Carmela De Gaetani

Cyclin D1 expression in papillary superficial bladder cancer: its association with other cell cycle-associated proteins, cell proliferation and clinical outcome.

Cyclin D1 contributes to regulate G1 progression by forming a complex with different cyclin‐dependent kinases. It has oncogenic properties and is frequently overexpressed in several human tumor types. In our study, expression of cyclin D1 and Ki67, a proliferation marker, was evaluated by immunohistochemistry in human papillary superficial (pTa‐pT1) bladder cancers and was correlated with p27Kip1, p21Waf1 and c‐erbB‐2 expression, with p53 gene status and protein expression, ploidy and cancer progression. Cyclin D1 expression was neither associated with tumor stage nor with tumor grade but high cyclin D1 expression (≥25% positive nuclei) was significantly associated with p53 gene mutation (p = 0.012), low p21Waf1 (p = 0.015) and high p27Kip1 (p = 0.016) protein expression. Ki67 expression was not associated with tumor stage but a high proliferation index (≥10% positive nuclei) was significantly associated with high tumor grade (p = 0.001) and with DNA aneuploidy (p = 0.005). There was no significant difference in proliferative activity between high and low cyclin D1 expressor tumors. Patients whose tumors showed high expression of cyclin D1 displayed a significantly longer disease‐free survival (p < 0.001 by log‐rank test). Increased Ki67 expression was significantly associated with shorter disease‐free survival (p = 0.003). Both cyclin D1 (p = 0.027; RR = 1.898) and Ki67 (p = 0.047; RR = 1.932) protein expressions were independent predictors of reduced disease‐free survival on a multivariate analysis that also included p27Kip1 expression and tumor stage. The simultaneous presence of low cyclin D1, low p27Kip1 and high Ki67 expression defined a “high‐risk” group of patients who displayed a significantly increased risk of recurrence (p < 0.0001). These results suggest that evaluation of cell cycle‐associated markers can help to identify high‐risk patients and may affect the management of patients with papillary superficial bladder cancer.

Primary mixed adenocarcinoma and small cell carcinoma of the appendix: a clinicopathologic, immunohistochemical, and molecular study of a hitherto unreported tumor.

Appendiceal carcinoids range from well-differentiated endocrine tumor to well-differentiated endocrine carcinoma, while poorly differentiated (small cell) carcinoma has not been described in this site. We report herein a case of mixed intestinal-type adenocarcinoma associated with a small cell carcinoma arisen in a 35-year-old woman and clinically presenting as an appendiceal abscess. The resected tumor histologically appeared as a biphasic lesion composed of a nonmucinous adenocarcinoma closely juxtaposed with a poorly differentiated (small cell) endocrine carcinoma. The subsequent right hemicolectomy was unremarkable, but one pericolic lymph node showed a metastatic deposit consisting of the adenocarcinoma only. The patient thus underwent a chemotherapeutic protocol for colorectal cancer, and she is alive and well at the 65-month follow-up. Immunohistochemically, the adenocarcinoma strongly stained for cytokeratin 20 and carcinoembryonic antigen, while the endocrine component displayed a dot-like positivity for pan-cytokeratins and chromogranin. Of note, both components did not stain with CDX2 and p53. At genotypic analysis by microsatellite instability, both components shared many microsatellite alterations as well as a normal p53 gene setup, although small cell carcinoma harbored additional alterations. Clinical and molecular findings led us to consider this lesion as a clonal tumor in which the endocrine component seems to derive from a progressive differentiation of the adenocarcinoma following a glandular-to-endocrine sequence.

Superficial papillary urothelial carcinomas in young and elderly patients: a comparative study

To compare the clinicopathological and immunohistochemical findings of superficial papillary transitional cell carcinomas in ‘young’ and ‘elderly’ patients, as the natural history and prognosis of bladder tumours in young patients remains a matter of debate.

Estrogens Modulate the Circadian Rhythm of Hypothalamic Beta-Endorphin Contents in Female Rats

The aim of the present study was to evaluate the changes in the diurnal rhythm of the hypothalamic beta-endorphin (beta-EP) contents in female rats as a function of circulating estrogens. With this purpose we evaluated the diurnal hypothalamic beta-EP changes (1) during the estrous cycle, and (2) in ovariectomized rats with and without acute and chronic estrogen replacement. Ovariectomized rats were treated either acutely with 10 micrograms of estradiol benzoate (EB) or chronically with 2 micrograms/day of EB for 15 days. beta-EP concentrations were measured in acid extracts of medial basal hypothalamus by a specific radioimmunoassay. During the estrous cycle, hypothalamic beta-EP concentrations showed a significant nocturnal increase, with no difference between the 4 days of the cycle. On the day of estrus, beta-EP concentrations between 12.00 and 18.00 h resulted significantly lower than in the other days of the cycle. After ovariectomy, the night-related changes in hypothalamic beta-EP disappeared. The acute administration of EB induced a significant increase in hypothalamic beta-EP after 21 h (18.00 h). On the other hand, the chronic replacement restored the nocturnal peak of hypothalamic beta-EP (18.00, 21.00, 24.00 h). The present data emphasize the role of central beta-EP in regulating the reproductive functions. Moreover, the effect of estrogen in modulating the circadian changes in hypothalamic beta-EP supports the important role of estrogens in brain function.

Melatonin treatment delays reproductive aging of female rat via the opiatergic system.

In female rat age-related reproductive decline is accompanied by progressive impairment of the neuroendocrine mechanisms that regulate LH secretion. The biosynthetic activity of the pineal gland is markedly depressed and the nocturnal secretion of melatonin decreases significantly. The aim of the present study was to evaluate whether the nocturnal administration of melatonin via the drinking water (0.4 micrograms/ml) throughout the course of aging from 14 to 24 months of age could (1) influence the age-related changes that occur in basal serum levels of LH and in the LH response to GnRH or to naloxone stimulation at 16, 18 and 20 months of age, and (2) delay the onset of the postreproductive constant estrous-anovulatory state as evaluated by the daily recording of vaginal smears and by occurrence of polyfollicular ovaries at 24 months of age. Our results demonstrate that melatonin replacement delays the increase in LH serum levels and the decrease in LH response to GnRH that occur in 18-month-old control animals. Furthermore, they show that melatonin treatment prevents the loss of LH response to naloxone manifested in control rats between 16 and 20 months of age. Melatonin also appears to prevent the progressive increase in the monthly occurrence of estrus phases as well as to decrease the number of rats with polyfollicular ovaries at 24 months of age in comparison to control animals. These results suggest that the age-related decrease in circulating melatonin during the night may contribute to the reproductive decline of aging, and that this effect may involve the central opioid system.

Does HPV play a role in the etiopathogenesis of ameloblastoma? An immunohistochemical, in situ hybridization and polymerase chain reaction study of 18 cases using laser capture microdissection.

Ameloblastomas are epithelial tumors of odontogenic origin, biologically characterized by local recurrence. Among different etiologic factors, HPV infection has been recently postulated to be somehow involved in ameloblastoma etiopathogenesis. To address this issue, we studied 18 ameloblastomas by means of immunohistochemistry, in situ hybridization (conventional and amplified), polymerase chain reaction and nested-polymerase chain reaction analyses using laser capture microdissection in order to detect the occurrence of HPV in this setting. No evidence of HPV infection was detected by morphological examination, immunohistochemistry, in situ hybridization and conventional polymerase chain reaction, while nested-polymerase chain reaction showed a weak positive band in two cases. However, the subsequent restriction enzyme analysis carried out from the nested-polymerase chain reaction amplification products of these two samples excluded the presence of HPV subtypes 16, 18, 31, 33, 35, 52, and 58. The search for HPV 6 and 11 in the same specimens was also negative. In conclusion, our data do not support an etiopathogenetic evidence for HPV in ameloblastoma.

Prevalence and prognostic significance of microsatellite alterations in young patients with bladder cancer

Mutations in microsatellite sequences are a hallmark of neoplastic transformation and have been reported in the majority of human cancers. Conflicting results have been reported on the role of microsatellite alterations in bladder tumorigenesis and it has been suggested that they might be mainly involved in the development of bladder cancers in young patients. In this study, DNA was extracted from laser-microdissected samples of 51 superficial papillary bladder urothelial carcinomas arising in young patients and was analyzed for the status of 19 microsatellite loci previously reported to be associated with bladder tumorigenesis. The occurrence and the pattern of microsatellite alterations, in form of loss or length variation, was evaluated and correlated with other clinicopathologic and molecular markers. The prognostic significance of these alterations was also evaluated. Loss of heterozygosity at one or more loci was detected in all 51 tumors analyzed. Length variation in at least one locus was observed in 48 (94%) of the cases. The microsatellite that was more frequently altered was D11S488 (69%), followed by D9S162 (61%), D3S3050 (55%), D3S1300 (51%) and D4S243 (51%), all the remaining being altered in less than 50% of cases. The occurrence of microsatellite alterations was not associated with tumor grade nor with tumor stage, the expression of p53, cyclin D1 or the cyclin-dependent kinase-inhibitor p27Kip1 while it was significantly more frequent in tumors with increased expression of the proliferation marker MIB-1 (P=0.003). The occurrence of alterations at the analyzed loci was associated with a reduced risk of tumor recurrence (P=0.04 by log-rank test) and disease progression (P=0.02) in a univariate analysis. These findings demonstrate that microsatellite alterations are frequent and early events and might have a prognostic significance in bladder cancers arising at young age.

p27Kip1 Expression and Survival in N0 Gastric Carcinoma

p27Kip1, a cyclin-dependent kinase inhibitor, is considered to be a tumor suppressor gene. Absent or reduced expression of the p27Kip1 protein has been reported being a negative prognostic marker in primary lung, breast, colon, bladder, and prostate carcinomas. p27Kip1 protein expression was evaluated in a series of 96 gastric carcinomas with no lymph node involvement (NO) to verify any impact on the clinical outcome. The analysis also considered the classic clinico-pathological parameters, such as age, sex, and depth of tumor invasion (pT). The most widely used classification systems for gastric carcinoma were adopted. The expression of p27pKip1 was related neither to the pT category nor to tumor histology. Kaplan-Meier analysis documented a significant impact of an advanced pT category (p < 0.0001) and p27Kip1-reduced expression (p < 0.0002) on survival. Multivariate analysis confirmed that the reduced p27Kip1 protein expression was a strong independent predictor of poor outcome, ranking second to the pT category only (p < 0.006 and p < 0.004 respectively). As reported for other neoplasms, the expression of p27Kip1 appears to be associated with the clinical outcome of gastric carcinoma.

Acetyl-l-carnitine restores the daily pattern of hypothalamic β-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.