Differences in gene expression in muscle-invasive bladder cancer: a comparison of Italian and American patients.

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.

The prognostic significance of S-phase analysis in stage Ta/T1 bladder cancer. A Southwest Oncology Group Study.

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.

Impact of hormone replacement therapy on the body mass and fat compositions of menopausal women: a cross-sectional study.

OBJECTIVE: The purpose of the study was to compare the body mass and fat compositions of menopausal women who were taking conventional doses of hormone replacement therapy (HRT) with that of menopausal women who were not taking any hormones. DESIGN: The body fat composition of 169 healthy postmenopausal women was measured using a noninvasive handheld machine, the Electrolipograph (BioAnalogics ELG, Beaverton, OR, USA). Impedance to electrical flow in tissues is lower with increasing water content of the tissue. Information on HRT, lifestyle, diet, smoking, and alcohol was obtained from the medical record and by a telephone interview before women were invited to participate. HRT and non-HRT groups were compared. Multivariate linear regression, which included age, years since menopause, type of menopause, and use of HRT, was performed for each of the two major outcomes: body mass index (BMI) and percentage of body fat. RESULTS: Comparisons between subgroups showed a large number of significant differences reflecting differences in age since menopause, baseline BMIs, and baseline waist to hip ratios. In the regression model, however, the only factor significantly associated with lower fat and BMI was the use of HRT. Women who were taking HRT had significantly lower percentages of body fat (-4.8%; p < 0.001) and BMI (-2.6 kg/m2; p < 0.001) compared with nonusers. Age and duration and type of menopause were not significant predictors of weight and BMI in this group of postmenopausal women. CONCLUSIONS: In this study, HRT seems to be associated with a significant reduction in postmenopausal weight and fat mass gains. This may be an important mechanism by which HRT exerts its beneficial long-term effects on cardiovascular health.

Use of a yeast assay to detect functional alterations in p53 in prostate cancer: review and future directions.

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.

Racial differences in clinically localized prostate cancers of black and white men.

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.

p53 and bcl-2 immunohistochemical alterations in prostate cancer treated with radiation therapy.

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.

Very frequent p53 mutations in metastatic prostate carcinoma and in matched primary tumors.

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.

Correlation of genetic and immunodetection of TP53 mutations in malignant and benign prostate tissues.

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.

Prognostic significance of DNA ploidy in Ta/Tl bladder cancer: A southwest oncology group study.

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.

False DNA aneuploidy in canine and human neoplasms.

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.

Significance of abnormal diploid DNA histograms in localized prostate cancer and adjacent benign prostatic tissue.

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.

Fixation method useful for cytologic examination and DNA flow cytometry of exfoliated bladder cells.

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.

Flow cytometry as a predictive modality in prostate cancer.

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.

Evaluation of DNA flow cytometry as a screening test for bladder 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)

Precision of DNA flow cytometry in inter-institutional analyses.

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)

DNA flow cytometric study of the hyperplastic and neoplastic canine prostate.

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.

Evaluation of DNA flow cytometry as a screening test for bladder cancer.

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.

Comparison of flow cytometry to routine testicular biopsy in male infertility.

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.