likely that these pathobiological characteristics contribute to the pathophysiology of this disease process.

Peyronie's disease cell culture models: phenotypic, genotypic and functional analyses.

Peyronie's disease is a fibromatosis of the tunica albuginea. While trauma is believed to be the inciting event, the exact pathophysiology of this condition is unknown. In vitro analysis of cell biology can shed light on the pathogenesis of medical conditions and has been used for many decades as a research tool. We have established a cell culture model, which we have used to study the pathobiology of cells derived from Peyronie's disease plaque tissue. In 10 separate cell cultures derived from different individuals, these cells have demonstrated consistent phenotypic, genotypic and functional alterations. In neither of the control cell cultures, neonatal foreskin fibroblasts and normal tunica-derived fibroblasts have any of the above aberrations been demonstrated. The cells studied have been shown to be fibroblasts in nature with a sub-population of myofibroblasts present in culture. The Peyronie's disease plaque tissue-derived fibroblasts have demonstrated (i) consistent morphologic transformation (ii) increased S-phase on flow cytometry (iii) decreased dependence on culture medium (iv) cytogenic instability (v) excess production of fibrogenic cytokines and (vi) stabilization and dysfunctionalization of p53. Further refinement of this model and future analyses may permit an increased understanding of the pathogenesis of this condition and allow the development of therapeutic strategies.

Basic fibroblast growth factor expression in Peyronie's disease.

PURPOSE: Peyronie's disease is a fibromatosis resulting in scarring of the tunica albuginea. While the inciting event is believed to be trauma to the erect penis, little is understood about the cascade of cellular events that leads to the formation of the plaque. Dysregulated wound healing serves as a paradigm for the study of this condition. Previous work has demonstrated a role for fibrogenic cytokines in wound healing, fibromatoses, including Peyronie's disease. We analyze the expression of the fibrogenic cytokine, basic fibroblast growth factor (FGF), by fibroblasts derived from Peyronie's disease tissue. MATERIALS AND METHODS: Patients with Peyronie's disease undergoing either penile prosthesis insertion or Nesbit penile plication surgery had biopsy specimens removed from the plaque and from normal tunical tissue remote from the plaque. Cell cultures were derived from these specimens. Cultured cells were characterized using immunofluorescence staining and immunosorbent digital imaging. The cell culture supernatants were analyzed using an enzyme-linked immunosorbent assay for the production of basic FGF. Foreskin tissue from men without Peyronie's disease was used as control cells. RESULTS: Five independent cell lines were established from plaque tissue and 4 independent cell lines were established from normal tunica from the same subjects. Intracellular antigen expression was consistent with the cells being myofibroblasts. Production of basic FGF by the plaque derived myofibroblasts was significantly greater compared to production by normal tunical myofibroblasts and foreskin fibroblasts. CONCLUSIONS: These data demonstrate the successful establishment of cell lines from plaque tissue and normal tunica from men with Peyronie's disease. The findings indicate a potential role for basic FGF over expression in the tunical fibrosis that occurs in this condition. This information may allow a better understanding of the basic mechanisms involved in the development of this disease. Furthermore, it may permit the elaboration of therapeutic strategies to prevent or reduce tunical scarring and plaque formation.

Perturbation of cell cycle regulators in Peyronie's disease.

Peyronie's disease is a fibromatosis of the tunica albuginea, characterized by development of a plaque consisting primarily of collagen. It has been suggested that trauma to the erect penis is the inciting event. More recent research has focused on the cellular events leading to the dysregulated wound healing and plaque formation. Previous work has shown chromosomal aneusomies and this combined with an increased S-phase in plaque derived cell cultures suggests a perturbation in the cell cycle in this condition. The p53 protein has been shown to be an important cell cycle regulator and pro-apoptotic factor. Aberrant p53 function leading to cell immortalization and proliferation has been implicated in several human malignancies. We hypothesized that abnormal p53 function may explain the high proliferative ability of fibroblasts derived from Peyronie's plaques. This study was undertaken to study the presence and function of p53 and its downstream elements (p21, mdm-2) in Peyronie's disease cell cultures. Plaque-derived fibroblasts have been established in culture and characterized. These cells and control neonatal foreskin fibroblasts were subjected to 5 Gy of gamma radiation to induce DNA damage. After fixation, antibodies to p53 and its transcriptional elements were used to stain irradiated and non-irradiated cells and levels of p53, p21 and mdm-2 were quantified using combined immunofluorescence and flow cytometry. Non-irradiated plaque fibroblasts demonstrated the presence of p53, p21 and mdm-2 at baseline. In control foreskin fibroblasts no p53 or mdm-2 were detectable at baseline. In irradiated foreskin-derived cells significant changes in all elements were demonstrated indicating a fully functional p53 pathway and cell cycle checkpoint system in these cells. In contrast, plaque-derived cells showed no such alterations in levels of cell cycle regulators following irradiation. This is highly suggestive of an aberration of the p53 pathway in plaque-derived fibroblasts. Peyronie's plaque-derived fibroblasts demonstrated stabilization and defunctionalization of p53 protein combined with appropriate responses of its transcriptional elements. These findings may explain the high cell proliferation rates in these cells and suggests a role for perturbation of the p53 pathway in the pathogenesis of Peyronie's disease.

Inhibition of Peyronie's plaque fibroblast proliferation by biologic agents.

Peyronie's disease is a fibromatosis of the tunica albuginea which affects up to 2% of men. Plaque development is believed to result, at least in part, from fibroblast proliferation and excess collagen deposition. Numerous oral and intralesional therapies have been used, including verapamil, colchicine and steroids. The purpose of this study was to investigate the in vitro effects of prostaglandin-E1 (PGE1), verapamil and colchicine on the proliferation rates of fibroblasts derived from Peyronie's disease tissue. Using tissue culture, multiple cell lines comprising fibroblasts from Peyronie's plaque, normal tunica and foreskin were established. Cells of low passage were removed from the parent culture and incubated with varying concentrations of PGE1 (0.1-10 mg/ml), verapamil (10-1000 mg/ml), and colchine (2.5 mg/ml). Proliferation was assessed at 48, 72 and 96 hours using the Vybrant MTT cell proliferation and then compared to control cells. Six plaque lines and 5 normal tunical cell lines were established. These cell lines exhibited excellent linear growth in culture media alone. Co-culture wih PGE1 resulted in no significant inhibition at 0.1 and 1 mg/ml, but a mean inhibition of 60.6+/-11.5% at a concenrtation of 10 mg/ml was noted. Similar inhibition was noted with verapamil at 100 and 1000 mg/ml with a mean inhibition of 65.2+/-10.6%. Colchicine resulted in a mean inhibition of 28% at a concentration of 2.5 mg/ml. Maximum inhibition occurred at 96 hours in all cases. There was no statisitically significant difference in proliferation rates between plaque and normal tunical cell lines. We have developed an in vitro model to assess the effects of biologically active agents on the growth of fibroblasts derived from Peyronie's disease tissue. Our data suggests that PGE1, verapamil, and colchicine inhibit in vitro proliferation of fibroblasts at specific concentrations. Refinement and application of this knowledge may allow the development of useful pharmacologic strategies for men with PD.

Comparative analysis of DNA flow cytometry and cytology of bladder washings: review of discordant cases.

DNA analysis is becoming an important diagnostic and prognostic adjunct test in urinary cytology. The aim of this study was to compare the results of DNA flow cytometry (FCM) with the cytologic diagnosis of bladder washings (BW). DNA ploidy was evaluated in 251 BW. In 65 cases, follow-up surgical biopsies were available. Cytology results were classified as positive and negative, and FCM results were categorized as diploid and aneuploid. Both tests were evaluated independently. Cases were defined as discordant if the cytology was negative and FCM was aneuploid, or if the cytology was positive and FCM was diploid. All discordant cases were reviewed, and positive predictive values (PPV) for FCM and cytology were calculated for cases with follow-up biopsy results. Cytologic evaluation classified 181 cases as negative, with 175 of them diploid and 6 aneuploid; and 70 as positive, with 53 of them diploid and 17 aneuploid. Overall, there were 59 discordant cases (23.5%, with a confidence limit of 18.2-28.8%). Of 6 aneuploid/cytology-negative cases, biopsies were available in 4 cases and showed one grade 1, two grade 2, and one grade 3 urothelial carcinoma (UC). Reanalysis of these 6 cytology specimens showed 1 case that should have been interpreted as positive (false negative), 4 true negatives, and 1 polyoma virus infection. Out of 53 diploid/cytology-positive cases, biopsies were available in 45 cases and showed nine grade 1, 14 grade 2, three grade 3 UCs, 11 UCs in situ, and eight negative biopsies. The PPV for cytology was 85%, and the PPV for FCM was 95%. We concluded that FCM, which requires a large number of cells, often cannot detect small aneuploid populations, which are present particularly in cases of UC in situ.

Cell cycle models for molecular biology and molecular oncology: exploring new dimensions.

Some cell cycle models assume that cells are normally in a quiescent state until they are stimulated to enter the cell cycle and proceed through an S phase of fixed duration. Other models assume that cells normally cycle rapidly until they undergo growth retardation, proceed through an S phase of longer duration, and then undergo apoptosis or cell differentiation preferentially. These seemingly contradictory model types can be reconciled by restricting the latter type to the transition from log phase to plateau phase growth, and the former type to the recruitment of slowly proliferating cells into rapid cycle. Both proliferative states can be unified in a single cell cycle model that recognizes differences in the behavior of rapidly dividing and slowly dividing cells in the same population. Rb appears to play a major role in protecting slowly proliferating cells from apoptosis, permitting them to differentiate or persist as reserve cells that can be recruited into rapid cycle under appropriate circumstances. We examine the mechanistic basis for the recruitment phenomenon in some detail. The mitogenic signaling pathway is divided into a proximal segment, which consists of growth factor-induced membrane signaling, commonly through ras, raf, and cyclin D/cdk Rb kinase activation, and is subject to checks and balances that are designed to limit the propagation of the mitogenic signal. ras and raf compete with wild-type p53 both with respect to mitogenic signal propagation at the Rb node, and, separately, with respect to apoptosis/anti-apoptosis. The distal segment of the mitogenic signaling pathway, which consists of Rb phosphorylation, the release of E2F, the induction of c-myc, cyclins E and A, and DNA synthesis, is distinguished by a multiplicity of nested positive feedback loops; these would be expected to drive a mitogenic signal that entered the distal segment through at least one round of DNA synthesis. Using this model, we can identify two separate mechanistic strategies for neoplastic transformation. Chronic mitogenic stimulation of slowly proliferating cells would appear to be a common feature of Rb +/+ tumors. Rb -/- tumors dispense with the early segment of the mitogenic signaling pathway and its anti-apoptotic features, and maintain rapid cell cycling to compensate for high apoptotic rates.

Telomerase activity, telomere length, and DNA ploidy in prostatic intraepithelial neoplasia (PIN).

PURPOSE: To investigate the relationship of telomerase activity, telomere length, and DNA ploidy in high grade prostatic intraepithelial neoplasia (PIN). MATERIALS AND METHODS: Tissue samples were carefully microdissected to obtain adenocarcinoma or PIN-containing tissue free of cancer. Telomerase activity was measured using the PCR-based telomeric repeat amplification protocol (TRAP). Telomere length was estimated from Southern blots of telomere restriction fragments (TRFs). DNA ploidy of PIN and carcinoma was determined by image analysis of adjacent Feulgen stained tissue sections. RESULTS: Telomerase activity was found in 4 of 25 samples (16%) of high grade PIN. All telomerase positive PIN foci had a diploid DNA content. Although 5 of 25 samples (25%) of high grade PIN foci analyzed were DNA aneuploid, none of these demonstrated telomerase activity. Telomerase positive foci of prostate carcinoma (69% of all cancer foci analyzed) displayed heterogeneity in TRF length, with a mean TRF length two kilobase pairs shorter than that of telomerase negative specimens. CONCLUSIONS: Telomerase activity is present in a low percentage of high-grade PIN foci, which are diploid by DNA content measurements.

Assessing genetic markers of tumour progression in the context of intratumour heterogeneity.

This is a report from the Kananaskis working group on quantitative methods in tumour heterogeneity. Tumour progression is currently believed to result from genetic instability and consequent acquisition of new genetic properties in some of the tumour cells. Cross-sectional assessment of genetic markers for human tumours requires quantifiable measures of intratumour heterogeneity for each parameter or characteristic observed; the relevance of heterogeneity to tumour progression can best be ascertained by repeated assessment along a tumour progressional time line. This paper outlines experimental and analytic considerations that, with repeated use, should lead to a better understanding of tumour heterogeneity, and hence, to improvements in patient diagnosis and therapy. Four general principles were agreed upon at the Symposium: (1) the concept of heterogeneity requires a quantifiable definition so that it can be assessed repeatably; (2) the quantification of heterogeneity is necessary so that testable hypotheses may be formulated and checked to determine the degree of support from observed data; (3) it is necessary to consider (a) what is being measured, (b) what is currently measurable, and (c) what should be measured; and (4) the proposal of working models is a useful step that will assist our understanding of the origins and significance of heterogeneity in tumours. The properties of these models should then be studied so that hypotheses may be refined and validated.

Common patterns of genetic evolution in human solid tumors.

Human solid tumors develop multiple genetic evolutionary abnormalities as they evolve. Studies that have focused primarily on early colorectal cancer have suggested that genetic instability is a prominent feature of preinvasive disease. At least two separate mechanisms for the generation of genetic instability have been identified. The first, which involves widespread microsatellite instability in near-diploid cells, affects less than one-fifth of colon cancers. The second form of genetic instability is characterized by the development of p53 gene abnormalities that result in gross aneuploidy and multiple structural chromosomal changes. p53/aneuploidy affects most colon cancers, breast cancers, and many other solid tumors. This genetic evolutionary change commonly occurs at the interface between severe dysplasia and invasive disease. Specific post-aneuploid sequences of genetic changes that are relevant to tumor progression often involve the accumulation of multiple gain-of-function abnormalities in individual cells. The co-occurrence of Her-2/neu overexpression and EGF receptor overexpression in the same aneuploid cells defines an adeno/squamous genetic evolutionary sequence that is common to ductal breast cancers, non-small cell lung cancers, and other solid tumors. Later steps in this sequence include ras and c-myc overexpression. The neuroendocrine genetic evolutionary sequence is a separate branch of the p53/aneuploidy sequence with distinctive features that include loss of Rb and raf1 overexpression. Her-2/neu overexpression is not characteristic of this sequence; c-myc amplification/overexpression is common to both p53-associated sequences. The neuroendocrine sequence is found in small cell carcinoma of the lung and in minor proportions of other solid tumors, including breast cancer. Multiparameter cell-based methods are especially well suited for elucidation in human solid tumors of the genetic evolutionary sequences that could provide a rational scientific basis for determining prognosis and for optimizing therapy in individual cancer patients.

Limited efficacy of preoperative computed tomographic scanning for the evaluation of lymph node metastasis in patients before radical prostatectomy.

OBJECTIVES: To evaluate the efficacy of preoperative computed tomographic (CT) scanning in patients with presumed localized prostatectomy prior to radical retropubic prostatectomy. METHODS: A retrospective study of 173 consecutive patients believed to be candidates for radical retropubic prostatectomy who underwent preoperative CT scanning regardless of preoperative prostate-specific antigen (PSA) value, clinical stage, or Gleason grade was undertaken. All patients underwent radical retropubic prostatectomy with bilateral pelvic lymph node dissection or aspiration needle biopsy of abnormal nodes on CT scanning. RESULTS: One hundred sixty-five of 173 patients (95.4%) were believed to have normal CT scans preoperatively. Of these 165 patients, 156 (94.5%) were found to have negative lymph nodes confirmed histologically at the time of lymphadenectomy. Nine patients (5.5%) were found to have lymph node metastases confirmed histologically, despite a negative CT scan. Computed tomographic scanning understaged 9 of 12 (75%) patients with proven metastases. Incidental abdominal pathology of clinical significance was documented in 4 patients (2.3%), including 2 with renal cell cancers, 1 with colon cancer, and 1 with a large (8 cm) abdominal aortic aneurysm. Prostate-specific antigen levels in patients with metastatic lymph nodes ranged from 0.7 to 130 ng/mL (Hybritech Tandem assay), with a mean level of 42 ng/mL. Although 9 of 33 patients (27.3%) with PSA levels greater than 25 ng/mL had node metastases, only 3 of these 33 patients (9.1%) were correctly diagnosed by CT scanning. CONCLUSIONS: Although additional numbers of patients with high PSA levels need to be evaluated, we could not find any justification for routine preoperative CT scanning in patients with a PSA of less than 25 ng/mL. These results suggest that significant savings can be realized by abandoning the practice of routine CT scanning for lymph node metastasis in all patients with newly diagnosed prostate cancer.

Aprotinin modulation of platelet activation in patients undergoing cardiopulmonary bypass operations.

BACKGROUND: Aprotinin significantly decreases postoperative blood loss, yet its exact mechanism of action remains unproven. METHODS: To study the cytoprotective effect on platelets, we collected blood samples from patients during cardiopulmonary bypass (CPB) operations performed with or without aprotinin. Analysis included whole-blood flow cytometry. RESULTS: The highest percentages of activated platelets (positive for GMP-140 expression) were bound to leukocytes and erythrocytes in all CPB patients. Platelet-platelet activation did not reveal any marked differences between groups. However, in the platelet-cell bound region, increased ristocetin-stimulated platelet activation was observed from 30 minutes on CPB to 90 minutes after CPB with aprotinin (11.9% +/- 5.1% to 33.1% +/- 8.6%; p < 0.05), but not without aprotinin (17.5% +/- 0.1% to 17.9% +/- 2.3%). Platelet autoactivation increased more in the untreated group with time on CPB. CONCLUSIONS: This study demonstrates that in the presence of aprotinin, platelets remain unstimulated during CPB and the von Willebrand GPIb-mediated activatability of platelets is preserved, thus maintaining a viable platelet population. Most important, this study reveals that these mechanisms are more related to platelet-leukocyte than to platelet-platelet interactions.

Genetic and phenotypic heterogeneity of human malignancies: finding order in chaos.

The presence of cellular heterogeneity within human tumors has been recognized for many years. Current concepts regarding the clonal origin of human neoplasms, and recent advances in the study of successive genetic changes that occur during tumor evolution may now make it possible to understand in greater depth the biological and clinical implications of intra-tumor heterogeneity at both the phenotypic and genotypic levels. In order to explore these concepts further, and to better identify the potential contributions that flow and image cytometry can make to our understanding of tumor heterogeneity, a session of the 1994 ISAC Congress was dedicated to plenary presentations on human cancer cell heterogeneity. Here, we provide a brief overview of the genetic evolutionary progression of human cancers, some considerations of clinically important phenotypic and genotypic markers, and an outline that might serve as a basis for framing relevant issues that are ammenable to further study. All Nature is but Art, unknown to thee; All Chance, Direction, which thou canst not see; All Discord, Harmony not understood: All partial Evil, universal Good. (Alexander Pope, Essay on Man, end of Epistle 1).

DNA ploidy and proliferation heterogeneity in human prostate cancers.

DNA ploidy determinations have been shown to have clinical application in predicting disease progression, survival, or response to anti-androgen therapies in prostate carcinomas. Since intra-tumor heterogeneity may have a profound effect on DNA measurements, we determined the frequency of DNA ploidy and proliferation (here S-phase fraction) heterogeneity in early prostatic carcinomas, and estimated the potential impact of heterogeneity on predicting disease course, survival, or response to therapy. Using image and flow cytometric analysis of archival, paraffin-embedded prostate tumors, we measured DNA ploidy in individual foci of prostatic carcinoma in stage T1a, T1b and T1c disease. Image analysis studies included the use of Feulgen stained tissue sections, and a comparison of these results with flow cytometric DNA ploidy determinations on nuclei isolated from the same tumor foci. Flow cytometry was also used to measure DNA Index and tumor S-phase fraction, in some cases using multiparameter analysis of isolated nuclei to determine DNA content and the level of the proliferation-associated antigen, p105. Our results indicate that DNA aneuploid foci of prostate carcinoma are infrequently seen in stage T1a disease (13% of the individuals studied), and that the presence of both DNA diploid and aneuploid foci in the same sample is seen in less than 10% of these individuals. Stage T1b and T1c tumors containing only DNA diploid nuclei are seen, though these are likely most common in low volume, low Gleason grade tumors. By using flow cytometry to compare these results with those using image analysis of the same tumor foci, we demonstrated that the majority (> 75%) of these aneuploid tumors are DNA tetraploid. Our data on prostate tumor S-phase fractions indicate that DNA diploid tumors generally have a lower S-phase than DNA aneuploid foci (including comparisons of DNA diploid and aneuploid foci in the same prostate tumor). These results support the model that early prostate tumors are DNA diploid and have a low S-phase, and that these tumors likely evolve to DNA tetraploid tumors with a similar low S-phase fraction.