Phase II randomized clinical trial of lycopene supplementation before radical prostatectomy.

An inverse association has been observed between dietary intake of lycopene and the risk of prostate cancer. We investigated the effects of lycopene supplementation in patients with prostate cancer. Twenty-six men with newly diagnosed, clinically localized (14 T(1) and 12 T(2)) prostate cancer were randomly assigned to receive 15 mg of lycopene (n = 15) twice daily or no supplementation (n = 11) for 3 weeks before radical prostatectomy. Biomarkers of differentiation and apoptosis were assessed by Western blot analysis on benign and malignant parts of the prostate gland. Prostatectomy specimens were entirely embedded, step-sectioned, and evaluated for pathological stage, Gleason score, volume of cancer, and extent of high-grade prostatic intraepithelial neoplasia. Plasma levels of lycopene, insulin-like growth factor-1 (IGF-1), IGF binding protein-3, and prostate-specific antigen were measured at baseline and after 3 weeks of supplementation or observation. Eleven (73%) subjects in the intervention group and two (18%) subjects in the control group had no involvement of surgical margins and/or extra-prostatic tissues with cancer (P = 0.02). Twelve (84%) subjects in the lycopene group and five (45%) subjects in the control group had tumors <4 ml in size (P = 0.22). Diffuse involvement of the prostate by high-grade prostatic intraepithelial neoplasia was present in 10 (67%) subjects in the intervention group and in 11 (100%) subjects in the control group (P = 0.05). Plasma prostate-specific antigen levels decreased by 18% in the intervention group, whereas they increased by 14% in the control group (P = 0.25). Expression of connexin 43 in cancerous prostate tissue was 0.63 +/- 0.19 absorbance in the lycopene group compared with 0.25 +/- 0.08 in the control group (P = 0.13). Expression of bcl-2 and bax did not differ significantly between the two study groups. IGF-1 levels decreased in both groups (P = 0.0002 and P = 0.0003, respectively). The results suggest that lycopene supplementation may decrease the growth of prostate cancer. However, no firm conclusions can be drawn at this time because of the small sample size.

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.

Maintenance bacillus Calmette-Guerin immunotherapy for recurrent TA, T1 and carcinoma in situ transitional cell carcinoma of the bladder: a randomized Southwest Oncology Group Study.

PURPOSE: Bacillus Calmette-Guerin (BCG) immunotherapy has been widely accepted as the optimal treatment for carcinoma in situ and high grade superficial transitional cell carcinoma. However, controversy remains regarding the role of maintenance therapy, and its long-term effect on recurrence and progression. MATERIALS AND METHODS: All patients in the study had transitional cell carcinoma of the bladder with carcinoma in situ or an increased risk of recurrence. The criteria for increased risk were 2 or more episodes of tumor within the most recent year, or 3 or more tumors within 6 months. At least 1 week following biopsy of carcinoma in situ and resection of any stage Ta or T1 transitional cell tumors 660 patients were started on a 6-week induction course of intravesical and percutaneous Connaught BCG. Three months following initiation of BCG induction therapy 550 consenting patients were stratified by purified protein derivative skin test and the presence of carcinoma in situ, and then randomized by central computer to receive BCG maintenance therapy (maintenance arm) or no BCG maintenance therapy (no maintenance arm). Maintenance therapy consisted of intravesical and percutaneous BCG each week for 3 weeks given 3, 6, 12, 18, 24, 30 and 36 months from initiation of induction therapy. The 384 eligible patients who were disease-free at randomization constitute the primary intent to treat analytic group because they could be followed for disease recurrence. All patients were followed for adverse effects of treatment, recurrence, disease worsening and survival. RESULTS: No toxicities above grade 3 were noted in the 243 maintenance arm patients. The policy of withholding maintenance BCG from patients with increased side effects may have diminished the opportunity to observe severe toxicity. Estimated median recurrence-free survival was 35.7 months (95% confidence interval 25.1 to 56.8) in the no maintenance and 76.8 months (64.3 to 93.2) in the maintenance arm (log rank p<0.0001). Estimated median time for worsening-free survival, defined as no evidence of progression including pathological stage T2 disease or greater, or the use of cystectomy, systemic chemotherapy or radiation therapy, was 111.5 months in the no maintenance and not estimable in the maintenance arm (log rank p = 0.04). Overall 5-year survival was 78% in the no maintenance compared to 83% in the maintenance arm. CONCLUSIONS: Compared to standard induction therapy maintenance BCG immunotherapy was beneficial in patients with carcinoma in situ and select patients with Ta, T1 bladder cancer. Median recurrence-free survival time was twice as long in the 3-week maintenance arm compared to the no maintenance arm, and patients had significantly longer worsening-free survival.

A novel hydroxamic acid compound, BMD188, demonstrates anti-prostate cancer effects by inducing apoptosis. II: In vivo efficacy and pharmacokinetic studies.

BACKGROUND: In the preceding paper, we demonstrated that, BMD188 [cis-1-hydroxy-4-(1-naphthyl)-6-octylpiperidine-2-one], a newly synthesized cyclic hydroxamic acid compound, induces potent apoptotic death of prostate cancer cells in vitro. In this project, we studied the in vivo pharmacokinetic behavior and anti-tumor efficacy of this novel compound. MATERIALS AND METHODS: A bioavailability/elimination study was first performed using radiolabeled BMD188 administered to rats through intraperitoneal (i.p.), intravenous (i.v). or oral (p.o.) routes. Based on these pharmacokinetic data as well as pilot experiments on in vivo toxicity, two sets of efficacy studies, with i.p. administered BMD188, were performed in SCID mice or athymic nude mice which had been orthotopically transplanted with Du145 human prostate cancer cells. Tumor growth rate was measured and the final tumor weights and sizes determined. Subsequently, histopathological data were obtained and tumor tissue sections were used for apoptosis (i.e., TUNEL) staining. RESULTS: The pharmacokinetic studies revealed low (approximately 8%) absorption through the p.o. route and high (approximately 70%) absorption through the i.p. route. The average plasma half life (T1/2) of BMD188 was approximately 50 h. Post-absorption, plasma elimination of radioactivity was similar to that in animals given [3H]-188 intravenously. The in vivo efficacy results indicate that i.p. administered BMD188 significantly inhibited the primary growth and local invasion of Du145 prostate cancer cells orthotopically implanted into SCID or athymic nude mice. The tumor-inhibitory effect of BMD188 was due to apoptosis induction in vivo, as revealed by histological analysis as well as TUNEL staining of the tumor tissue sections. CONCLUSION: Collectively, the preceding in vitro and the current in vivo studies suggest that BMD188 and its analogs may find clinical applications in the treatment of prostate cancer patients by inducing apoptotic death of prostate cancer cells.

Matrix metalloproteinase-9 expression in bladder washes from bladder cancer patients predicts pathological stage and grade.

The matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and MMP-9) have been associated with tumor cell invasion and metastasis in many human cancers. Here we examined the expression of proMMP-2 (gelatinase A) and proMMP-9 (gelatinase B) proteins in the cellular component of bladder washes obtained from 65 patients. Twenty-six patients had active bladder cancer, 24 had a history of bladder cancer but no evidence of active disease at the time of cystoscopy (recurrence-free), and 15 patients had lesions other than bladder cancer (controls). The results were correlated with the cytological findings of the bladder wash and the histopathological results of the tumor resection when performed. In patients with active transitional cell carcinoma of the bladder, 71 and 38% had expression and overexpression of the latent form of MMP-9 (proMMP-9), respectively. In contrast, neither latent nor active MMP-2 could be detected in any of the samples examined, regardless of tumor status. Overexpression of proMMP-9 correlated with higher grade (P = 0.003) and pathological stage (P = 0.04) of disease in the active bladder cancer group. No significant gelatinase expression was detected in the recurrence-free and control cases. Compared with urine cytology, proMMP-9 expression had an overall higher sensitivity for bladder cancer identification (71 versus 54%, P = 0.11). Detection of proMMP-9 in bladder washes may be a novel approach for the identification of patients with more aggressive forms of bladder cancer.

BMD188, A novel hydroxamic acid compound, demonstrates potent anti-prostate cancer effects in vitro and in vivo by inducing apoptosis: requirements for mitochondria, reactive oxygen species, and proteases.

A newly synthesized cyclic hydroxamic acid compound, BMD188 [cis-1-hydroxy-4-(1-naphthyl)-6-octylpiperidine-2-one], was found to induce the apoptotic death of cultured prostate cancer cells by activating caspase-3. Orally administered BMD188 significantly inhibited the primary growth of prostate cancer cells (Du145) orthotopically implanted into SCID mice. Mechanistic studies indicated that BMD188 did not alter the protein levels of several Bcl-2 family members. In contrast, the BMD188 effect required three essential factors: reactive oxygen species (ROS), the mitochondrial respiratory chain function, and proteases. First, the apoptosis-inducing effect of BMD188 could be blocked by ROS scavengers such as Desferal. Second, both BMD188-induced PARP cleavage as well as PC3 cell apoptosis could be dramatically inhibited by several complex-specific mitochondrial respiration blockers. The involvement of mitochondria was also supported by the observations that BMD188 dramatically altered the mitochondrial distribution and morphology without affecting the cellular ATP levels. Finally, the apoptosis-inducing effect of BMD188 in PC3 cells could be significantly inhibited by serine protease inhibitors (TPCK and TLCK) as well as by caspase inhibitors (zVAD-fmk and DEVD-CHO). Collectively, the present study suggests that BMD188 and its analogs may find clinical applications in the treatment of prostate cancer patients by inducing apoptotic death of prostate cancer cells.

Inhibition of pancreatic tumor cell growth in culture by p21WAF1 recombinant adenovirus.

New innovations are needed for the treatment of pancreatic cancer, as current treatments do not offer significant improvements in overall survival. p21WAF1--a tumor suppressor gene--acts as a downstream effector of p53 function and mediates G1 cell cycle arrest by inhibiting cyclin-dependent kinases, which promote cell growth. p21 expression has also been shown to increase more than 20-fold in senescent cells in culture. The replication-defective recombinant adenoviral system (rAd), a major innovation in gene transfer technology, has recently been used in gene therapy applications for various malignancies but not for pancreas cancer. In this study we used rAd-p21 in cell growth inhibition studies of pancreatic tumor cell lines in vitro to explore its potential as a prospective gene therapy for pancreatic adenocarcinoma. We studied two pancreatic cell lines in culture, HPAC and Hs766T. HPAC revealed higher endogenous levels of p21 gene expression at the protein and RNA levels compared to Hs766T. p21 induction was tested using different doses of rAd-p21 to establish an optimum dose for significant induction of p21 gene expression. Tumor cell growth in culture following rAd-p21 infection was also analyzed in both cell lines. HPAC and Hs766T cell lines showed a significant dose-dependent increase in p21 protein expression when infected with rAd-p21. Both cell lines showed significant growth arrest, but Hs766T showed less cell growth inhibition than HPAC cells. Flow cytometric cell cycle analysis of rAd-p21-infected cells showed a statistically significant increase in the number of cells in G0/G1 in HPAC cells. Similar results were also obtained in Hs766T cells, however, the data were not statistically significant. In conclusion, pancreatic tumor cell growth can be inhibited by rAd-p21 in vitro, with significant numbers of tumor cells reverting from S to G0/G1. Thus rAd-p21 may be effective as a candidate gene therapy for pancreatic cancer and should be further evaluated with in vivo studies.

Histopathologic and flow-cytometric analysis of neoplastic and benign "background" tissue in breast carcinoma resections.

Two-color, multiparametric synthesis phase fraction (SPF) analysis of cytokeratin-labeled epithelial cells was flow cytometrically performed on both benign (SPFb) and malignant tissue samples (if available, SPFt) from 132 mastectomy/lumpectomy specimens. These data were then correlated with clinicopathologic features, including (1) tumor differentiation, (2) the proportion of tumor comprised of duct carcinoma-in situ (DCIS), and (3) the histology of accompanying benign breast tissue, classified by predominant microscopic pattern as intact, normal terminal duct lobular units (NTDLU, 34% of cases), atrophic (AT, 33% of cases), proliferative fibrocystic (PFC, 26% of cases), and non-proliferative fibrocystic (NPFC, 7% of cases). SPFt was inversely correlated with extent of DCIS (DCIS = 0-20% tumor volume - 12.7% mean SPFt, vs. DCIS >20% tumor volume - 6.4% mean SPFt, p = 0.001). SPFt also correlated with the histology of background benign breast tissue (NTDLU - 14.8% mean SPFt vs. AT - 6.9% mean SPFt vs. PFC - 12.7% mean SPFt, p = 0.05) but it did not correlate with patient age or SPFb (overall mean = 0.73%). SPFb was correlated with patient age (>56 yr - 0.59% mean SPFb vs. <56 yr - 0.84% mean SPFb, p = 0.02), with background histology (NTDLU - 1.1% mean SPFb vs. AT - 0.43% mean SPFb vs. PFC - 0.70% mean SPFb, p < 0.02) and with the grade of the neoplasm (well/moderate - 0.58% mean vs. poorly differentiated - 0.85% mean, p = 0.04). Patients having a background of PFC were significantly older than patients with a background of NTDLU (45.2 yr vs. 60.2 yr, p = 0.01). We conclude: (1) breast carcinomas arising from a background of more actively cycling pre-involutional or proliferative fibrocystic epithelium have a greater proliferative fraction than tumors arising from atrophic epithelium, implying that the differentiation status of target cells may impact the effect(s) of tumorigenic events; (2) PFC may represent delayed, abnormal or interrupted involution rather than a hyperproliferative state relative to NTDLU, suggesting that it facilitates neoplasia by extending the period of exposure to promoter agents such as endogenous hormones, and (3) lower SPFt in breast neoplasia with more abundant "residual" DCIS may reflect a lengthier pre-invasive disease interval due to intrinsically less aggressive phenotype.

The clinical significance of p21(WAF1/CIP-1) and p53 expression in pancreatic adenocarcinoma.

BACKGROUND: Wild-type p53 protein activates the WAF1/CIP-1 (p21) gene, leading to G1 arrest after DNA damage. The authors investigated the relation of p21 and p53 expression in pancreatic adenocarcinomas to disease stage, overall patient survival, and survival when chemotherapy or radiation therapy was given. METHODS: Paraffin embedded tissue sections of 75 ductal adenocarcinomas of the pancreas were immunostained for p53 and p21. Nuclear expression was scored as absent, focal (<10%), moderate (10-50%), or strong or diffuse (>50%). RESULTS: The median survival of patients whose pancreatic tumors expressed the p21 protein (43 of 75 cases, 57%) was better than that for patients whose tumors were p21 negative (32 of 75 cases, 43%) (median survival, 13.5 vs. 9.8 months, respectively; P = 0.23). No difference in survival was found with regard to p53 protein expression (43 of 75 cases, 57%); however, strong p53 expression was significantly associated with advanced disease stage (70% in Stage IV vs. 13-28% in lower stages). Expression of p21 correlated with earlier clinical stage. Stage specific comparisons showed a trend toward increased survival among p21 positive tumor patients diagnosed at clinical Stages I and III but not among those diagnosed at Stage IV. Adjuvant chemotherapy or radiation improved survival significantly if tumors expressed p21 or no p53. CONCLUSIONS: Expression of p21 is significantly associated with earlier clinical stage in pancreatic adenocarcinoma, perhaps accounting for the better survival observed in this patient group than among those whose tumors were p21 negative. Improved survival with either chemotherapy or radiation therapy was observed for patients whose tumors were p21 positive or p53 negative.

Relationship of family cancer history to the expression of p53, p21WAF-1, HER-2/neu, and K-ras mutation in pancreatic adenocarcinoma.

CONCLUSION: In our series of 81 cases, a history of family cancer was present in 52% of patients (42/81) with pancreatic cancer. Nine percent (7/81)had a family history of pancreatic cancer. Our studies suggest a possible relationship of family cancer history to the expression of p53 and p21WAF in pancreatic tumors, but show no relationship to the expression of HER-2/neu or to the prevalence of K-ras mutations. A lower incidence of p53 expression observed in patients with a family history of cancer suggests normal p53 protein is present in a majority of patients who develop pancreatic tumors related to other--as yet unidentified-inherited or familial risk factors. There was no significant difference in survival of pancreas cancer patients with and without a family history of cancer. However, survival in pancreas cancer patients may be influenced (improved) by p21WAF-1 expression. BACKGROUND: Pancreas cancer is the fifth leading cause of cancer deaths (27,800 deaths/yr) in the United States. Various risk factors, including cigarette smoking, high-fat diet, DDT exposure, chronic pancreatitis, and diabetes mellitus, have been associated with pancreatic carcinoma. A few studies have suggested a genetic predisposition or increased risk for pancreatic cancer within families, but the exact etiology is largely unknown. In a series of 81 patients with pancreatic carcinoma, we analyzed the status of K-ras gene mutations and the expression of P21WAF-1, p53, and HER-2/neu protein to identify possible molecular associations in pancreas cancer cases of these molecular markers to family histories of cancer and pancreas cancer. METHODS: Paraffin-embedded tissue sections from 81 cases of pancreatic adenocarcinoma were used for DNA extraction and immunohistochemical staining. K-ras mutation was studied by single-stranded conformation polymorphism (SSCP) and slot-blot allele-specific oligonucleotide (ASO) hybridization of PCR-amplified DNA product. Overexpression (aberrant expression) of p53, p21WAF-1, and HER-2/neu was documented by scoring nuclear localized p53, p21WAF-1 protein and cell membrane expression of HER-2/neu after immunostaining with gene product-specific monoclonal antibodies (MAbs). RESULTS: Forty-two (42) of 81 patients studied in this series had a history of cancer in their families (52%). Seven of those 42 had a history of pancreatic carcinoma (17% or 9% of total cases). The incidence of K-ras mutation and the expression of p21WAF-1 and HER-2/neu in patient groups with and without a family history of cancer was not statistically different (83 vs 74%, p = 0.416; 57 vs 41%, p = 0.184; and 83 vs 81%, p = 1.000, respectively). However, the incidence of p53 expression was significantly lower in patients with a family history of cancer (40 vs 72%, p = 0.007). There was no statistical difference in survival of patients with a family history of cancer in relation to either K-ras mutation, p53 expression, p21, or HER-2/neu expression. However, patients lacking a family history of cancer showed improved survival trends in relation to p21 expression (median survival of 16 vs 8 mo, p = 0.029).

Prevalence and clinical significance of combined K-ras mutation and p53 aberration in pancreatic adenocarcinoma.

CONCLUSION: This study could not attribute survival differences to the coincident acquisition of two common genetic alterations, K-ras mutation and p53 overexpression in pancreatic adenocarcinoma patients. Additionally, our data indicate the converse to be true: Those patients lacking both K-ras mutation and aberrant p53 expression showed the shortest survival when compared with cases showing either alteration or both. This study also showed the negative effect of K-ras mutation and p53 expression on pancreas cancer patients' survival after treatment with either radiation therapy or chemotherapy. BACKGROUND: Mutations of the oncogene K-ras at codon 12 are reported to be the most common genetic alteration in pancreatic carcinoma, whereas either overexpression or mutation of the tumor suppressor p53 gene is considered the most common genetic alteration in neoplasia of all types. p53 overexpression has been attributed to survival differences in pancreatic carcinoma, but such association is still controversial. No studies have fully documented the combined incidence of K-ras and p53 alterations in pancreatic adenocarcinoma, or their combined effect on patient survival in a large case series. The influence of radiation or chemotherapy in groups showing both, either, or neither mutation is also undocumented. METHODS: Paraffin-embedded tissue sections from 76 cases of pancreatic adenocarcinoma were cut for DNA extraction for K-ras analysis and immunohistochemical staining for aberrant p53 expression. K-ras mutation was determined by single-strand conformation polymorphism (SSCP) and slot-blot allele-specific oligonucleotide (ASO) hybridization of PCR-amplified DNA product p53 expression was scored on the basis of percent nuclear staining with the MAb DO7. RESULTS: Sixty-four of 76 cases (84%) showed K-ras mutation, p53 expression, or both, K-ras was mutated in 55 of 76 cases (72%). p53 was expressed in 33 of 76 cases (43%). Twenty-four of 76 cases (31%) showed both K-ras mutation and p53 expression. The presence of both alterations was not related to significant differences in tumor grade, stage, or survival compared to either alteration alone. A sizable subset (16% of cases) lacked either alteration, and surprisingly, this group showed the shortest median survival compared to those with K-ras mutation, p53 expression, or both (p = 0.024). Patients whose tumors were K-ras-negative showed the greatest difference in median survival with radiation therapy (median survival 30.8 mo vs 7.8 mo with no radiation, p = 0.005).

HER-2/neu expression in pancreatic adenocarcinoma: relation to tumor differentiation and survival.

HER-2/neu expression in pancreatic adenocarcinoma has been inconsistently reported and has not been fully evaluated with respect to histologic grade and tumor grade heterogeneity. We studied HER-2/neu expression in a series of 79 primary pancreatic carcinomas using immunohistochemical methods, with expression scored for each histologic grade represented in each tumor. We found significantly lower expression of HER-2/neu in poorly differentiated (PD) portions of tumors-those areas lacking glandular differentiation-compared to well-differentiated (WD) and moderately differentiated (MD) portions of tumors. Forty-two of 68 (62%) invasive tumors with WD or MD glands showed moderate or strong expression of HER-2/neu in WD/MD areas; only 6 of 32 (19%) invasive tumors with PD areas showed similar expression in PD. In mutually exclusive patient sets, we also found a statistically different prevalence of HER-2/neu expression in patients with PD (6/32 cases; 19%) and without PD (29/47 cases; 62%) tumors (p < 0.001). Twenty-three cases had directly comparable areas of PD versus MD or WD. In 19 of 23 cases HER-2/neu expression was graded comparatively lower (or negative) in areas of PD than in MD or WD. Overall 46 of 79 cases (58%) showed moderate to strong HER-2/neu expression inclusive of all histologic grades, and 63 of 79 (80%) cases were HER-2/neu positive, if including weak or focal staining. There was no significant difference in the survival of patients with HER-2/neu-positive versus-negative tumors or in patients with versus without PD tumors. We have confirmed that although HER-2/neu gene expression is common to many pancreatic carcinomas, it is not common to tumors lacking glandular differentiation. HER-2/neu gene expression could not be related to survival differences--perhaps due to overall poor survival within adenocarcinomas of the pancreas--but the pattern of HER-2/neu expression suggests a relationship to glandular differentiation and early oncogenesis.

Evaluation of MYC and chromosome 8 copy number in breast carcinoma by interphase cytogenetics.

We used fluorescence in situ hybridization (FISH) to determine MYC and chromosome 8 copy number on whole nuclear imprint preparations of 24 breast carcinomas, seven benign breast samples, and two phyllodes tumors. None of the benign tissues and neither of the phyllodes tumors demonstrated an increased copy number for MYC or chromosome 8, which was defined as greater than two signals in > 10% of nuclei. In contrast, 22 of 24 carcinomas demonstrated an increased MYC copy number. The modal numbers of MYC copies/nucleus were 0-2 in seven cases (29%), 3-5 in seven cases (29%), 6-9 in five cases (21%), and > 9 in five cases (21%). An increased chromosome 8 copy number was observed in 21 of 22 carcinomas with MYC gain, and the modal number of signals/nucleus was either identical to (n = 14; 64%) or less than (n = 8; 36%) the number of MYC copies. The number of MYC copies correlated with cellular DNA content, as determined by using flow cytometry. In peridiploid tumors (DNA index 0.9-1.2; n = 7), the MYC copy numbers/nucleus were 0-2 in five cases and 3-5 in two cases. In contrast, the modal MYC copy numbers/nucleus among the 11 hyperdiploid tumors (DNA index 1.3-1.9) were 0-2 in one case, 3-5 in four cases, 6-9 in five cases, and > 9 in one case. All three tetraploid/hypertetraploid carcinomas exhibited > 9 MYC copies/nucleus. We conclude that an increased MYC copy number, as detected by using interphase cytogenetics, is extremely frequent in human breast carcinomas. However, in most cases, MYC gene duplication is probably secondary to polysomy of chromosome 8 and/or genomic endoreduplication (i.e., DNA aneuploidy).

Cell cycle analysis of normal, atrophic, and hyperplastic breast epithelium using two-color multiparametric flow cytometry.

We performed two-color flow cytometric synthesis phase fraction (SPF) determinations on cytokeratin-labeled benign epithelial populations from 142 breast specimens (41 mastectomy, 70 diagnostic biopsy, 31 reduction mammoplasty). There was wide variability of SPF, ranging from 0.1 to 3.5%, with a frequency distribution skewed to higher values (mean 0.75%, median 0.5%). The mean SPE for women less than 29 years was 0.91%, vs. 0.89% for 30-42 years, 0.66% for 43-49 years, and 0.56% for > or = 50 years (P = 0.05). Histologically atrophic tissue samples exhibited a mean SPF approximately half that of morphologically normal tissue from premenopausal age women (0.79% vs. 0.36%, P = 0.02). Tissues showing histologically proliferative fibrocystic features had a greater mean SPF than non-proliferative fibrocystic tissues (0.59% vs. 0.92%); however, due to the wide spread of values within each of these categories, this difference was not statistically significant and neither group was significantly different from 'normal' tissue samples. Patients with histologically normal breast tissue, though, were significantly younger (mean = 34.6 years) than those with fibrocystic changes (non-proliferative mean = 53.4 years vs. proliferative mean = 42.8 years, P = 0.005). Synchronous right- and left-sided specimens obtained from reduction mammoplasty demonstrated significantly correlated SPF determinations (R = 0.77). We conclude that selective analysis of epithelial populations using two-color flow cytometry provides cell cycle information in benign breast tissue which is analogous to that obtained by labor-intensive nucleotide labeling studies. This study also confirms the biologic variability and age-dependence of breast epithelial proliferation. Finally, the data imply that derangements of cell proliferation in fibrocystic conditions are heterogeneous, complex and incompletely correlated with histologic parameters such as hyperplasia.

Correlation between p53 immunostaining patterns and gene sequence mutations in breast carcinoma.

We performed p53 immunostaining in 82 invasive breast carcinomas by using two commercially available antibodies, one of which (DO7) was employed in formalin-fixed paraffin-embedded sections. The other antibody (PAb1801) was evaluated in corresponding acetone-fixed cryostat sections. A greater percent of cases were immunostained with DO7 compared to PAb1801 (52% vs 33%); however, the staining was more often heterogeneous (6-50% cells positive) or focal (< or = 5% cells positive) with DO7 (9% vs 31%). To investigate the genetic relevance of p53 immunostaining, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were performed on exons 2-11 by using archival tissue samples of 18 cases that were selected on the basis of certain immunostaining patterns. Two (33%) of six tumors with negative staining for DO7 had gene sequence mutations; however, one of these mutations was a base-pair deletion that caused a reading-frame shift and the other was a base-pair insertion that resulted in a stop codon. Both of these tumors exhibited immunostaining with PAb1801, although it was weak and cytoplasmic in one case. Conversely, three (30%) of 10 tumors showing immunoreactivity in 6-100% of cells with both reagents lacked a gene sequence mutation. Of the remaining seven tumors that were positive by SSCP, six contained a point mutation resulting in a base-pair substitution. Despite repeat analyses, one of the cases positive by SSCP failed to demonstrate a mutation in the sequenced exons. Four (80%) of five cases with heterogeneous DO7 immunoreactivity (that is, 6-50% of nuclei positive) were positive for gene sequence mutation. Neither of two cases showing focal DO7 nuclear staining in < 5% of tumor cells contained a mutation in the sequenced exons, and neither of these cases was strongly positive with PAb1801. Staining for either antibody was significantly associated with adverse outcome, as determined by disease recurrence at 52 months median follow-up (DO7, p = 0.01; and PAb1801 p = 0.002, chi-squared test). We conclude that a variety of factors may account for discrepancies when immunohistology is used to evaluate p53 status. These include fixation artifacts, differing epitope specificities of monoclonal reagents, presence of immunohistologically "silent" mutations and, possibly, aberrant overexpression of wild-type protein.

Tumor suppressor p53 gene mutation in squamous cell carcinoma of the larynx.

Using polymerase chain reaction (PCR)-SSCP (single-strand conformation polymorphism) and PCR-DNA sequencing analysis, we screened 61 cases of laryngeal squamous cell carcinoma for mutations in exons 5-8 of the p53 gene. Mutations were found in 31.3% (19 of 61) of the laryngeal cancers. Seventeen of 19 (84.2%) cases showing p53 gene mutations were stage III and IV, which suggests that p53 gene mutation is a rather late event in tumor development and is involved in the progression of laryngeal squamous cell carcinoma. A high frequency of G:C to T:A transversion (50%, seven of 14), especially G to T (35.7%, five of 14), was noted in laryngeal carcinoma samples in our study. This finding may point toward an environmental carcinogen (such as tobacco smoke) as an important agent in the genesis of laryngeal squamous cell carcinoma. Most of the p53 gene mutations (18 of 19) found in our studies could change the protein and thus may cause an inactivation of the p53 tumor suppressor gene, strongly suggesting that p53 gene mutation plays a crucial role in the progression of laryngeal carcinoma.

Prostate cancer old problems and new approaches : Part I. epidemiology, incidence and genetic alterations.

Rates of prostate cancer (PCa) have increased so dramatically over the last decade that the age adjusted incidence rate for PCa is now greater than that any other cancer among men in the United States. This review, published as a three part series, provides a state-of-art assessment of the PCa problem in its divergent aspects.Part 1 covers epidemiology, incidence and progression. Several epidemiological studies have demostrated that first degree male relatives of men with PCa are at increased risk of developing the disease. Familial and genetic factors as well as medical, anthropometric, dietary, hormonal and occupational factors involved in PCa are discussed. Postmortem examination of the prostate in men without evidence of PCa documented a high frequency of adenocarcinoma. Latent disease occurred as early as the second decade of life. Although there is no significant difference in incidence between Caucasian and African-American males, high grade prostatic intraepithelial neoplasia (HGPIN) is higher in the latter group. While dietary fat, androgens and certain environmental factors may be determinants for PCa, the exact mechanism of tumorigenesis is still relatively unknown. The current thinking of the role of genomic instability, chromosomal alterations, tumor suppressor genes and the androgen receptor are explored.