Search

Objective: To determine the frequency and predictive value of p53 mutations in localized prostate cancer, comparing the accuracy of detection using immunohistochemistry (IHC) with a modified yeast assay, on archival tissue samples., Materials and Methods: Prostate cancer tissue was obtained from 98 patients who had >/= 2 years of clinical follow-up after radical prostatectomy. DNA sequencing was used to verify the presence of p53 mutations in samples that were immunopositive or that gave evidence for p53 alterations using the yeast assay. The IHC and yeast findings were compared with patient outcome to determine the predictive value of these two test types., Results: Fifty-five tumours (57%) were immunopositive, and 58 (59%) were positive using the yeast assay. Sequence-confirmed p53 mutations occurred in 44 (45%) cases. The IHC protocol generated 49% (27/55) false-positive and 36% (15/42) false-negative results, and was 65% sensitive and 50% specific, with an overall accuracy of 57%. The yeast assay resulted in 24% (14/58) false-positive results with a specificity of 74% and an accuracy of 86%. When the p53 status of these patients was correlated with their clinical outcome, patients who had sequence-confirmed p53 mutations had a 2.6-fold greater failure rate (P = 0.026) and a 2.5-fold greater risk of dying from prostate cancer (P = 0.05). Notably, mutations in exon 6 predicted a six-fold increase in treatment failure (P = 0.043) and a 5.3-fold increase in the chance of dying from prostate cancer (P = 0.009). Abnormal yeast-assay findings gave similar predictive results to those obtained for DNA sequencing, while immunopositivity did not correspond to patient outcome., Conclusions: Mutations of p53 occurred in 45% of localized prostate cancers. These alterations have important prognostic implications. The yeast assay was more accurate for detecting p53 mutations than the IHC protocol used and, unlike IHC, the results of the yeast assay were predictive of patient outcome.

Detection and functional evaluation of mutant p53 alleles using a yeast assay could yield significant information for predicting the prognosis of patients with prostate cancer (CaP). Since the current version of this yeast assay is not applicable to archival tissues, we developed a modified assay for use on formalin-fixed, paraffin-embedded tissue and have applied it to the study of patient samples. Using this modified assay, we examined archival CaP samples from 10 patients for mutations in exons 5-8 of p53 gene. Mutations were detected in four samples: three resulted in the formation of red yeast colonies indicating complete loss of function, while one gave pink yeast colonies, indicating that this mutant retained partial function. In parallel, we analyzed these samples for p53 abnormalities using a single-strand conformational polymorphism (SSCP) approach. Only three of the four yeast-positive samples gave abnormal SSCP bands. In each case where abnormal p53 was found by both methods, DNA sequencing revealed the identical base change. These results suggest that the modified yeast assay may be more sensitive than SSCP for detection of p53 mutations, and demonstrate that the modified method can be used to detect and evaluate the function of p53 mutants present in archival tissue.

Background: Few studies have used multiple assays to examine the functionality of mutant p53 in prostate cancer (CaP). We employed seven functional assays to study 16 representative mutant p53 alleles, six from localized and ten from metastatic CaP., Methods: Yeast assays were employed to determine loss of function (LOF), partial function (PF), and dominant-negative status. Assays using p53-null Saos2 cells were used to determine whether mammalian cells transfected with mutant p53 could up-regulate the MDR-1 or PCNA promoters, alter IL-6 expression or confer the ability to grow in soft agar. As a further test of gain of function (GOF), p53-null PC3 cells stably transfected with these mutant p53 alleles were examined for cell cycle distributions., Results: All 16 mutant p53 alleles demonstrated either total or partial LOF. All but one allele also had at least one gain of function; however, the pattern of GOF was different for each mutant allele. Alleles derived from both localized and metastatic CaP had similar GOF characteristics; however, only alleles from metastatic disease had significantly increased S-phase fractions., Conclusions: Different mutant p53 alleles from CaP had different, complex functional profiles. The lack of predictable patterns for these alleles suggest that each mutation may uniquely affect p53 function., (Copyright 2002 Wiley-Liss, Inc.)

Objective: To seek differences in gene expression in the primary muscle-invasive bladder cancers of two cohorts of patients having different survival rates. An Italian group treated by transurethral resection of the bladder tumor (TURBT) and neo-adjuvant chemotherapy using methotrexate, vinblastine, adriamycin and cisplatin (M-VAC) followed by TURBT, partial cystectomy or radical cystectomy (75% 3-year survival) was compared to an American cohort treated by radical cystectomy (51% 3-year survival)., Methods: Immunohistochemistry was used to examine the protein expression levels of three genes that act at the G1/S cell cycle checkpoint, p53, p21/waf-1/cip1 (a downstream effector gene in the p53 pathway) and Rb, plus a major inhibitor of apoptosis, Bcl-2., Results: For the bladder cancers of the Italian patient cohort, there was a significantly higher rate of p53 immunopositivity (93 vs. 63%, p = 0.002) and a significantly lower rate of Rb loss (25 vs. 54%, p = 0.009). In bivariate analysis, 72% of Italian tumors were immunopositive for both p53 and p21 (p53+/p21+) vs. 49% for the American tumors. The subset of Italian patients with p53+/p21+ tumors were more frequently disease-free (stage pT0) following chemotherapy and were less likely to fail therapy than those with p53+/p21- tumors (p = 0.0357). Loss of Rb staining was associated with a decreased 5-year survival in the Italian, but not in the American patients., Conclusions: (1) Significant differences in the expression of the p53, p21 and Rb genes were found between the 2 groups of patients. (2) Italian patients with p53+/p21+ tumors had significantly lower recurrence rates after TURBT and chemotherapy than those having p53+/p21- tumors. (3) Absence of p21 immunopositivity in the Italian tumors may identify alterations in the p53 pathway that predict poor outcome.

Objectives: An intergroup study (SWOG 8795) comparing two forms of adjunctive therapy (immuno and chemo), bacillus Calmette-Guerin (BCG) and mitomycin C (MMC), furnished preregistration index tumors for 244 patients with superficial, papillary stage Ta/T1 TCC. These were examined by flow cytometry to learn whether DNA ploidy or proliferation (low vs high S-phase fraction (SPF) helped to predict disease recurrence or progression., Methods: Cell cycle analysis using commercially available (Multicycle) programs was performed on 249 Ta/T1 bladder cancers. Tumor grade, available for 223 cases, was assigned by a single study pathologist. The SWOG statistical office reviewed follow-up information and other data and performed statistical analysis., Results: Disease recurrence occurred in half the cases studied. The most parsimonious model predictive of recurrence included only treatment arm and tumor grade, with the MMC arm and tumor grade greater than I indicating worse prognosis (p = 0. 014). Neither ploidy nor SPF predicted recurrence-free survival or contributed prognostic information that was additive to tumor grade. Within 5 years of follow-up, disease progression or death from bladder cancer occurred for 29/223 (13%) of patients. The most parsimonious model for progression-free survival included only grade greater than I (p<0.001) and high SPF (p = 0.029) (relative risk: tumor grade, 4.3, high SPF, 1.9)., Conclusions: Knowledge of tumor proliferation (low versus high SPF) contributes prognostic information about tumor progression that is additive to tumor grade.

Background: While many studies have suggested that p53 mutations are common in human cancers, the functional activity of these mutant alleles has not yet been fully addressed. We believe that information about the functional status of individual p53 mutants will prove to be important for a better understanding of the role of p53 in tumor development and progression. Ultimately, this information could also influence treatment decisions for individual cancer patients., Methods: A recently developed yeast functional assay can be used to assess the transactivational activity of p53 mutants. Furthermore, this assay is more sensitive than single strand conformational polymorphism (SSCP) for detection of p53 mutations. In this review, we summarize the mechanism of this new technique and describe its applications in cancer research, with an emphasis on prostate cancer., Results: The use of the yeast functional assay provides a simple, sensitive, and reproducible method for detecting p53 mutations and for determining the transactivational activity and dominant-negative role of individual p53 mutants., Conclusions: This method may be adapted to analyze other transcriptional factors, including the human androgen receptor., (Copyright 1999 Wiley-Liss, Inc.)

Background: The frequency of mutant p53 in bone marrow metastases of patients with carcinoma of the prostate (CaP) and in matched sets of metastatic and primary lesions from the same patients was investigated. The data were examined in relation to prior treatment with androgen ablation (AA) therapy and were compared with the frequency of mutant p53 reported for primary CaP., Methods: Seventeen patients with M1b (bone metastasis: TNM Stage IV) CaP had either unilateral or bilateral bone marrow biopsies taken for these studies. Specimens were divided and the outer one-third examined histologically to confirm the presence of CaP cells. Immunohistochemical (IHC) staining for accumulated p53 protein was performed by an antibody cocktail technique. RNA was extracted from the remaining portion of the biopsy, and p53 transcripts were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and screened for base sequence changes in the exon 4-11 region using nonisotopic single-strand conformation polymorphism (SSCP) analysis and direct DNA sequencing., Results: Ten of 17 metastases (59%) demonstrated accumulation of p53. Six of 7 (86%) of the p53 IHC positive bone marrow samples contained RT-PCR-SSCP abnormalities, as did 2 of 3 IHC negative samples. Overall, 12 of 17 metastases (71%) contained mutant p53. Four of 7 biopsies (57%) retrieved prior to AA contained mutant p53, whereas 8 of 10 post-AA biopsies (80%) contained mutant p53. One patient showed identical SSCP abnormalities in right and left iliac crest metastases after therapy, and in this patient DNA sequencing demonstrated a missense mutation at codon 126 (TAC --> GGC, Tyr --> Gly). Archival primary cancers from seven patients were retrieved. All seven were IHC positive for p53 accumulation., Conclusions: p53 mutations are associated with increased metastatic potential of CaP. Abnormalities are found at approximately twice the frequency in metastases than in unselected samples of primary CaP, whereas in matched specimens there is a high rate of consonance. Mutant p53 may contribute to systemic therapy resistance, due to increased association with post-AA CaP specimens.

Purpose: Tumor grade, deoxyribonucleic acid (DNA) ploidy, proliferation, p53 and bcl-2 expression were examined in clinically localized prostate cancers of black and white American men to learn whether these features showed racial differences., Materials and Methods: A total of 117 prostate cancers (43 black and 74 white patients) obtained at radical prostatectomy for clinically localized disease were assigned Gleason scores by a single pathologist. Enzymatically dissociated nuclei from archival prostate cancers were examined by DNA flow cytometry using propidium iodide staining and the multicycle program to remove debris and sliced nuclei and to perform cell cycle analysis. For immunostaining after microwave antigen retrieval we used a DO-1/DO-7 monoclonal antibody cocktail for p53 and the clone 124 antibody for bcl-2., Results: Significantly more black than white men had Gleason score 7 tumors. The DNA ploidy distribution of Gleason 6 or less tumors was similar for both races. As anticipated, the ploidy distribution of higher grade prostate cancer in white men was more abnormal but, unexpectedly, this was not found for higher grade prostate cancer in black men. No significant racial differences were found in S phase fractions, p53 or bcl-2 immunopositivity. However, for prostate cancer in black men there was a significant association between bcl-2 immunopositivity and higher S-phase fractions., Conclusions: The aggressive prostate cancers of black men may be characterized by the 2 features of high proliferation and a block to programmed cell death.

Objectives: Radiation therapy is definitive treatment for localized prostate cancer. It causes cellular deoxyribonucleic acid (DNA) damage, which, if irreparable, results in apoptosis or programmed cell death. Overexpression of mutant p53 and/or bcl-2 proteins prolongs cell survival despite exposure to damaging agents. We examined whether abnormal expression of either gene could help to explain radiation therapy failures in prostate cancer., Methods: Archival tissue from patients who had failed radiation therapy as treatment for prostate cancer was obtained before and after treatment. These cancer samples were examined immunohistochemically for accumulation of p53 and bcl-2 proteins. Comparison was made with specimens from patients who had no evidence of recurrent or persistent disease at least 3 years following radiation therapy., Results: High rates of p53 immunopositivity were found in the prostate tissue from all groups studied. More patients who had failed radiation therapy were found to have bcl-2 immunopositive specimens than were those without evidence for recurrent disease (41% preradiation and 61% postradiation versus 8%, P <0.05). More patients who failed radiation therapy had both p53 and bcl-2 immunopositive prostate tissue than did those who were treated successfully (32% preradiation and 48% postradiation versus 8%)., Conclusions: bcl-2 immunopositivity, with or without concomitant detection of p53, was found in significantly more cancers of patients who failed radiation therapy. Positive staining for bcl-2 may serve as a marker for determining the radiation sensitivity of a tumor and thus may help to guide treatment options. It is also notable that a high proportion of the prostate cancers examined were immunopositive for p53.

The prognostic value of the p53 gene (TP53), the most commonly mutated gene in human cancers, has been well established for several cancer types. However, because varying frequencies of TP53 mutations have been identified in prostatic adenocarcinoma (CaP) by genetic and immunohistochemical (IHC) studies, the role of TP53 in CaP tumorigenesis is currently unresolved. These experimental discrepancies could be caused by tissue heterogeneity within prostatic neoplasms, variations in experimental protocols, or other factors. Thus, the goal of this study was to develop a reliable IHC approach for the detection of p53 in archival prostate tissue. The authors evaluated four p53 antibodies, CM-1, 1801, DO-1, and DO-7, for their ability to reveal p53. They chose two reference CaP cell lines, 26 patient specimens (including eight benign prostatic hyperplasias (BPHs), 16 CaPs, and two lymph node metastases), one prostate and nine kidney cell lines for p53 analysis. The TP53 status of these samples was characterized using single-strand conformational polymorphism (SSCP) analysis of RNA/PCR products and sequencing. IHC detection of p53 was markedly enhanced by using the combination of microwave heat-induced antigen unmasking and a cocktail of the DO-1 and DO-7 antibodies. This approach identified 14 of 15 (93%) cell lines and patient samples having TP53 missense mutations in the exons 5 to 8 region. Of the 21 patient samples and cell lines that were either normal by SSCP or expressed p53 mutations that are not expected to stain, 18 (86%) were immunonegative. Because of this good correlation between molecular and IHC analysis, this approach may help to resolve the uncertainty about TP53 in CaP tumorigenesis.

While 80% of transitional cell carcinomas (TCC) present as Ta Tl lesions, they account for only 15% of deaths caused by TCC. We have evaluated the ability of DNA ploidy analysis to predict outcome in 228 patients with Ta Tl TCC. All patients were judged to be at increased risk for tumor recurrence due to having two occurrences of Stage TI tumor within 56 weeks, or three or more tumors presenting simultaneously within 16 weeks of registration. Concurrent carcinoma in situ was acceptable. All patients were treated with either bacillus Calmette Guerin (BCG) immunotherapy or mitomycin-C (MMC) intravesical chemotherapy. Patients with nondiploid tumors had higher hazard rates for both tumor progression and death (p = 0.007 and p = 0.016, respectively); however, the prognostic information of DNA ploidy was not additive to tumor grade.

In most cases, the appearance of aneuploid peaks in DNA histograms may be an artefact of tissue preparation or it may reflect non-stoichiometric dye binding of a cellular subpopulation rather than true DNA aneuploidy. This report reviews how false DNA aneuploidy can be recognized and eliminated from sample submitted for DNA flow cytometric analysis.

To clarify the relationship of DNA ploidy to tumor grade and volume, 32 clinical Stage B prostate cancers, with low and high Gleason scores and small and large tumor volumes, were compared with adjacent histologically normal prostate tissue and with samples from benign prostatic hyperplasia (BPH). All 22 samples from benign glands were diploid, with 2.7 +/- 1.2% tetraploid (4C) cells. Samples from cancer-bearing glands were considered diploid (normoploid) if they had a major diploid (2C) peak and a small 4C peak with the percentage of cells falling within 3 standard deviations of the figure found for BPH. Abnormal ploidy included abnormal diploid (6.3-14.9% 4C), tetraploid (> or = 15% 4C), and aneuploid samples (peaks not at 2C or 4C). Abnormal DNA ploidy was found to be related to tumor volume. All five tumors smaller than 0.4 cm3 and their adjacent benign tissue were normoploid; however, 10 of 13 cancers with volumes of 0.4-1 cm3 had abnormal ploidy (9 abnormal diploid, 1 tetraploid) and 6 of 9 of the adjacent benign tissue samples also were abnormal diploid. All larger tumors (> 1 cm3) showed abnormal ploidy (7 abnormal diploid, 3 tetraploid, 5 aneuploid). For large tumors, abnormal ploidy was present in 10 of 13 of the adjacent benign areas (8 abnormal diploid, 2 benign areas that were clearly aneuploid). Abnormal diploid cancers are intermediate forms between diploid and tetraploid tumors, as defined above. Although they have fewer 4C cells than tetraploid cancers, they have equivalent numbers of hypertetraploid cells (BPH: 1.3 +/- 0.9%; abnormal diploid: 10.8 +/- 5.4%; tetraploid: 11.1 +/- 6.8% hypertetraploid cells). Thus, the authors propose that abnormal diploid cancers represent an early stage in ploidy progression. DNA ploidy abnormalities also occur in benign prostatic tissue adjacent to many prostate cancers, consistent with the concept that human prostatic cancer is a field-change disease.

Single-institution studies have shown that DNA flow cytometry is superior to routine cytologic evaluation of following patients for bladder cancer recurrence. For 15 urine and 15 bladder washing specimens, we evaluated a fixative employing methanol plus acetic acid (MA), freshly mixed 20:1 (vol/vol). Routine cytologic evaluation following Papanicolaou staining, and DNA flow cytometry were performed. Paired aliquots from the same washings and urines were processed as fresh spray-fixed samples and MA-fixed samples. The majority of the MA-fixed specimens showed good nuclear preservation when assessed for chromatin texture, presence of distinct nuclear envelope, and clarity of nucleolus, while only a minority of the fresh urine and washing samples showed these features. Cytoplasmic degeneration was seen only in fresh specimens. The presence of aneuploidy and the percentage of hyperdiploid cells could be reliably determined in the MA-fixed samples. This fixation protocol is recommended for the transport of urine and bladder washing specimens to centralized laboratories for both cytologic and flow cytometric evaluation.

Clinical staging and histologic grading do not have sufficient predictive value to determine the response to therapy of any given prostate cancer. A review of the findings from the largest prospective study of patients with localized and locally advanced prostate cancer and from retrospective flow cytometric studies of specific disease stages suggests that DNA flow cytometry offers additional prognostic information for this disease. However, for the individual patient, this added information may have limited value, since approximately 15% of those with diploid disease will experience disease progression within 5 years as compared with half of those with nondiploid disease. We have found that ploidy does not predict length of survival once prostate cancer becomes disseminated, nor does it predict those who will benefit from receiving definitive radiation therapy for localized prostate cancer. On the other hand, for those who have persistent tumor, we have frequently found increased ploidy abnormalities in the tumor sampled after radiation therapy and are currently correlating this finding with clinical outcome. We have also found that DNA flow cytometry can be used to predict tumor volume. For larger, grade-matched diploid tumors, there are significant increases in the proliferation of both the tumor and the adjacent benign tissue, which we take to be evidence of "field effects" in this disease. An even more obvious manifestation of the same phenomenon is seen in the occurrence of aneuploidy in benign tissue near high-grade, large-volume prostate cancer. It is concluded that DNA flow cytometry has much to tell us about the natural history and biologic behavior of prostate cancer.

At this present time, we feel that there is no role for DNA flow cytometry (FCM), or indeed DNA studies by any other method, to be used as a screening procedure for patients with no prior history of bladder cancer due to the high false-positive rate found when monitoring exfoliated urothelial cells. On the other hand, for patients who have had a superficial transitional cell carcinoma (TCC), which has a documented 50% recurrence rate, and depending on pathological features, a progression rate from 7 to 45%, DNA FCM provides a sensitive method to predict future disease recurrence. It provides an extremely effective way to predict future progression and further acts as a method to monitor changes in the malignant potential of the patient's disease. For those patients with a past history of superficial TCC who develop abnormal ploidy without any overt tumor, 80% will, within the next four years, suffer a disease recurrence. For the patient who has a Ta TCC and receives intravesical Bacillus Calmette-Guerin (BCG), the development of abnormal ploidy in bladder washing specimens is the single best indicator for future disease recurrence. Similarly, a negative DNA FCM of a bladder washing at six months after intravesical therapy is an excellent predictor of no further occurrence. In patients with superficial TCC, ploidy of the initial and recurrent tumor predicts for future progression. Half of those patients with stage Ta bladder cancer with two successive aneuploid bladder tumors develop muscle invasive disease within one year, while three-fourths develop advanced disease within two years after recurrence of their second aneuploid lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric DNA measurements were carried out on 45 formalin-fixed, paraffin-embedded canine prostate tissues. The tissues were categorized as normal, hyperplastic, or neoplastic on the basis of light microscopic examination, and DNA ploidy was compared with histologic classification. Ten normal prostate samples showed diploid DNA histograms, but with proliferation greater than that found in the normal human prostate. Thirteen specimens of benign prostate hyperplasia showed diploid or near-diploid DNA histograms. Of 22 prostatic carcinomas, 12 were diploid and 10 were aneuploid, with the majority of the aneuploidy being near-triploid. The frequency of DNA aneuploidy recognized in canine prostatic carcinoma is similar to findings in human prostatic carcinoma if all their grades of malignancy are included.

A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter- and intra-laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter- and intra-laboratory differences. Results were evaluated by a two-way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra- and inter-laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated DNA flow cytometry of urine histogram profiles as a screening procedure for bladder cancer. To establish the false-positive rate, we first studied a low-risk group of 90 patients with no history of bladder cancer: 24 with benign prostatic hyperplasia and 66 with upper tract stones, and found 4.4% unsatisfactory and 15.1% abnormal histograms. Urine samples were then obtained for 54 additional patients (most being evaluated for benign prostatic hyperplasia) whose selection was based on their smoking habits. These included 20 nonsmokers, 10 who had not smoked for at least 20 years (mean: 18 packs/year) and 24 current smokers (mean: 71 packs/year). Abnormal histograms were obtained for 14% of nonsmokers and for 36% of smokers, a group known to be at risk for developing bladder cancer. Although abnormal histograms are more frequent for smokers, the background false-positive rate remains unacceptably high for general screening purposes.

We describe a method to fix exfoliated bladder cells that is suitable for followup of bladder cancer patients by deoxyribonucleic acid flow cytometry. After fixation with room temperature methanol plus acetic acid (20:1, volume:volume) urine and bladder washing samples from these patients can be stored at room temperature for 3 to 7 days and then assessed reliably for the presence of aneuploidy and the percentage of hyperdiploid cells. For those with active transitional cell carcinoma diagnostic accuracy comparing fresh to fixed specimens was improved from 58 to 92% with urine and from 50 to 100% with washing samples. For patients with a history of transitional cell carcinoma who currently are free of disease the false positive rate remains unchanged after fixation. The procedure described is suitable for use in the outpatient clinic and should permit shipping of samples without refrigeration to a central flow cytometry facility for analysis.

We compared DNA flow cytometry to morphologic evaluation of routine testicular biopsies as methods of monitoring spermatogenesis. The study group consisted of 14 azoospermic men and 5 others who underwent testicular surgery unassociated with fertility problems. The findings for both studies were divided into three groups: normal, moderately abnormal, and markedly abnormal. Correlations between the findings from routine biopsy and flow cytometry were good. Of 9 patients having normal testicular morphology, 7 had normal ploidy classes by DNA flow cytometry while 2 had moderately abnormal histograms. Of 5 cases with moderately abnormal morphology, 1 had normal, 1 had moderately abnormal, and 3 had markedly abnormal ploidy distributions. In 5 cases described as Sertoli cell only, all DNA histograms were markedly abnormal, consisting almost exclusively of diploid cells. DNA flow cytometry of testicular biopsies and aspirates has been demonstrated to be a rapid, reproducible, and objective approach in evaluating the infertile male and is a promising method to investigate spermatogenesis in an outpatient clinic in lieu of formal testis biopsy.

The potential ability of cytoreductive surgery to increase the effectiveness of chemotherapy (vindesine) was tested utilizing male Wistar Lewis rats transplanted simultaneously with intraperitoneal and flank implants of a spontaneously arising renal adenocarcinoma. Cytoreduction was accomplished in some animals by removing the flank tumor 5-7 weeks following implantation; all animals received vindesine (IP injection of 0.5 mg/kg on two successive weeks). While vindesine reduced tumor growth, in no case did the addition of cytoreductive surgery enhance the effect of chemotherapy. The addition of cytoreductive surgery to marginally effective chemotherapy was found to be ineffective or even detrimental.

Flow cytometry can be performed on testicular aspirates of vasovasostomy candidates preoperatively. On the basis of ploidy ratios and debris components, DNA histograms can be classified as normal or abnormal. Using this method, the likelihood of the presence of sperm may be predicted.

Analysis of DNA histograms obtained from rat renal cancer cells stained with propidium iodide and submitted to flow cytometry revealed a tumor cell population with prominent 2C and 4C peaks and with usually less than 10% each of S-phase or hyper-4C cells. The presence of an increased proportion of 4C cells was found to depend on the age and size of the tumor nodule. Sampling replicate portions of the same tumor or different tumors of the same size and age frequently revealed highly variable 4C:2C ratios. Treatment of animals bearing this tumor with a single i.p. injection of cyclophosphamide (CY), under conditions known to reduce the tumor burden by 80% within 1 week, or with 5-fluorouracil (FUra), which is ineffective against this tumor, in many cases did not yield changes in DNA histograms that permitted one to distinguish the effective drug. Either no marked difference in histogram shape occurred after therapy, or FUra induced more striking differences in cell cycle position than did CY. In tumor generations with greater than 15% S-phase cells, treatment with CY resulted in multiple effects on DNA histograms. These included detecting increased numbers of moribund cells (hypo-2C), a decrease in 4C cells, and an increase in hyper-4C cells. These changes did not occur with the ineffective agent FUra. The tumors grown in vitro show no evidence of replication of 4C cells. The DNA histograms of late-log-phase cultures have a major 2C peak and a minor S plus G2 hump. Since neither the untreated tumor in vivo nor that grown in vitro has a major hyper-4C cell population, it is probable that the tumor stem cells are chiefly 2C (diploid-hyperdiploid). Treatment in vitro of late-log-phase cultures with CY or FUra produces DNA histograms which permit identification of the effective agent. After CY, a major part of the cell population was hypo-2C (moribund) cells.

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.

HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2-0.5 microg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G(1) are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G(2). CD inhibits transport of [(14)C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.

Several physical and biochemical parameters of a rapidly growing, hormonally responsive, poorly differentiated strain of Dunning R3327 rat prostatic adenocarcinoma (the CP strain) were monitored for 1 month during growth in control and hormonally manipulated male Fischer X Copenhagen rats. The tumor was implanted into control rats and into rats 1 month following orchiectomy. Twenty-nine days following tumor implantation, 1 group of unoperated rats was orchiectomized while the rats implanted subsequent to orchiectomy were repleted with pharmacological doses of testosterone. At 2 and 4 weeks following treatment, half the original number of rats from each group were sacrificed and the growth rate (doubling time), per cent of aneuploid cells and androgen receptor levels (total, cytoplasmic and nuclear) were determined for each tumor. Orchiectomy increased tumor doubling time, while testosterone repletion decreased it, demonstrating the hormonal dependence of this tumor strain. Orchiectomy also decreased the levels of aneuploid cells in the tumor; however, repletion of testosterone to rats orchiectomized prior to implantation did not restore the aneuploid cell number to control levels. A sensitive indicator of the hormonal status of the tumor was the per cent of androgen receptors in the nucleus. Tumors grown in rats orchiectomized after implantation had the lowest percentage of androgen receptor in the nucleus while orchiectomized rats repleted with testosterone had the highest percentage. Comparison of the levels of androgen receptors in the tumors from the various groups (androgen receptor per gram of tissue) unexpectedly revealed that tumors grown in the orchiectomized rats had slightly higher total receptor levels than did control tumors, while the tumors of orchiectomized rats repleted with testosterone had lower amounts than did the control tumors. In contrast to these findings, the prostates of orchiectomized rats replenished with testosterone had higher levels of total androgen receptor than did the prostates of control rats.

Deoxyribonucleic acid flow cytometry was performed on aspirated prostatic cells from 198 patients who had benign cytological or histological findings. Unsatisfactory acellular histograms were obtained from 10.6 per cent of the cases. Three-fourths of the satisfactory samples (more than 5,000 cells after subtracting debris) showed the expected single peak deoxyribonucleic acid diploid to near diploid histograms. Unexpectedly, the remaining samples were deoxyribonucleic acid aneuploid, most having 2 peridiploid peaks (deoxyribonucleic acid index 0.82 to 1.31). Usually, proliferation was low with less than 20 per cent hyperdiploid cells and with 2.5 +/- 1.5 per cent G2 cells. In 10 per cent of the single peak histograms there was evidence of inflammation, identified as an increase in hyperdiploid cells without an increased percentage of G2 cells but with a tail of high channel values. The aforementioned histogram features were considered to be benign findings. Seven per cent of the samples had deoxyribonucleic acid histograms suggestive of prostate cancer. Of these samples 7 had diploid or peridiploid aneuploid histograms with high proliferation (more than 20 per cent hyperdiploid cells with 8.5 +/- 3.8 per cent G2 cells), while 5 had histograms with deoxyribonucleic acid aneuploidy other than peridiploidy.

Quantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant of the relatively low signal-to-noise ratios often found in DNA distributions obtained from clinical samples. Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S. A computer-based method has been developed for extraction of the above-mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight-line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer-based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer-based method. Correlation of percent hyperdiploid cell fraction was also excellent.(ABSTRACT TRUNCATED AT 250 WORDS)

For the neurologically impaired patient electroejaculation with a rectal probe can furnish sperm for artificial insemination. However, documentation of pregnancies in this patient group has been limited. Deoxyribonucleic acid flow cytometry has been shown to be a reproducible quantitative method with which to evaluate spermatogenesis. We assessed the ability of deoxyribonucleic acid flow cytometry on testicular aspirates to predict the quality of sperm production in 13 men undergoing electroejaculation. Semen analysis was performed on antegrade and retrograde specimens. Deoxyribonucleic acid histograms from testicular aspirates were evaluated for the relative proportions of haploid, diploid and tetraploid cells. These ratios were compared to the means reported for our own series of 25 patients who at vasovasostomy had sperm intraoperatively and to the similar figures from a group of 10 accident victims. Five patients had normal testicular ploidy compartments defined as within 2 standard deviations from control means. Of the 5 patients 4 had normal sperm counts and motilities (30 per cent or greater). Two patients have contributed sperm for artificial insemination; 1 has resulted in pregnancy. Three patients had moderately abnormal (2 to 4 standard deviations) testicular ploidy classes. These patients had adequate sperm counts but low motilities (5 per cent or less). Of the 5 patients with markedly abnormal (greater than 4 standard deviations) histograms 4 remained azoospermic despite repeated attempts at electroejaculation. Our data support the use of deoxyribonucleic acid flow cytometry on testicular aspirates as a predictor of which patients might succeed and those who are unlikely to succeed in an electroejaculation program.

Flow cytometric DNA analysis of human urinary bladder specimens may be clinically useful for prognosis in transitional cell (urothelial) carcinoma and for detecting recurrence after treatment. However, many important methodological differences exist among institutions which have described this technique, and it has not previously been shown that data from different institutions are comparable. The National Cancer Institute has created a Flow Cytometry Network to address the need for technology assessment of flow cytometry. This report describes the independent flow cytometric analysis and interpretation of "unknown" paraffin-embedded bladder tumor specimens by the five Network institutions. Although important differences in method existed among the institutions, substantial agreement was achieved in actual data generated and their interpretation. This suggests that a consensus regarding acceptable laboratory performance of this technique could be reached, which should facilitate its more widespread clinical implementation.

The projection of knobby protuberances at the cell surface (zeiosis) is a general cellular response to cytochalasin D (CD), resulting from herniation of endoplasm through undefended places of the cortex during cell contractions and displacement of microfilaments induced by CD. Zeiosis is prevented by agents that interfere with the contractile response to CD, such as inhibitors of energy metabolism or cyclic AMP. The developed protrusions, which remain relatively stable in the presence of CD, contain chiefly mono- or subribosomes, and occasionally other organelles normally resident in endoplasm; compact microfilament felt occupies their bases and extends into their proximal stalks. Protein synthesis in the knobs is less than half of that in the polyribosome-containing endoplasm residual in the main body of the cell. Knobs first protrude singly near the margin of the contracting cells and rapidly cluster into small groups in the periphery even at lower temperature. The clusters then migrate centripetally and coalesce into a large aggregate near the apex of the immobilized and retracted cell: this movement is energy- and temperature-dependent. Aggregation is more prominent and stable in cell lines of epithelial derivation than in fibroblastic or other lines in which nuclear extrusion occurs more readily. The latter is regarded as a special manifestation of zeiosis. Macromarkers, such as latex spherules, migrate like the zeiotic knobs on the cell surfaces in the presence of CD. The aggregated knobs, although persistent for days in the presence of CD, are rapidly recessed after withdrawal of the agent as ruffling is resumed and the cells spread. These movements are discussed in terms of current concepts of mobility of the cell membrane.

Ethanol preservation of voided and catheterized urine has long been the standard for urinary cytologic study. In this report ethanol preservation of bladder irrigation specimens was evaluated for flow cytometric analysis in a multiinstitutional study requiring specimen transport. Specimens from ten patients obtained at one center were preserved for varying periods of time in 50% ethanol, then distributed by express mail to four other participating centers, up to 3000 miles distant. On receipt, from 1 to 3 weeks after collection, the samples were processed and examined by flow cytometric study using propidium iodide as the fluorochrome. Forty-six of the 50 (92%) analyzed specimens gave satisfactory histograms. In 41 of the 46 adequate samples (89%), an aneuploid peak in the alcohol preserved (propidium iodide stained) specimen correlated well with the fresh (acridine orange stained) specimen. However, there was variable loss of DNA stainability with a broadening of the coefficient of variation in some alcohol-preserved specimens and an increase in cellular debris so that measurements of DNA index and percent hyperdiploid cells were considered unreliable. The authors conclude that ethanol preservation of bladder irrigation specimens for short periods of time may be a feasible alternative when flow cytometric analysis cannot be carried out on fresh specimens, but that this is not optimal fixation for specimens that must be transported and further studies of other fixatives are recommended.

The levels of the low-molecular weight keratin 18 appearing in the voided urine of 130 healthy volunteers was studied using a new immunoradiometric assay for keratin 18 (IRMAK-18). Keratin 18 was detected in 79 urines with an average level of 2.5 +/- 4.7 ng./ml. The assay does not detect complex epidermal keratins or non-keratin intermediate filaments. Only 50% of the purified keratin added to urine could be recovered. The recovered keratin had the same molecular weight as the input molecules, implying that substances in the urine physically inhibited the assay rather than destroyed the keratin. The independent variables of cell count, dead cells, and protein concentration were not found to be important in evaluating the results. However, the urine concentration, as measured by creatinine, might be an important consideration for assessing the levels of keratin detected.

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.

Two blind retrospective flow cytometric studies were performed on archival stage O (Ta) papillary grades 1 to 2 transitional cell carcinomas of the bladder to learn whether deoxyribonucleic acid histogram patterns predict disease recurrence and progression. All patients with aneuploid histograms as predicted experienced recurrent disease. In 20 of 28 cases of recurrent disease the ploidy pattern changed during recurrence. Because of this finding we predicted disease progression if 2 or more histograms in the sequence were aneuploid. Two-thirds (8 of 12) of those having more than 1 aneuploid tumor incident experienced invasion, while only approximately a third (6 of 16) lacking repetitive aneuploidy had invasion. Furthermore, of the patients with 2 aneuploid lesions half had progression within 1 year and two-thirds within 2 years after the second aneuploid lesion. Therefore, it appears that in this disease deoxyribonucleic acid histograms can provide prognostic information beyond that obtained from tumor grade and stage.

Unilateral testicular torsion may result in contralateral testicular alterations which appear immunologically mediated and avoidable by immunosuppression or orchiectomy of the twisted testicle within 24 hours. This study was instituted to assess three temporal aspects of these observations: (1) The effect of prepubertal torsion was studied. It was found that after prepubertal torsion, the contralateral testicle underwent normal development. (2) The duration of torsion necessary to result in contralateral testicular alterations in adult rats was studied. Deoxyribonucleic acid (DNA) histograms were utilized to assess disturbed spermatogenesis. After greater than 8 hours of torsion, detorsion offered no protection to the contralateral testicle. Marked alterations occurred in DNA histograms of 60% to 80% of the animals. (3) The duration and significance of these alterations were assessed. The alterations persisted in the contralateral testis 6 months, and the fertility rates were significantly lower than for control animals.

This study evaluates the ability of DNA histograms obtained by flow cytometry to detect and quantify reversible alterations in spermatogenesis induced by cyclophosphamide, a known inhibitor of spermatogenesis. Evaluation of per cent of cells in each of the haploid (lc), diploid (2c), and tetraploid peaks (4c) as determined by flow cytometry in treated and control Balb/C mice over a six-week period, and comparison with routine histologic evaluation have led us to conclude that DNA histogram evaluation is a rapid and accurate means of identifying testicular damage and recovery. This technique may be useful in sequential monitoring of the effects of malignancy and/or treatments applied on spermatogenesis in young men.

Deoxyribonucleic acid flow cytometry of bladder washings has proved to be a valuable procedure for the diagnosis and followup of patients with transitional cell carcinoma. However, for this procedure to gain maximal acceptance, it should be possible to use voided urine specimens instead of bladder washings. To evaluate this possibility we compared histogram findings for 114 bladder washings and 122 concomitantly obtained urine samples (voided and catheterized) from 89 consecutive patients who had active or a history of transitional cell carcinoma. Unsatisfactory histograms were obtained in 4 per cent of the urine samples and in 2.6 per cent of the bladder washing samples. The satisfactory rate for voided or catheterized urine samples was the same. We conclude that in this patient population satisfactory deoxyribonucleic acid histograms can be obtained from samples of voided urine.

A human cell line has been established from a renal adenocarcinoma rib metastasis of a 58-y-old male. This cell line has been maintained in continuous culture for 20 mo. through more than 50 passages. It displays simultaneous expression of the intermediate filaments cytokeratin and vimentin. Flow cytometric analysis of DNA content reveals a major hyperdiploid population.

The ability to detect and monitor the course of transitional cell carcinoma (TCC) of the bladder using DNA histograms obtained from flow cytometry was studied. Voided urine and barbotage specimens were collected from patients with active TCC or a past history of TCC. These specimens were submitted to routine cytologic and flow cytometric analyses. Samples were considered to be positive if they met one or more of three criteria: if they had aneuploid or tetraploid peaks, if the DNA index of the major G1 peak was shifted more than 10 per cent from that of diploid cells, or if 15 per cent or more of the cells fell to the right of the major diploid G1 cell population thereby constituting a significant hyperdiploid cell population. Using these methods for patients with active disease, the detection rate was 91 per cent. In patients with a past history of TCC, positive histograms preceded the appearance of visible tumor in one third of the cases. Flow cytometry proved to be an excellent way of following patients with a past history of TCC or of screening patients suspected of having active disease. Following this protocol, few biologically active tumors go undetected. However, in 112 patients without a history of bladder cancer, the false positive or suspicious rate was 38 per cent. Before flow cytometry can be recommended as a widespread screening method for patients thought to be at risk of TCC of the bladder developing, this suspicious group will have to be eliminated.

Canine mandibular salivary glandular epithelial cells were established in culture and passaged more than 250 times. The cultivated cells show epithelial morphology, and ultrastructurally, show desmosomes, desmosome-like specializations (punctae adhaerens), cytoplasmic tonofilaments, and neoluminae suggestive of glandular epithelium. The cells showed modest PAS reactivity and were negative for alcian blue. Using immunohistochemistry, the 563 cells were shown to express cytokeratins, vimentin and epithelial membrane antigen. Chromosome analysis of the cells revealed a modal number of 76 plus 2 sex chromosomes, and the DNA histogram showed major peaks at 2 C (diploid) and 4 C (tetraploid). Attempts to demonstrate a retrovirus by particle labelling with 3H-uridine or by isolation of 70S RNA gave negative results. A nucleic acid hybridization test for mycoplasma contamination gave negative results. It is anticipated that this established cell line derived from normal canine glandular epithelium will prove useful for canine virologic studies, or as a substrate for the growth of type C retroviruses from heterospecies.