Non-neoplastic jaw cysts: a 30-year epidemiological study of 2150 cases in the Italian population.

Non-neoplastic jaw cyst (NJC) is one of the most common lesions in oral cavity, but there are only few detailed and extended epidemiological data based on the 2017 WHO classification. The aim of this study was to perform an epidemiological analysis of all NJCs treated from 1990 to 2019 at the Marche Polytechnic University, and to compare these data with those published in the literature. This retrospective study considered 2060 patients treated from 1990 to 2019. The NJCs were classified according to the 2017 WHO classification, and the main clinicopathological variables were analysed (sex, age, diagnosis, site of onset, size, and recurrences). Of 2150 total lesions, there were 2095 primary cysts and 55 recurrences; men are more frequently affected than women (M/F ratio of 1.73:1). The mean age of occurrence was 46.6 years, with a peak of frequency in the fifth decade. The mandible was more frequently involved than the maxilla, with a mean size of 1.9cm. Radicular cyst was the most frequently diagnosed cyst (56.6%), followed by dentigerous cyst (23.4%) and odontogenic keratocyst (12.9%). This is the first epidemiological study on NJCs in the Italian population according to 2017 WHO classification.

High dose irradiation after pleurectomy/decortication or biopsy for pleural mesothelioma treatment.

PURPOSE: The role played by radiation therapy after pleurectomy/decortication or surgical biopsy in malignant pleural mesothelioma is uncertain. We treated patients with accelerated hypofractionated radiotherapy using helical tomotherapy and intensity-modulated arc therapy in an attempt to keep lung toxicity to a minimum. The present study reports the feasibility and toxicity of this approach. MATERIAL AND METHODS: Between 2008 and 2012, 36 patients with malignant pleural mesothelioma underwent accelerated hypofractionated radiotherapy to the hemithorax after pleurectomy/decortication (19 patients) or biopsy (17 patients). The prescription dose was 25Gy in five fractions over 5 consecutive days. RESULTS: We observed three patients with G3 pneumonitis, five cases of grade 2 dyspnea and six cases of grade 2 cough. The median follow-up was 37 months (range: 3-54 months). The median overall survival for patients who underwent pleurectomy/decortication followed by radiotherapy was 21.6 months [95% confidence interval (95% CI): 15.5-24.1] compared to 19.4 months for patients not submitted to surgery. CONCLUSION: Treatment of intact lung with pleural intensity-modulated arc irradiation in malignant pleural mesothelioma patients with malignant pleural mesothelioma proved safe and feasible, with an acceptable rate of pneumonitis. Survival rates were encouraging for both biopsy-only and pleurectomy/decortication groups. We are currently conducting a phase II dose escalation trial in a similar patient setting to prospectively evaluate the impact of radiotherapy on toxicity, disease-free survival and overall survival.

In vitro and in vivo antitumor efficacy of docetaxel and sorafenib combination in human pancreatic cancer cells.

The response of pancreatic cancer to treatments remains unsatisfactory, highlighting the need for more effective therapeutic regimens. Sorafenib, an orally available multikinase inhibitor, is active against different tumors, including pancreatic cancer. We studied the antitumor efficacy of sorafenib in combination with different antitumor drugs currently used in clinical practice in in vitro and in vivo experimental models of human pancreatic cancer. The cytotoxic effect of sorafenib and conventional antitumor drug combinations was evaluated in vitro in human pancreatic cancer cell lines and the efficacy of the most active combination was tested on tumor-bearing mice. Flow cytometric, Western blot and immunohistochemistry analyses were performed to investigate the mechanisms involved in the activity of single drugs and in their interaction when used in combination. Sorafenib showed a strong sequence-dependent synergistic interaction in vitro with docetaxel, which was highly dependent on the drug sequence employed. In vivo, human pancreatic cancer-xenografted mice treated with docetaxel followed by sorafenib reduced and delayed tumor growth, with complete tumor regression observed in half of the mice. This marked antitumor effect resulted in an overall increase in mouse survival of about 70% and in a complete cure in 3 of the 8 treated mice. The strong activity was also accompanied by marked apoptosis induction, inhibition of tumor angiogenesis and downregulation of ERK signalling. Our results show that the docetaxel and sorafenib combination exerts high therapeutic efficacy in experimental models of human pancreatic cancer, indicating a promising antitumor strategy for clinical use.

Isolation of stem/progenitor cells from normal lung tissue of adult humans.

OBJECTIVES: This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults. MATERIALS AND METHODS: Cultures of multicellular spheroids were obtained from human lung tissue specimens after mechanical and enzymatic digestion. Tissue-specific markers were detected on their cells by immunohistochemical and immunofluorescent techniques. Ultrastructural morphology of the spheroids (termed as bronchospheres) was evaluated by electron microscopy, gene expression analysis was performed by reverse transcription-polymerase chain reaction, and gene down-regulation was analysed by an RNA interference technique. RESULTS: Bronchospheres were found to be composed of cells with high expression of stem cell regulatory genes, which was not or was only weakly detectable in original tissues. Morphological analysis showed that bronchospheres were composed of mixed phenotype cells with type II alveolar and Clara cell features, highlighting their airway resident cell origin. In addition to displaying specific pulmonary and epithelial commitment, bronchospheres showed mesenchymal features. Silencing of the Slug gene, known to play a pivotal role in epithelial-mesenchymal transition processes and which was highly expressed in bronchospheres but not in original tissue, led bronchospheres to gain a differentiated bronchial/alveolar phenotype and to lose the stemness gene expression pattern. CONCLUSIONS: Ours is the first study to describe ex vivo expansion of stem/progenitor cells resident in human lung epithelia, and our results suggest that the epithelial-mesenchymal transition process, still active in a subset of airway cells, may regulate transit of stem/progenitor cells towards epithelial differentiation.

Role of p53 codon 72 arginine allele in cell survival in vitro and in the clinical outcome of patients with advanced breast cancer.

BACKGROUND: The p53 codon 72 polymorphism, which results in either an arginine or proline residue, plays a different role in vitro and in vivo in cell survival and drug resistance. We verified, in vitro, the impact of the arginine allele on cell survival under normoxia and hypoxia, and investigated in vivo the role of p53 codon 72 arginine homozygosity in the clinical outcome of advanced breast cancer patients. METHODS: Tumors at advanced stages grow in vivo in a hypoxic environment, and we mimicked such conditions in vitro using p53 null breast cancer cells transfected with either the arginine or proline allele. We also analyzed in vivo the p53 codon 72 genotype status of advanced breast cancer patients. RESULTS: In vitro transfection of the arginine allele induced higher cell death under normoxia, whereas cell death was greater in proline-transfected cells under hypoxia. The arginine allele upregulated BCRP-I, a hypoxia response gene, which increases drug resistance. Metastatic breast cancer patients homozygous for arginine had a significantly shorter time to progression and overall survival than those with heterozygous arginine/proline tumors. CONCLUSION: We provide a molecular explanation for the association of the arginine allele with tumor aggressiveness and treatment resistance in advanced breast cancer.

Pro-apoptotic activity of cyclopentenone in cancer cells.

Studies on cyclopentenone prostaglandins (CPPGs), clavulones and other cyclopentenones have shown that these compounds have a significant anticancer activity mediated by their cyclopentenone (CP) chemical moiety. In this study the cytotoxicity against cancer cells of the model compound cyclopent-2-en-1-one (2CP) was investigated. Being a highly water soluble small molecule, 2CP could be an ideal candidate to overcome pharmacological issues related to drug delivery and penetration. Its cytotoxic activity was tested on various melanoma and lung cancer cells. Interestingly, 2CP was both cytotoxic and pro-apoptotic, more pronounced on melanoma cells, at concentrations in the sub-micromolar range. On melanoma cells its mechanism of action was mediated by the mitochondria and the activation of caspase 3.

Phase I study of paclitaxel and uracil plus tegafur combination in patients with pretreated metastatic breast cancer: drug sequencing based on preclinical modelling studies.

OBJECTIVE: Taxanes and fluoropyrimidines are active in metastatic breast cancer (MBC), and their combination has proven effective in anthracycline-refractory patients. We conducted a phase I study to determine the maximum tolerated dose (MTD) of uracil plus tegafur (UFT) given in combination with leucovorin (LV) and paclitaxel (Pacl) in patients with refractory MBC. METHODS: Pacl was infused at a fixed dose of 150 mg/m2 on day 1. UFT, at doses escalated by 50 mg/m2 starting from 200 mg/m2 . day, and LV, at a fixed dose of 90 mg/day, were given orally every 8 h for 11 days (days 3-13). Cohorts of at least 3 patients were treated at each dose level, and if 1 experienced dose-limiting toxicity (DLT), a maximum of 3 additional patients were added at the same dose level. MTD was reached if 2 out of the 6 patients experienced DLT. RESULTS: Sixteen patients were enrolled in the study. The most important toxicity observed was hematological. Nonhematological toxicities were paresthesia and myalgia, asthenia, nausea, and mucositis. DLT occurred in only 1 patient (grade 3 hepatic toxicity). CONCLUSIONS: The recommended dose for a subsequent phase II trial is Pacl 150 mg/m2 on day 1, and UFT 300 mg/m2 and LV 90 mg on days 3-13, every 2 weeks.

Cytotoxicity of different selective serotonin reuptake inhibitors (SSRIs) against cancer cells.

Cell membrane ion transporters expression and activity are altered in cancer cells and these phenotypic alterations offer potential targets for cancer therapies. Among the therapeutic agents affecting cell membrane transporters, serotonin reuptake inhibitors (SSRIs) have been shown to have anticancer potential. In this work, we have compared two SSRIs, one very specific for serotonin reuptake transporters (paroxetine) and another which also inhibit norepinephrine and dopamine transporters (venlafaxine), for their ability to counteract growth of various murine and human cancer cell lines. We found that paroxetine has cytotoxic activity against tumor cells, both of murine or human origin in the micromolar concentration range, whereas venlafaxine has not. A neurotransmitter receptor mediated mechanism of action appears thus unlikely for SSRIs cytotoxicity on cancer cells. With ranges of SSRIs cytotoxicity on cancer cells defined, limits in their possible applicability in cancer therapy is discussed.

Pro-apoptotic effect of a nitric oxide-donating NSAID, NCX 4040, on bladder carcinoma cells.

Nitric oxide-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs) are a promising class of compounds that cause cell cycle perturbations and induce apoptosis in cell lines from different tumors. We investigated the activity of a recently developed NO-NSAID (NCX 4040) in bladder cancer cell lines (HT1376 and MCR). Cells were treated with different drug concentrations for different exposure times. Cytostatic and cytocidal activity was tested by SRB assay and apoptosis was evaluated by TUNEL analysis, ANNEXIN V assay and fluorescence microscopy. To further investigate the cell death-inducing mechanisms of NCX 4040, we analyzed gp-170, caspase expression and mitochondrial membrane potential (Delta Psi) depolarization. NCX 4040 showed a striking cytocidal activity in both cell lines, reaching LC(50) at a 10-microM and 50-microM concentrations in HT1376 and in MCR cells, respectively, after an exposure of only 6 h followed by an 18-h washout. Apoptosis was triggered in up to 90% of cells and was associated with active caspase-3 expression and Delta Psi depolarization in both cell lines after a 6-h exposure. In conclusion, NCX 4040, which probably causes apoptosis via a mitochondrial-dependent mechanism, could prove to be a useful agent for improving bladder cancer treatment.

Modulation of drug cytotoxicity by Iressa (ZD1839) in pancreatic cancer cell lines.

High expression of the epidermal growth factor receptor (EGFR) family confers a growth advantage on malignant cells in various tumor types. Most pancreatic cancers express EGFR, which seems to play an important role in the acquisition of aggressive clinical behaviour and in tumor invasion. Iressa (ZD1839), a quinazoline tyrosine kinase inhibitor selective for the EGF receptor, has shown good anti-tumor activity in both preclinical and clinical studies. Using two pancreatic cancer cell lines that express different EGFR and ErbB-2 levels, we analyzed the activity of Iressa and evaluated its modulation effect on four conventional cytotoxic drugs: gemcitabine, oxaliplatin, docetaxel and SN38. Iressa was tested at scalar doses up to the plasma peak level concentration and showed a similar weak cytostatic effect in both cell lines. Conversely, an additive or weak synergistic effect was observed when the drug was administered simultaneously with or following cytotoxic drugs. Our data show that Iressa has only a weak activity at doses within the plasmatic peak concentration and that its effect is independent of EGFR and p42/p44 expression and phosphorylation levels. This is in agreement with recent literature data that attribute an essential role to a specific EGFR mutation in mediating response to Iressa. This mutation was absent in both pancreatic cell lines tested.

Potentiation of antiproliferative drug activity by lonidamine in hepatocellular carcinoma cells.

The ability of lonidamine (LND), a derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of anticancer drugs was investigated in two human hepatocarcinoma (HCC) cell lines. The cytotoxicity of drugs used singly, in association or in sequence was evaluated using the Sulforhodamine B (SRB) assay. LND did not appreciably potentiate the effect of antitumor drugs when given before or simultaneously, in either cell line. Conversely, a synergistic interaction was observed in both cell lines when LND was given after conventional drugs. LND produced a moderate decrease in S-phase cell fraction and did not induce apoptosis. Conversely, paclitaxel (TAX) induced an important block in G2 and an increase in apoptosis. Following a 48-h TAX wash out, a progressive passage of cells from G2 to M phase was observed with a corresponding increase in apoptotic cells. Post-treatment with LND increased the cytotoxicity of some antitumor drugs, especially TAX, in hepatocarcinoma cells, possibly by preventing, as an energolytic drug, cell damage repair or by producing an additional effect on microtubule stabilization.

Measurements and modeling of acoustic scattering from partially and completely buried spherical shells.

The use of low-frequency sonars (2-15 kHz) is explored to better exploit scattering features of buried targets that can contribute to their detection and classification. Compared to conventional mine countermeasure sonars, sound penetrates better into the sediment at these frequencies, and the excitation of structural waves in the targets is enhanced. The main contributions to target echo are the specular reflection, geometric diffraction effects, and the structural response, with the latter being particularly important for man-made elastic objects possessing particular symmetries such as bodies of revolution. The resonance response derives from elastic periodic phenomena such as surface circumferential waves revolving around the target. The GOATS'98 experiment, conducted jointly by SACLANTCEN and MIT off the island of Elba, involved controlled monostatic measurements of scattering by spherical shells which were partially and completely buried in sand, and suspended in the water column. The analysis mainly addresses a study of the effect of burial on the dynamics of backscattered elastic waves, which can be clearly identified in the target responses, and is based on the comparison of measurements with appropriate scattering models. Data interpretation results are in good agreement with theory. This positive result demonstrates the applicability of low-frequency methodologies based on resonance analysis to the classification of buried objects.

Flow-cytometric determination of tumor cells in lymph nodes.

In solid tumors, metastasis occurs through the dissemination of tumor cells in the bloodstream and the lymphatic system. In particular, lymph node infiltration gives useful prognostic information and represents one of the most important factors for selecting the type of clinical treatment in disease management. Furthermore, the analysis of lymph node infiltration has become important for identifying patients with breast cancer or malignant melanoma who may be candidates for regional lymph node dissection. Tumor cells in lymph nodes are currently identified in tissue sections using morphological and immunohistochemical analyses, but these approaches are time-consuming, and micrometastases may escape detection. The aim of the present study was to define the potential of a flow cytometric (FCM) determination based on cell size and autofluorescence to shorten the time required for lymph node analysis. The sensitivity of the FCM approach, defined on mixtures of tumor cells from established cell lines and peripheral blood lymphocytes (PBL(s)) at different concentrations, was 1 tumor cell/1,000 PBL(s). FCM analysis was performed on 89 lymph nodes, 29 from breast, 41 from lung and 19 from colon cancer patients. Agreement between FCM and morphological results, used as gold standard, was observed in 83% of the cases, and there was a 90% sensitivity to the FCM approach for each tumor type. Disagreement was observed for 15 lymph nodes and was due, in the majority of cases (80%), to FCM-positive and morphologically negative results. A large number of patients and a more accurate pathological examination of consecutive histological sections of lymph nodes are needed to further evaluate the validity of the FCM approach.

In vitro preclinical models for a rational design of chemotherapy combinations in human tumors.

Today, drug combinations are frequently used in the treatment of cancer to increase therapeutic efficacy. Currently used clinical protocols for cancer combination therapies are mainly obtained empirically or on the basis of results from previous clinical trials. Information obtained from clinical protocols is invaluable, but it is time-consuming, expensive and does not provide data on the biochemical and molecular mechanisms of interaction of the drugs used in combination treatments at cellular level. Therefore, in vitro drug combination studies on established cell lines or primary cell cultures play an important role in designing and optimising combination protocols. A variety of in vitro assays and different mathematics models have been developed to investigate cytotoxic effects and to analyse the type of drug interactions. Increased knowledge of the cellular targets of traditional and new drugs and the development of new technologies have resulted in a new role for the in vitro tests which are no longer used only to evaluate the cytotoxic effects of drugs, but also to investigate the interference on cell cycle, induction of apoptosis and molecular or biochemical interactions. A review on in vitro preclinical tests used to evaluate the effects of drug combinations and to design the rationale of combined chemotherapy protocols is presented.

Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells.

Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. Flow cytometry is a widely-used technique for checking apoptosis, permitting a multiparametric analysis. It is possible to follow the alterations occurring in the nucleus, mitochondria and plasmatic membrane during the different apoptotic stages using probes such as LDS-751, JC-1 or Annexin V. The potential of these probes to identify the early or late stages of apoptosis has been widely investigated in cells growing in suspension. In order to assess apoptosis in adherent cells, we tested a combination of fluorescein diacetate (FDA), a substrate for non specific esterase whose activity decreases during the early phase of apoptosis, and trypan blue in MCF-7 human breast cancer cells. Apoptotic cells showed a decrease in the green fluorescence emitted by fluorescein, the product of FDA hydrolysis, whereas necrotic cells emitted a red fluorescence due to the trypan blue staining. FDA-trypan blue double-staining was used to investigate the different kinetics of apoptosis induced by taxol, camptothecin and UV-B irradiation in MCF-7 cells. This method is rapid and simple, and can be used for monitoring the process of apoptosis from early stages in adherent cells, for the physical separation of apoptotic and live cells, and for immunophenotyping, including Fas expression.

Lanreotide-induced modulation of 5-fluorouracil or mitomycin C cytotoxicity in human colon cancer cell lines: a preclinical study.

The effect on growth of the long-acting somatostatin analogue lanreotide (LAN), alone or in combination with 5-fluorouracil (5-FU) and mitomycin C (MIT), was investigated in three human colon cancer lines. Cell survival inhibition induced by LAN alone, as evaluated by sulforhodamine B assay, ranged from 20% to 40% as a function of cell line and concentration. The IC50, the concentration inhibiting cell survival by 50%, was never reached. The antiproliferative effect produced by a 48 h exposure to 5-FU or MIT was synergistically enhanced in all cell lines by a subsequent 48 h exposure to LAN. The synergistic interaction was not related to specific cell cycle perturbations or to the somatostatin receptor 2 (sst2) mRNA abundance. In conclusion, our study seems to indicate that LAN is a potentially useful modulating agent for enhancing 5-FU and MIT activity in colorectal cancer patients.

Combined 4-hydroxy-ifosfamide and vinorelbine treatment in established and primary human breast cell cultures.

BACKGROUND: Vinorelbine and ifosfamide are active drugs against breast cancer, but the best treatment schedule has yet to be defined by preclinical or clinical studies. The antitumor activity of 4-hydroxy-ifosfamide (4-OH-IF), the active form of ifosfamide, and vinorelbine (VNB) and their interaction were investigated in two established breast cancer cell lines (MCF-7 and BRC-230) and in 10 primary breast cancer cultures. MATERIALS AND METHODS: Cytotoxic activity was evaluated by a highly efficient clonogenic assay (HECA). The median-effect principle was applied to evaluate synergistic and antagonistic interactions and the corresponding combination index values were calculated. Cell cycle perturbations were analysed by flow cytometry. RESULTS: In MCF-7 and BRC-230 cell lines the sequence VNB for 4 hours followed by 4-OH-IF for 24 hours produced an antagonistic effect. Conversely, the inverse sequential scheme, 4-OH-IF-->VNB provided synergistic effects on both cell lines. The synergism was associated with a strong block in the G2-M phase. Synergistic activity of 4-OH-IF-->VNB sequence was confirmed in 7 of 10 primary breast cancer cultures. CONCLUSIONS: In conclusion, the sequence 4-OH-IF-->VNB appeared to be the most effective scheme both in established cell lines and in primary breast cancer cultures.

Docetaxel and gemcitabine activity in NSCLC cell lines and in primary cultures from human lung cancer.

The activity of the following drugs was investigated in two established NSCLC cell lines: docetaxel, gemcitabine, vinorelbine, paclitaxel, doxorubicin (0.01, 0.1, 1 microg ml(-1)), cisplatin, ifosfamide (1, 2, 3 microg ml(-1)) and carboplatin (2, 4, 6 microg ml(-1)). The cytotoxic activity was evaluated by the sulphorhodamine B assay. The two most active drugs, docetaxel and gemcitabine, used singly and in association, were investigated as a function of treatment schedule. The sequence docetaxel-->gemcitabine produced only a weak synergistic interaction in RAL but a strong synergism in CAEP cells. The synergistic interaction increased in both cell lines after a 48-h washout between the drug administrations. Flow cytometric analysis showed that in docetaxel-->gemcitabine sequence, docetaxel produced a block in G2/M phase and, after 48 h, provided gemcitabine with a large fraction of recovered synchronized cells in the G1/S boundary, which is the specific target phase for gemcitabine. Conversely, simultaneous treatment induced an antagonistic effect in both cell lines, and the sequential scheme gemcitabine-->docetaxel produced a weak synergistic effect only in RAL cells. Moreover, the synergistic interaction disappeared when washout periods of 24 or 48 h between two drug administrations were adopted. The synergistic activity of docetaxel-->48-h washout-->gemcitabine was confirmed in 11 of 14 primary cultures, which represents an important means of validating experimental results before translating them into clinical practice.

Mimetic gain in batesian and Müllerian mimicry.

Starting from field investigations and experiments on mimetic butterfly populations a model for two mimetic species is developed. The model comprises various features such as the growth rates and carrying capacities of the two species, their unpalatability to predators, the recruitment and the training of the predators and, most important, the similarity of the two mimetic species. The model ranges from pure Batesian to pure Müllerian mimicry over a spectrum of possible cases. The mimetic gain is introduced as the relative increase in equilibrium density in a mimetic situation as compared to a situation where mimicry is not present. The dependence of this quantity on parameters as growth rate, carrying capacity, unpalatability, and similarity is investigated using numerical methods.