Gabriele Cevenini

Automated diagnosis of pigmented skin lesions.

Since advanced melanoma remains practically incurable, early detection is an important step toward a reduction in mortality. High expectations are entertained for a technique known as dermoscopy or epiluminescence light microscopy; however, evaluation of pigmented skin lesions by this method is often extremely complex and subjective. To obviate the problem of qualitative interpretation, methods based on mathematical analysis of pigmented skin lesions, such as digital dermoscopy analysis, have been developed. In the present study, we used a digital dermoscopy analyzer (DBDermo‐Mips system) to evaluate a series of 588 excised, clinically atypical, flat pigmented skin lesions (371 benign, 217 malignant). The analyzer evaluated 48 parameters grouped into 4 categories (geometries, colors, textures and islands of color), which were used to train an artificial neural network. To evaluate the diagnostic performance of the neural network and to check it during the training process, we used the error area over the receiver operating characteristic curve. The discriminating power of the digital dermoscopy analyzer plus artificial neural network was compared with histologic diagnosis. A feature selection procedure indicated that as few as 13 of the variables were sufficient to discriminate the 2 groups of lesions, and this also ensured high generalization power. The artificial neural network designed with these variables enabled a diagnostic accuracy of about 94%. In conclusion, the good diagnostic performance and high speed in reading and analyzing lesions (real time) of our method constitute an important step in the direction of automated diagnosis of pigmented skin lesions.

Delayed risk stratification, to include the response to initial treatment (surgery and radioiodine ablation), has better outcome predictivity in differentiated thyroid cancer patients

INTRODUCTION After initial treatment, differentiated thyroid cancer (DTC) patients are stratified as low and high risk based on clinical/pathological features. Recently, a risk stratification based on additional clinical data accumulated during follow-up has been proposed. OBJECTIVE To evaluate the predictive value of delayed risk stratification (DRS) obtained at the time of the first diagnostic control (8-12 months after initial treatment). METHODS We reviewed 512 patients with DTC whose risk assessment was initially defined according to the American (ATA) and European Thyroid Association (ETA) guidelines. At the time of the first control, 8-12 months after initial treatment, patients were re-stratified according to their clinical status: DRS. RESULTS Using DRS, about 50% of ATA/ETA intermediate/high-risk patients moved to DRS low-risk category, while about 10% of ATA/ETA low-risk patients moved to DRS high-risk category. The ability of the DRS to predict the final outcome was superior to that of ATA and ETA. Positive and negative predictive values for both ATA (39.2 and 90.6% respectively) and ETA (38.4 and 91.3% respectively) were significantly lower than that observed with the DRS (72.8 and 96.3% respectively, P<0.05). The observed variance in predicting final outcome was 25.4% for ATA, 19.1% for ETA, and 62.1% for DRS. CONCLUSIONS Delaying the risk stratification of DTC patients at a time when the response to surgery and radioiodine ablation is evident allows to better define individual risk and to better modulate the subsequent follow-up.

The prognostic role of Beclin 1 protein expression in high-grade gliomas.

High Grade Gliomas (HGG) have a poor outcome, however, prognostic sub-groups of patients may be individuated by some clinico-biological parameters. It was recently demonstrated that the main response of HGG to therapy is autophagic death. Autophagy is involved in tumor suppression, and is defective in HGG, in which we previously found an underexpression of beclin 1 autophagic gene protein product. Underexpression of Beclin 1 protein has been correlated to poor patient outcome in other tumor types. In this paper, the prognostic role of beclin 1 expression in HGG patients was investigated. We firstly evaluated the tumor cell cytoplasmic expression of Beclin 1 protein (BPCE), in a sample of 76 HGG by immunohistochemistry, and compared it with cell proliferation and apoptosis. We found high BPCE score positively correlated with apoptosis, and negatively with cell proliferation (p < 0,05). We then correlated BPCE score with survival and other prognostic parameters (histological grading , MGMT gene methylation status, age, patient performance status according to the Karnofski classification (KPS), extent of surgery, radiation therapy (RT) modality, temozolomide chemotherapy (TMZ CHT), and optimal/suboptimal post-surgical treatment). Forty-seven (61.8%) and twenty-nine (38.2%) patients showed high and low BPCE scores, respectively. BPCE showed statistically significant correlations with survival both at the univariate (p = 0.03) and multivariate analysis (p = 0.037). High BPCE was also positively correlated with high KPS values (p = 0.023), and with the accomplishment of an optimal post-operative therapy (p = 0.037). Furthermore, among patients showing a MGMT methylated gene, survival was significantly higher in cases with a higher BPCE score. BPCE score might be added to pathological evaluation of HGG for prognostic purposes.

Beclin 1 and LC3 autophagic gene expression in cutaneous melanocytic lesions.

Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was designed to assess the expression of Beclin 1 and LC3 in cutaneous melanocytic lesions, in which they have not yet been investigated. In melanoma, we correlated their expression with conventional histopathologic prognostic factors. In 149 lesions, including benign nevi, dysplastic nevi, radial growth phase melanomas, vertical growth phase melanomas, and melanoma metastases, proteins were evaluated by immunohistochemistry, and, in representative cases of benign nevi, vertical growth phase melanomas and melanoma metastases were evaluated by Western blotting. In most lesions, messenger RNA level was also assessed by real-time reverse transcriptase polymerase chain reaction. Both genes were expressed in all the investigated conditions. Beclin 1 cytoplasmic protein and messenger RNA, as well as LC3 messenger RNA, significantly decreased with tumor progression (P < .05). The percentage of cases with high cytoplasmic expression of beclin 1 from 100% in benign nevi declined to 86.4% in dysplastic nevi, 54.5% in radial growth phase melanomas, 54.3% in vertical growth phase melanomas, and 26.7% in melanoma metastases. The lowest expression of LC3 II protein was observed in melanoma metastases (53.3% of cases) (P < .05); LC3 II protein overexpression was, however, found in several nonbenign lesions, with the highest percentage (45.5%) in radial growth phase melanomas. LC3 II protein expression was inversely correlated to thickness, ulceration, and mitotic rate. In a multivariate analysis, messenger RNAs for both genes discriminated between nonmalignant (benign and dysplastic nevi) and malignant (radial, vertical growth phase melanomas, and melanoma metastases) lesions. Our results, therefore, indicate that beclin 1 and LC3 II autophagic gene expression is altered also in melanocytic neoplasms.

Genetic Alterations of the Retinoblastoma-Related Gene RB2/p130 Identify Different Pathogenetic Mechanisms in and among Burkitt’s Lymphoma Subtypes

Alterations of cell cycle-associated genes probably contribute to the pathogenesis of Burkitts Lymphoma (BL), in addition to c-myc translocation. Mutations disrupting the nuclear localization signal of the retinoblastoma-related gene RB2/p130 have been documented recently in BL cell lines and primary tumors. Given the importance of the RB2/p130 gene in controlling cell growth, mutations of this gene may result in uncontrolled cell proliferation. We tested the expression and genomic organization of the RB2/p130 gene in relation to the proliferative features of a series of BL samples collected from the endemic and sporadic regions, regardless of whether the samples were acquired immune deficiency syndrome (AIDS)-related. The expression of the Rb2/p130, p107, and cell proliferation-related proteins (cyclin A and B) was determined by immunohistochemistry. The structures of exons 19 through 22 of the RB2/p130 gene, encoding for the B domain and C terminus, were analyzed by polymerase chain reaction (PCR) analysis and single-strand conformation polymorphism (SSCP) technique. The direct PCR products were sequenced to identify the actual mutations. Our results suggest that BL is composed of a mixture of molecular types with distinct genetic and phenotypic patterns, probably resulting from different pathogenetic mechanisms. In endemic BL, the RB2/p130 gene is mutated in most of the cases, and the protein is restricted to the cytoplasm. In AIDS-related BL, high levels of nuclear expression of the wild-type pRb2/p130, p107, and cell proliferation-related proteins were detected. This finding is in line with the molecular mechanisms observed in virus-linked oncogenesis. Sporadic BLs were mainly characterized by the low nuclear values of the wild-type pRb2/p130 and, conversely, the high values of p107. The increased cell proliferation due to different alterations of cell growth control by Rb-related proteins may be the first step in lymphomagenesis, during which additional genetic changes, including missense mutations of c-myc, may subsequently occur.

Immunohistochemical analysis of pRb2/p130, VEGF, EZH2, p53, p16INK4A, p27KIP1, p21WAF1, Ki-67 expression patterns in gastric cancer†

Although the considerable progress against gastric cancer, it remains a complex lethal disease defined by peculiar histological and molecular features. The purpose of the present study was to investigate pRb2/p130, VEGF, EZH2, p53, p16INK4A, p27KIP1, p21WAF1, Ki‐67 expressions, and analyze their possible correlations with clinicopathological factors. The expression patterns were examined by immunohistochemistry in 47 patients, 27 evaluated of intestinal‐type, and 20 of diffuse‐type, with a mean follow up of 56 months and by Western blot in AGS, N87, KATO‐III, and YCC‐2, ‐3, ‐16 gastric cell lines. Overall, stomach cancer showed EZH2 correlated with high levels of p53, Ki‐67, and cytoplasmic pRb2/p130 (P < 0.05, and P < 0.01, respectively). Increased expression of EZH2 was found in the intestinal‐type and correlated with the risk of distant metastasis (P < 0.05 and P < 0.01, respectively), demonstrating that this protein may have a prognostic value in this type of cancer. Interestingly, a strong inverse correlation was observed between p27KIP1 expression levels and the risk of advanced disease and metastasis (P < 0.05), and a positive correlation between the expression levels of p21WAF1 and low‐grade (G1) gastric tumors (P < 0.05), confirming the traditionally accepted role for these tumor‐suppressor genes in gastric cancer. Finally, a direct correlation was found between the expression levels of nuclear pRb2/p130 and low‐grade (G1) gastric tumors that was statistically significant (P < 0.05). Altogether, these data may help shed some additional light on the pathogenetic mechanisms related to the two main gastric cancer histotypes and their invasive potentials. J. Cell. Physiol. 210: 183–191, 2007.

Post-surgical thyroid ablation with low or high radioiodine activities results in similar outcomes in intermediate risk differentiated thyroid cancer patients

BACKGROUND In differentiated thyroid cancer (DTC) patients at intermediate risk of recurrences, no evidences are provided regarding the optimal radioactive iodine (RAI) activity to be administered for post-surgical thyroid ablation. METHODS This study aimed to evaluate the impact of RAI activities on the outcome of 225 DTC patients classified as intermediate risk, treated with low (1110-1850  MBq) or high RAI activities (≥3700  MBq). RESULTS Six to 18 months after ablation, remission was observed in 60.0% of patients treated with low and in 60.0% of those treated with high RAI activities, biochemical disease was found in 18.8% of patients treated with low and in 14.3% of patients treated with high RAI activities, metastatic disease was found in 21.2% of patients treated with low and in 25.7% of patients treated with high RAI activities (P=0.56). At the last follow-up (low activities, median 4.2 years; high activities, median 6.9 years), remission was observed in 76.5% of patients treated with low and in 72.1% of patients treated with high RAI activities, persistent disease was observed in 18.8% of patients treated with low and in 23.5% of patients treated with high RAI activities, recurrent disease was 2.4% in patients treated with low and 2.1% in patients treated with high RAI activities, deaths occurred in 2.4% of patients treated with low and in 2.1% of patients treated with high RAI activities (P=0.87). CONCLUSION Our study provides the first evidence that in DTC patients at intermediate risk, high RAI activities at ablation have no major advantage over low activities.

CADCS simulation of the closed-loop cardiovascular system

A pulsatile simulator of the closed-loop cardiovascular system, designed to solve simulation, identification and control problems in a research and education context, is presented. Its implementation makes use of a command-driven interactive program for simulation of non-linear ordinary differential equations. The flexibility of the simulator is demonstrated by the results presented which refer to a basal steady-state circulatory condition as well as a transient induced by an abrupt change in peripheral resistance.

Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments.

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio‐chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin‐1 and Atg‐5 expression increase, LC3‐I to LC3‐II conversion and LC3B‐GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG‐7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin‐mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy‐associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment. J. Cell. Biochem. 113: 2308–2318, 2012.

Activated Akt as an indicator of prognosis in gastric cancer

The immunohistochemical expression of phosphorylated (activated) Akt (pAkt) in 50 advanced gastric carcinomas has been analyzed and the results correlated with age, sex, location in the stomach, histotype, stage, survival, mitotic and apoptotic index, some cell cycle regulators (cyclin D1, cyclin E, p34/cdc2, p27/kip1), and cell proliferation. There was a statistically significant direct correlation between pAkt expression (both cytoplasmatic and nuclear) and depth of infiltration of the tumor, number of infiltrated lymph nodes and p34/cdc2 expression, and between prevalently nuclear pAkt and cyclin D1 and cyclin E. Conversely, there was a significant inverse correlation between nuclear pAkt and apoptotic index and between cytoplasmatic and nuclear pAkt and patient survival. No correlation was found between pAkt and sex, age, tumor location, histotype, mitotic index, and cell proliferation. These findings suggest that pAkt may be considered an indicator of tumor progression and patient survival in gastric cancer.