Loss of P27Kip1 expression correlates with tumor grade and with reduced disease-free survival in primary superficial bladder cancers.

p27Kip1 is a member of the Cip1/Kip1 family of cyclin-dependent kinase inhibitors and is a potential tumor suppressor gene. We previously reported a deregulated expression of p27Kip1 in a series of human cancer cell lines and in primary breast and colon cancers. Moreover, p27Kip1 has been reported as an important prognostic factor in primary lung, breast, colon, and prostate cancers. In this study, we evaluated the prognostic value of p27Kip1 in a series of 96 superficial (pTa-1) human bladder carcinomas. High (>50% positive cells), moderate (25-50%), and low (<25%) p27Kip1 staining was observed in 39 (41%), 19 (20%), and 38 (39%) of the 96 primary superficial bladder cancers, respectively. No significant association was found between the expression level of p27Kip1 and tumor stage. Decreased p27Kip1 staining correlated with higher tumor grade (P = 0.001). Interestingly, a significant association was observed between increased expression of p27Kip1 and positivity for p53 (>20% positive cells; P < 0.001). A significant correlation was also observed between low expression of p27Kip1 and decreased disease-free survival (P = 0.0003 by log-rank test) and overall survival (P = 0.01 by log-rank test). Furthermore, on multivariate analysis, low p27Kip1 protein expression was an independent predictor of reduced disease-free survival (P = 0.018; relative risk = 1.95) second only to tumor stage. These data indicate that p27Kip1 protein is frequently expressed at low level in poorly differentiated tumors and suggest that this protein might represent a useful prognostic marker for disease recurrence and overall survival in superficial bladder carcinomas.

Loss of p21Waf1 expression is a strong predictor of reduced survival in primary superficial bladder cancers.

p21Waf1 is a downstream effector of p53 and belongs to the Cip1/Kip1 family of cyclin-dependent kinase inhibitors. Thus, it is a potential tumor suppressor gene and likely plays an important role in tumor development. Moreover, reduced expression of p21Waf1 has been reported to have prognostic value in several human malignancies. In this study, we evaluated the prognostic value of p21Waf1 in bladder cancer compared with other clinicopathological features and with p27Kip1 and p53 expression. A total of 96 superficial (pTa-1) human bladder carcinomas were immunohistochemically stained for p21Waf1 protein expression. Positive p21Waf1 staining (> or =5% positive nuclei) was observed in 68 of the 96 (71%) tumors. p21Waf1 expression was neither associated with tumor stage (P = 0.9) nor with tumor grade (P = 0.18) but was significantly associated with both p53 protein expression (> or =20% positive nuclei; P = 0.007) and with p53 gene mutations (P = 0.017). A significant correlation was also observed between positivity for p21Waf1 and high (>50% positive cells) p27Kip1 expression (P = 0.04). With regard to prognosis, patients whose tumors showed absence of p21Waf1 staining displayed a significantly shorter overall survival (P = 0.01 by log-rank test). However, p21Waf1 expression did not correlate with disease-free survival (P = 0.15 by log-rank test). On a multivariate analysis that also included p53 and p27Kip1 expression, negative p21Waf1 staining was an independent predictor of reduced overall survival (P = 0.004; relative risk, 5.32), stronger than age and tumor stage. These data indicate that expression of p21Waf1 protein strongly correlates with survival and might represent a useful prognostic marker in primary superficial bladder carcinomas.

Communication: expression of the novel inhibitor of apoptosis survivin in normal and neoplastic skin.

Apoptosis plays a fundamental part in epidermal homeostasis, and apoptotic cells have been detected in normal and diseased skin. Little is known, however, on the inhibitory mechanisms of apoptosis at the skin level. In addition to bcl-2, a novel inhibitor of apoptosis designated survivin and structurally analogous to IAP apoptosis inhibitors has been recently identified. The expression of survivin in normal and pathologic skin was investigated. Immunohistochemical studies revealed that survivin is expressed in basal keratinocytes, but not in suprabasal epidermal layers, with a pattern similar to bcl-2. In western blots, the anti-survivin antibody recognized a single band of 16.5 kDa in protein extracts from normal human keratinocytes in culture, in agreement with the predicted size of survivin. In addition, survivin immunoreactivity was detected in benign and malignant melanocytic lesions, with strong expression in invasive lesions of melanomas. Whereas survivin staining was undetectable in benign epithelial tumors, such as seborrheic keratoses, it was observed in all epidermal layers in Bowen's disease. Interestingly, at variance with bcl-2, survivin was markedly expressed in squamous cell carcinoma, but virtually lacking in basal cell carcinoma, suggesting that these two apoptosis inhibitors may act through different anti-apoptotic pathways. Deregulation of survivin may influence both epidermal homeostasis and the development of melanoma and nonmelanoma skin cancer.

Argyrophilic nucleolar organizer regions and bromodeoxyuridine and 3[H]-thymidine labelling indices in colorectal cancer.

The count of argyrophilic nucleolar organizer regions (AgNORs) has been proposed as a useful method for evaluating cell replication in human tumours. The current study was undertaken to compare AgNOR values in colorectal cancers with two better established methods for investigating cell proliferation such as bromodeoxyuridine (BrdUrd) and 3[H]-thymidine (3[H]dT) labelling indices (LIs). Because some concern still exists regarding accuracy and reproducibility of AgNOR quantifying methods, we carried out a control study by independently repeating the same measurements (number, area and area per silver-stained NOR particle) in two centres with different operators and computer-assisted image analysers on 40 colorectal carcinomas. AgNOR values recorded in the two centres were strictly correlated (r = 0.75; P < 0.001 for number; r = 0.62, P < 0.01 for area; r = 0.63, P < 0.001 for area per silver-stained NOR particle) and the range of values were almost identical. Then, AgNOR values were compared with BrdUrd and 3[H]dT LIs, respectively obtained by in vivo incorporation and in vitro incubation in the same series of colorectal carcinomas. No correlation was found between AgNOR values and BrdUrd or 3[H]dT LIs. BrdUrd and 3[H]dT LIs were instead reciprocally significantly correlated. No evident correlation was seen between LIs or AgNOR values and clinico-pathological parameters of the tumour. In conclusion, in colorectal neoplasms, AgNOR values did not appear to relate with more direct parameters of cell proliferation. It follows that AgNOR reliability as a biomarker of cell proliferation remains questionable.

Acetyl-L-carnitine effect on pituitary and plasma beta-endorphin responsiveness to different chronic intermittent stressors.

The aims of the present study were: 1) to compare the effect of two different chronic intermittent stressors i.e. cold-swimming versus ether, on the pituitary opioidergic system; 2) to evaluate the response of pituitary and plasma beta-endorphin (beta-EP) to an acute stress in chronically stressed rats; and 3) to evaluate the effect of acetyl-l-carnitine treatment (10 mg/day/rat per os at night) on pituitary and plasma beta-EP changes induced by two different types of chronic stress. The stressors were applied twice a day for 10 days. Rats were killed either before, during or after the last swimming or ether stress session. beta-EP was measured by radioimmunoassay in anterior pituitary and in neurointermediate lobe extracts and in plasma. The following observations were made: 1) Chronic intermittent cold-swimming stress increased anterior pituitary contents and plasma beta-EP levels; 2) both chronic intermittent cold-swimming stress and ether stress caused an increase of neurointermediate lobe beta-EP contents; 3) as in control animals, rats exposed to chronic intermittent swimming stress reduced pituitary beta-EP contents and raised plasma beta-EP levels in response to the last acute swimming stress; 4) in contrast to control animals, rats exposed to chronic intermittent ether stress did not show any significant response of the pituitary-plasma opioidergic system to the last acute ether session; 5) the acetyl-l-carnitine treatment counteracted the changes evoked by chronic intermittent cold-swimming stress on the pituitary and plasma beta-EP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of human papillomavirus DNA in urinary bladder carcinoma by in situ hybridisation.

AIMS: To investigate the sensitivity of an in situ hybridisation system to detect human papillomavirus (HPV) infection in transitional cell bladder cancer and to evaluate the advantages of analysing multiple biopsies; to examine the correlation between HPV tumour infection detected by in situ hybridisation and the presence of serum anti-HPV antibodies detected by enzyme linked immunosorbent assay (ELISA); and to relate the presence of viral infection to grade, stage, and follow up in cases of bladder cancer. METHODS: The in situ hybridisation technique was used with broad spectrum and type specific (6/11, 16/18, 31/33/35) probes against HPV DNA in formalin fixed, paraffin embedded tissues from 43 cases of bladder cancer. The results were analysed for the presence and type of papillomavirus and correlated with clinicopathological variables. RESULTS: The presence of HPV DNA was identified by the in situ hybridisation technique in 17 of 43 cases of bladder cancer; 12 of these were serum antibody positive and 10 had had multiple biopsies. Fifteen of the cases that were negative for HPV DNA by in situ hybridisation had positive serum serology when tested by ELISA. In 14 cases, the HPV was either types 16/18 or types 31/33/35, both of which carry high oncogenic risk. The stage (p < 0.05) and grade (NS) of the tumour and the outcome on follow up (p < 0.05) were correlated with the presence of HPV infection. CONCLUSIONS: ELISA is not useful in identifying patients with HPV positive bladder cancer, but the use of several probes and multiple biopsies increases the detection rate of HPV in neoplastic tissues. The association between tumour virus infection and high grade/high stage tumours and worse outcome suggests that HPV infection of neoplastic tissue has a negative effect on the behaviour and evolution of transitional cell bladder carcinoma.

p53 gene mutations in medulloblastoma. Immunohistochemistry, gel shift analysis, and sequencing.

Medulloblastoma (MB), the most common malignant tumor of the CNS in children, bears a loss of the short arm of chromosome 17 in almost half of the cases. The tumor suppressor gene p53 is located on this chromosome and its role in the pathogenesis of this primitive tumor is controversial. Twenty-two MBs were analyzed by single-strand conformation polymorphism (SSCP) of polymerase chain reaction-amplified conserved exons. Fragments displaying a gel mobility shift were subsequently analyzed by direct sequencing. Immunohistochemistry for p53 was performed in all cases; three had cytogenetic analysis. Two cases (9%) were found to harbor a mutation: one homozygous and one heterozygous. The latter showed focal p53 immunostaining. None of the cases with chromosome 17p abnormality by cytogenetic analysis were found to have a mutation in the remaining allele. Loss of heterozygosity (LOH) of 17p, however, was found in four cases (one by SSCP and three by cytogenetic analysis). Together with the homozygous deletion in one case, the overall incidence of p53 allelic involvement in MB is 23%. Although LOH for the p53 gene may confer a selective advantage to tumor cells harboring mutations with dominant negative oncogenic effect, the infrequent occurrence of p53 mutations in face of frequent LOH for this gene supports the previously formulated hypothesis of a novel tumor-related locus distal to p53 on chromosome 17p.

Acetyl-l-carnitine restores the daily pattern of hypothalamic beta-endorphin in rats exposed to continuous light.

With the aim of evaluating the effect of acetyl-l-carnitine (ALC) on the daily pattern of hypothalamic beta-endorphin (beta-EP), we studied the effect of chronic treatment with ALC on hypothalamic beta-EP contents after suppression of the dark-phase of the light-dark cycle in female rats. We evaluated the hypothalamic content of beta-EP immunoreactivity every 3 h for 24 h in: (1) female rats treated with ALC for 15 days; (2) female rats treated with ALC for 15 days and exposed to continuous light for 24 h. The concentration of beta-EP immunoreactivity in tissue extracts was measured by radioimmunoassay. The results demonstrate that concentrations of beta-EP immunoreactivity in the medial basal hypothalamus show a circadian rhythm, with beta-EP immunoreactivity levels being higher during the night than during the rest of the day. Exposure to continuous light for 24 h abolished the nocturnal increase in hypothalamic beta-EP immunoreactivity. Rats treated with ALC showed a daily pattern in the beta-EP content of the medial basal hypothalamus similar to that of control rats. These data emphasize the possible role of ALC in restoring or maintaining the endogenous rhythmicity of central beta-EP.

Expression of p53 and bcl-2 in clinically localized prostate cancer before and after neo-adjuvant hormonal therapy.

The prognostic significance of p53 and bcl-2 expression in prostate carcinoma is currently under investigation. The aim of the present study was to analyze their expression in diagnostic biopsies and in prostatectomies performed after neo-adjuvant hormonal therapy to investigate their role in hormone resistance. One hundred and six patients with advanced prostate carcinoma were treated for 3 months with LHRH analogues before radical surgery. The expression of p53 and bcl-2 was analyzed by immunohistochemistry in all cases of prostatectomy and in available biopsies obtained before treatment, and was correlated with clinicopathologic parameters and follow-up. A significant increase in p53 expression was found following hormonal therapy, whereas no changes were observed in the expression of bcl-2. The increase in p53 did not correlate with the presence of therapy-induced morphological changes in prostate cancers, but it did correlate significantly with histologic grade and pathologic stage, biochemical progression of the disease, and short overall survival. At multivariate analysis, only grade and stage proved to be independent predictors of shorter survival. There were no correlations between bcl-2 and clinicopathologic variables whether in biopsies or in prostatectomies. The unfavorable clinical course associated with p53-positive carcinomas suggests that neo-adjuvant hormonal therapy may cause the selection of minor p53 mutated clones, rather than the induction of wild-type p53. In any case, the enhanced expression of p53 could label hormone-resistant cancers for further adjuvant therapy.

Apolipoprotein E polymorphism and central nervous system tumors: correlation with cell proliferation indices and clinical outcome.

AIMS: This study was designed to determine whether the polymorphism of apolipoprotein E (apoE), one of the key regulatory proteins in cholesterol metabolism, is related to varying susceptibility to central nervous system (CNS) neoplasms, and to evaluate any possible interaction between this polymorphism and tumor cell proliferation or clinical outcome. METHODS AND RESULTS: 53 CNS tumors were selected. Follow-up and survival data were available for 36 patients. ApoE genotypes and cell proliferation indices (nucleolar organizer regions, MIB-1, PCNA, p53) were determined from paraffin-embedded tissue by standard methods. Each of the indices of cell proliferation correlated positively with tumor grade and negatively with duration of clinical follow-up and survival. There was a non-significant trend for apoE epsilon2 allele carriers to have high-grade tumors and apoE epsilon4 allele carriers to have low-grade tumors. Possession of apoE epsilon4 was associated with a more advanced age of disease presentation (p < 0.01) and a longer duration of follow-up (p < 0.04). No significant correlations were found between possession of either apoE epsilon2 or apoE epsilon4 alleles and indices of cell proliferation. CONCLUSIONS: These preliminary findings suggest that possession of apoE epsilon4 allele may correspond to a more favorable clinical course in terms of more advanced age of disease presentation, and longer duration of follow-up and survival in patients with CNS neoplasms.

Medullary carcinoma of the thyroid gland with sustentacular cell-like cells in a patient with multiple endocrine neoplasia, type IIA. Report of a case with ultrastructural and immunohistochemical studies.

We report a case of a medullary carcinoma of the left lobe of the thyroid gland that occurred in a 57-year-old woman. The patient had undergone surgery for treatment of a bilateral-functioning pheochromocytoma when she was 39 years old. A medullary carcinoma of the thyroid gland and/or a pheochromocytoma had also been diagnosed in other family members. The tumor was composed of cells arranged in nests and large sheets separated by fibrous stroma that contained amyloid deposits. Elongated cells with thin, branched cytoplasmic projections that were strongly reminiscent of sustentacular cells usually found in paragangliomas were seen among the neoplastic cells. Immunohistochemical study showed a diffuse positive reaction for calcitonin and low-weight keratins (CAM 5.2) in neoplastic cells, whereas the sustentacular cell-like cells were positive for S100 protein. The reaction for thyroglobulin was negative. Electron microscopy disclosed large numbers of typical neurosecretory granules in the cytoplasm of tumor cells. The sustentacular cell-like cells showed elongated cytoplasmic processes and lacked neurosecretory granules. We concluded that the finding of sustentacular cell-like cells in a medullary carcinoma of the thyroid gland made its differential diagnosis from paraganglioma more problematic.

Effect of chronic intermittent stress on rat pregnancy and postnatal development.

The present study evaluates the effect of chronic intermittent cold-swimming stress on body weight gain of pregnant rats and subsequent development of the offsprings after birth, till peripubertal stage. When stress was administered during the first half (1-11 days) of gestation, weight gain of pregnant rats was significantly lower at the 9th and 11th days (P < 0.05 vs. control, respectively). No differences of weight gain in comparison with control rats were found at term gestation in pregnant rats exposed to stress continuously. Similarly, stress administered, starting from 12th day till term gestation, had no effect on weight gain. Even though weight gain of pregnant rats during the second half of pregnancy in group stress 1-11 was restored to normal values, a high mortality rate of neonates 1, 2 and 3 weeks after birth was found in this group (P < 0.02, 0.01 and 0.001 vs. controls). There was no significant difference between stressed and control groups with respect to the number or body weight of litters, as well as weight gain of neonates during the first 21 days of life. In addition, in offsprings from all stressed groups, a high number of small for date animals was found after 14 days of life, and 74.4% of these small for date animals died during the peripubertal period. The present data demonstrate that the exposure to stress in utero may induce damaging effects on postnatal development.

Detection of Chlamydia trachomatis in Papanicolaou-stained cervical smears: control study by in situ hybridization.

Ninety-one Papanicolaou-stained vaginopancervical smears were destained and subjected to in situ hybridization with Chlamydia trachomatis DNA probe. At cytologic examination (Pap test), 71 smears showed changes suggestive of chlamydial infection, while remaining 20 were negative. At the control by in situ hybridization, the results of Pap test were confirmed in 85 out of 91 cases, two false-positive and four false-negative cases being detected. The sensitivity and specificity of Pap test, compared with in situ hybridization, were 95% and 89%, respectively. Like some recent reports, the present study confirms the reliability of Pap test in the detection of Chlamydia trachomatis and its possible relevant role in reducing the diffusion of the infection, when properly applied to mass-screening program.

Does p53 immunostaining improve diagnostic accuracy in urine cytology?

The frequent change of the transitional cell carcinoma of the urinary tract accounts for the fact that cytological abnormalities in urinary specimens are often not sufficient to enable a definitive diagnosis of malignancy. The purpose of this work was to evaluate the possible use of p53 protein in increasing the diagnostic accuracy of urinary cytology. The expression of p53 was investigated by immunocytochemistry in two groups of urinary specimens, one cytologically positive and the other cytologically negative for cancer. Immunostaining was carried out using a monoclonal antibody to p53. In the positive group, in which bladder cancer was confirmed by cystoscopy and biopsy (31 cases), positive reaction for p53 was found in 55% of the cases (17 cases). In the negative group (92 cases), presence of cancer was histologically ascertained in 64 cases and in this group 15 cases (23.4%) showed positive p53 staining. In the remaining 28 cases of this group, where TCC was not present, 7 cases showed p53 positivity in non-neoplastic urothelial cells. This result shows that, while immunocytochemical detection of p53 in urinary specimens may be used for prognostic evaluation of patients with bladder cancer, it does not contribute to the diagnostic accuracy in cases with morphologically inconclusive or negative cytology. The sensitivity and specificity of the method in detecting bladder carcinoma were 23.5 and 75%, respectively.

Gastric angioendothelioma: a cytologic evaluation.

A rare case of gastric angioendothelioma is presented in which diagnosis was performed preoperatively by an endoscopic technique. Exfoliative cytology using a brush under direct visual control revealed characteristics, which may well be diagnostic. Data which favor the diagnosis of angioendothelioma are: 1) elongated or polygnal cells with ill-defined cytoplasmic borders and small cytoplasmic vacuoles; 2) ovoid or rounded, nucleolated nuclei with fine chromatin network; 3) concentric disposition of the cells and, above all, the presence of small capillary-like structures.

Atypical hyperplasia of the prostate. A pitfall in the cytologic diagnosis of carcinoma.

The cytomorphologic features of atypical hyperplasia were studied in fine-needle aspiration biopsies of the prostate. Atypical hyperplasia material was found in 31 cases. The most usual and distinctive cytologic features of atypical hyperplasia were clustering of poorly-differentiated epithelial cells with acinic pattern and fairly regular nuclei. Another relevant feature was the lack of cytoplasmic fluorescence after acridine orange staining. These features make possible the diagnosis of atypical hyperplasia and the distinction from poorly-differentiated carcinoma. The recognition of atypical hyperplasia cells in fine-needle aspiration biopsies of the prostate is important in the avoidance of cytodiagnostic errors.

Chlamydia trachomatis infections in asymptomatic women. Results of a study employing different staining techniques.

A total of 300 cervical smears randomly collected from asymptomatic women in a mass-screening program for the detection of cervical carcinoma was investigated for Chlamydia trachomatis infection by the use of Papanicolaou and immunofluorescence staining. Features of chlamydial infection detected in 18 cases by Papanicolaou-stained smears were confirmed in 11 cases with immunofluorescence; not a single case that was negative in the Papanicolaou-stained smears was positive by immunofluorescence. The presence of Chlamydia in the Papanicolaou-stained smears in ten cases, including two cases that were negative by immunofluorescence, was also proven by either immunoperoxidase staining or in situ hybridization. On the other hand, either immunoperoxidase or in situ hybridization gave false-negative results in two of the ten cases. Therefore, the combined use of different techniques demonstrated that false-negative results occurred with all techniques, except with Papanicolaou-stained smears, whose sensitivity is apparently the highest.

[Molecular biology in bladder carcinoma: contributions of immunohistochemistry]. / Biologia molecolare nel carcinoma vescicale: contributi di immunoistochimica.

Despite the insights genetics and molecular biology have given to a better understanding of the mechanisms which lead to the onset and development of bladder carcinoma, the factors that influence its unpredictable and, at times, particularly aggressive outcome are still largely unknown. Also in bladder carcinoma the study of cellular differentiation markers has been replaced by that of genotypic alterations, and, mainly with the help of immunohistochemistry, of the expression of genes involved in cell proliferation and death, such as MTS1, TP53, Rb, c-myc, Bcl-2, c-erb-B2. So far, anyway, no independent and reliable indicator able to predict the outcome of the single tumour has been identified, and this issue seems to be best addressed by studies of the altered expression of more than one oncoprotein simultaneously. Fairly identical is the question arised by TP53 mutations, which, while worsening the evolution of advanced muscle-infiltrating tumours, hold a still unclear and debated meaning in superficial tumours. It is anyway clear that molecular analysis only may enable to reliably detect the presence of any TP53 mutations. As a matter of fact, the multiplicity of genetic mutations, the frequent transcript variations and the intrinsic limits of immunohistochemistry may explain the discrepancy between immunohistochemical and molecular analysis results, with specificity and sensitivity levels clinically not acceptable. To date, anyway, the biological and clinical meaning of this discrepancy has still to be clarified, as well as the clinical meaning, if any, of p53 overexpression in the absence of gene mutations.