The role of CTLA-4 in negative regulation of T-cell mediated immune response is particularly well established. Much less is known about its expression and function in tumour cells, and to our knowledge, no data are available on its possible impact on prognosis of NSCLC patients. We investigated CTLA-4 expression and prognostic role in 81 patients with radically resected stage I-III NSCLC. The analysis was performed by tissue microarray immunohistochemistry, and the median H-score of 20 was used as a threshold to define CTLA-4 overexpressing tumours. Correlation with standard prognostic factors was performed by using absolute and relative fold change indexes. Hazard ratios (HR) and corresponding 95% confidence limits (95% CL) were computed through the Cox model. A higher frequency of CTLA-4 overexpression (>20) was found in non-squamous than in squamous NSCLC (52.8 vs. 35.7%) and in Ki67 ≤ 15 expressing tumours, as compared to those with Ki67 > 15 (51.5 vs. 38.7%). A reduced death rate was found in CTLA-4 overexpressing tumours (HR = 0.60, 95% CL = 0.28/1.23), and a further decrease was observed when considering tumours with CTLA-4 > 20 and Ki67 ≤ 15, in comparison with tumours with CTLA-4 ≤ 20 and Ki67 > 15 (HR = 0.41; 95% CL = 0.15/1.13). Our observational and exploratory study provides a first and promising indication for an independent prognostic effect of CTLA-4 overexpression in radically resected NSCLC. We presume that this effect relies on modulation of the interaction of microscopic disease with CTLA-4-ligands expressing cells leading to NSCLC cell death.

The potential role of p16INK4a methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16INK4a by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16INK4a promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16 INK4apromoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma.

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Abstract. Many studies have indicated that nuclear DNA content evaluation can be used to predict biological behavior of transitional cell carcinoma (TCC) of the bladder. Some authors also indicated that static cytometry is more useful in DNA content analysis than flow cytometry. The aim of the present study was to evaluate the prognostic significance of DNA ploidy in TCC of the bladder, performed by using static cytometry with an image analyzer, and monitoring patients at 10 years follow-up. Thirty-one consecutive patients underwent transurethral or open surgery for bladder tumors, and neoplastic tissue samples taken from each patient were imprinted on glass slides and sent for histopathological and DNA content evaluation. DNA ploidy evaluation was performed using a CAS 200 image analyzer. Nuclear DNA content evaluation was compared to patient follow-up on recurrence, progression or survival performed 10 years after surgery. Pathological evaluation demonstrated the presence of superficial TCC in 23 patients, while 8 had an invasive bladder tumor. Twenty-nine tumor samples were adequate for DNA content measurement, with 13 showing diploid DNA content and 16 with aneuploid DNA content. At 10 years follow-up, all patients with aneuploid DNA content demonstrated a lower survival time (p=0.049) and higher recurrence rate (p=0.0346). A log-rank test demonstrated that stage, grade and nuclear DNA content are the most useful prognostic parameters for predicting the biological behavior of TCC of the bladder. These results confirm that static cytometry is a good and reliable method to evaluate DNA tumor content and considered a useful prognostic parameter for predicting recurrence rate, disease progression or survival in patients affected by bladder tumors. IntroductionTransitional cell carcinoma of the bladder is a tumor with variable biological potential (1); the recurrence rate is 50% to 70% (2) and as many as 19% of pTa and 34% of pT1 tumors will progress to muscle-invasive features (3). The ability to predict true tumor biological potential could facilitate patient treatment selection and improve the patient survival rate and quality of life.Various pathological and clinical parameters, such as tumor grade, stage, multifocality, size, rate and pattern of recurrence, association with Cis and other dysplastic urothelial lesions have been associated with a poor clinical outcome (4). Histopathological stage and grade are conventional prognostic factors for predicting biological behavior (5).To increase understanding of the cellular mechanisms underlying the development of bladder carcinoma and its natural history, many groups of potential molecular markers and quantitative morphometric methods have been described over the years (6). DNA ploidy identification has been proposed (since the late 1970s) as a diagnostic (7) and prognostic method to evaluate the malignant potential in several neoplasms, such as ovarian (8), endometrial (9) or colorectal cancers (10). Moreover, other studies demonstrated that nuclear DNA content...

Cytofluorometric analyses of the macronuclear DNA content (MDC) and total protein content (TPC) of Paramecium primaurelia stock 90 were carried out in cells at successive ages, from 10 to 26 fissipns . after conjugation, during immaturity and transition to maturity. Between the 10th and the 20th exconjugant fission within the immaturity period, MDC decreases to a minimum whereas TPC remains constant. Following the 20th fission during the transition period TPC decreases, and from the 22nd to the 26th fission both MDC and TPC remain constant. These results suggest both that the MD0 decresases in the course of the immaturity period and that the sharp decrease in TPC observed during the transition period may be relevant to the onset of maturity.KEY WORDS: Ciliates -Cell life cycle stages -DNA and protein quantitative variations. ACKNOWLEDGEMENTS