Giuseppe Privitera

A 62 MeV proton beam for the treatment of ocular melanoma at Laboratori Nazionali del Sud-INFN (CATANIA)

At the INFN Laboratori Nazionali del Sud in Catania (Italy) the first Italian protontherapy facility, named CATANA (Centre di AdroTerapia e Applicazioni Nucleari Avanzate) has been realized in collaboration with the University of Catania. It is based on the use of the 62 MeV proton beam delivered by the K = 800 superconducting cyclotron installed and working at LNS since 1995. The facility is mainly devoted to the treatment of ocular diseases like uveal melanoma. A beam treatment line in air has been realized together with a dedicated positioning patient system. The facility is in operation since the beginning of 2002 and 52 patients have been successfully treated up to now. The main features of CATANA together with the clinical and dosimetric features will be extensively reported; particularly, will be described the proton beam line, that has been entirely realized at LNS, with all its elements, the experimental transversal and depth dose distributions of the 62 MeV proton beam obtained for a final collimator of 25 mm diameter and the experimental depth dose distributions of a modulated proton beam obtained for the same final collimator. Finally, the clinical results over one year of treatments, describing the features of the treated diseases will be reported.

Specific Alterations of MicroRNA Transcriptome and Global Network Structure in Colorectal Carcinoma after Cetuximab Treatment

The relationship between therapeutic response and modifications of microRNA (miRNA) transcriptome in colorectal cancer (CRC) remains unknown. We investigated this issue by profiling the expression of 667 miRNAs in 2 human CRC cell lines, one sensitive and the other resistant to cetuximab (Caco-2 and HCT-116, respectively), through TaqMan real-time PCR. Caco-2 and HCT-116 expressed different sets of miRNAs after treatment. Specifically, 21 and 22 miRNAs were differentially expressed in Caco-2 or HCT-116, respectively (t test, P < 0.01). By testing the expression of differentially expressed miRNAs in CRC patients, we found that miR-146b-3p and miR-486-5p are more abundant in K-ras–mutated samples with respect to wild-type ones (Wilcoxon test, P < 0.05). Sixty-seven percent of differentially expressed miRNAs were involved in cancer, including CRC, whereas 19 miRNA targets had been previously reported to be involved in the cetuximab pathway and CRC. We identified 25 transcription factors putatively controlling these miRNAs, 11 of which have been already reported to be involved in CRC. On the basis of these data, we suggest that the downregulation of let-7b and let-7e (targeting K-ras) and the upregulation of miR-17* (a CRC marker) could be considered as candidate molecular markers of cetuximab resistance. Global network functional analysis (based on miRNA targets) showed a significant overrepresentation of cancer-related biological processes and networks centered on critical nodes involved in epidermal growth factor receptor internalization and ubiquitin-mediated degradation. The identification of miRNAs, whose expression is linked to the efficacy of therapy, should allow the ability to predict the response of patients to treatment and possibly lead to a better understanding of the molecular mechanisms of drug response. Mol Cancer Ther; 9(12); 3396–409.

Specific alterations of the microRNA transcriptome and global network structure in colorectal cancer after treatment with MAPK/ERK inhibitors

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has a master control role in various cancer-related biological processes as cell growth, proliferation, differentiation, migration, and apoptosis. It also regulates many transcription factors that control microRNAs (miRNAs) and their biosynthetic machinery. To investigate on the still poorly characterised global involvement of miRNAs within the pathway, we profiled the expression of 745 miRNAs in three colorectal cancer (CRC) cell lines after blocking the pathway with three different inhibitors. This allowed the identification of two classes of post-treatment differentially expressed (DE) miRNAs: (1) common DE miRNAs in all CRC lines after treatment with a specific inhibitor (class A); (2) DE miRNAs in a single CRC line after treatment with all three inhibitors (class B). By determining the molecular targets, biological roles, network position of chosen miRNAs from class A (miR-372, miR-663b, miR-1226*) and class B (miR-92a-1*, miR-135b*, miR-720), we experimentally demonstrated that they are involved in cell proliferation, migration, apoptosis, and globally affect the regulation circuits centred on MAPK/ERK signaling. Interestingly, the levels of miR-92a-1*, miR-135b*, miR-372, miR-720 are significantly higher in biopsies from CRC patients than in normal controls; they also are significantly higher in CRC patients with mutated KRAS than in those with wild-type genotypes (Wilcoxon test, p < 0.05): the latter could be a downstream effect of ERK pathway overactivation, triggered by KRAS mutations. Finally, our functional data strongly suggest the following miRNA/target pairs: miR-92a-1*/PTEN-SOCS5; miR-135b*/LATS2; miR-372/TXNIP; miR-663b/CCND2. Altogether, these results contribute to deepen current knowledge on still uncharacterized features of MAPK/ERK pathway, pinpointing new oncomiRs in CRC and allowing their translation into clinical practice and CRC therapy.

Cellular and molecular effects of protons: Apoptosis induction and potential implications for cancer therapy

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.

Oxidation rate enhancement of SiGe epitaxial films oxidized in dry ambient

We present a study on thin oxides obtained by rapid thermal oxidation of Si1−xGex epitaxial layers. The oxidation processes were performed in dry O2 at 1000 °C for times up to 600 s. Our data show an oxide growth rate enhancement with respect to pure Si. Except for a very small amount of GeO2 that is found at the surface, all the Ge is rejected towards the SiO2/SiGe interface, forming a Ge-enriched layer free of extended defects. The comparison of our results for dry processes with those reported in the literature for wet ambient supports the idea that the kinetics of SiGe oxidation is controlled by similar mechanisms in both cases, in contrast with models and interpretations so far proposed.

RECURRENT INTRACRANIAL HEMANGIOPERICYTOMA WITH EXTRACRANIAL AND UNUSUAL MULTIPLE METASTASES: CASE REPORT AND REVIEW OF THE LITERATURE

Hemangiopericytoma is a rare tumor with uncommon location in the central nervous system. It has only recently been included (WHO classification 1993) in a specific group of CNS tumors and subsequently (WHO classification 1997 and 2000) as a group by itself, while before it was confused with meningeal tumors. We report on a case of a 48-year-old woman affected by this tumor. The neoplasm was located in the posterior fossa. The patient underwent primary surgery in 1990, not followed by any adjuvant therapy because of the histopathological diagnosis of meningioma. After being free from disease for eight years she developed a local recurrence in 1998. Subtotal excision of the tumor, which was finally identified as a hemangiopericytoma, was carried out, followed by adjuvant radiotherapy (64 Gy). After six months multiple metastases were found in the liver and right kidney. A radical metastasectomy was performed, followed by systemic chemotherapy. One year later (2001) the tumor recurred again intracranially and a metastases was detected in the right breast, so the patient again underwent cranial irradiation (40 Gy) and second-line chemotherapy. She died in September 2002, 12 years after the diagnosis. We may conclude that, despite the tumors natural tendency to recur several times and the ability of intracranial hemangiopericytoma to spread outside the CNS, it is possible to ensure a long survival time.

Laser-ultraviolet-A-induced ultraweak photon emission in mammalian cells

Photobiological research in the last 30 yr has shown the existence of ultraweak photon emission in biological tissue, which can be detected with sophisticated photomultiplier systems. Although the emission of this ultraweak radiation, often termed biophotons, is extremely low in mammalian cells, it can be efficiently increased by ultraviolet light. Most recently it was shown that UV-A (330 to 380 nm) releases such very weak cell radiation in differentiated human skin fibroblasts. Based on these findings, a new and powerful tool in the form of UV-A-laser-induced biophotonic emission of cultured cells was developed with the intention to detect biophysical changes between carcinogenic and normal cells. With suspension densities ranging from 1 to 8 x 10(6) cells/mL, it was evident that an increase of the UV-A-laser-light induced photon emission intensity could be observed in normal as well as melanoma cells. Using this new detection procedure of ultraweak light emission, photons in cell suspensions as low as 100 microL could be determined, which is a factor of 100 lower compared to previous procedures. Moreover, the detection procedure has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 micros instead of 150 ms, as reported in previous procedures. This improvement leads to measurements of light bursts up 10(7) photons/s instead of several hundred as found with classical designs. Overall, we find decreasing induction ratings between normal and melanoma cells as well as cancer-prone and melanoma cells. Therefore, it turns out that this highly sensitive and noninvasive device enables us to detect high levels of ultraweak photon emission following UV-A-laser-induced light stimulation within the cells, which enables future development of new biophysical strategies in cell research.

Discrimination between normal and cancer cells by using spectral analysis of delayed luminescence.

In our present studies, the time-resolved emission spectrum of delayed luminescence of cell cultures of human fibroblast and human melanoma have been measured using a sophisticated single photon device. Noticeable differences have been found both in the emission spectra, which are time dependent, and in the timing aspects of the different spectral components. This powerful and noninvasive technique can be applied in all fields of skin research, such as the investigation of skin abnormalities and to test the effect of products involved in regeneration, antiaging, and UV-light protection in order to prevent skin cancer.

Clinical application of proton beams in the treatment of uveal melanoma: the first therapies carried out in Italy and preliminary results (CATANA Project).

Background The first Italian proton therapy facility was realized in Catania, at the INFN-LNS. With its energy (62 MeV proton beam), it is ideal for the treatment of shallow tumors like those of the ocular region: uveal melanoma, first of all (the most common primary intraocular malignancy of adults) and other less frequent lesions like choroidal hemangioma, conjunctiva melanoma, and eyelid tumors. Material and methods The first patient was enrolled in February 2002, and to date 30 patients have been treated. All patients had a localized uveal melanoma, with no systemic metastases, and had specific indications for proton beam radiation therapy: lesions between 5–25 mm basal diameter, not exceeding 15 mm thickness, absence of total retinal detachment or glaucoma. According to the tumor dimensions, 2 patients had a small lesion or T1 (6%), 3 had a medium-sized lesion or T2 (10%), 14 had a large lesion or T3 (47%), and 11 had an extra-large lesion or T3 (37%); no patient had extrascleral invasion or T4 of the TNM-AJCC Staging System. In most cases, the tumor infiltrated only the choroid (14 patients, 47%) or the choroid plus the ciliary body (14 patients, 47%). We also treated a primitive iris melanoma, without diffusion to the ciliary body. The target volume was defined as the tumor plus a safety margin of 2.5 mm, laterally and antero-posteriorly; this margin was increased to 3 mm if ciliary body involvement was present. The treatment was carried out in 4 fractions on 4 consecutive days to a total dose of 54.5 Gy (single fraction 13.6 Gy), which corresponds to 60 CGE (Cobalt Gray Equivalent; single fraction 15 CGE), because the relative biological effectiveness is 1.1. Results The first follow-up is planned at 6–8 months after the end of the treatment, and our clinical end points are local control (defined as cessation of growth or tumor shrinkage), eye retention, and maintenance of a good visual function. At the time of this writing, we had preliminary results from 13 patients. Nine patients showed tumor shrinkage (69%), 3 a substantially stable dimension (23%), but almost all patients presented an increased ultrasound reflectivity (a surrogate for tumor control). Discussion and conclusions The literature data show that charged particle therapy has allowed an optimal local control in the treatment of uveal melanomas (about 96% in the different series, superior to that obtained with plaquetherapy [between 83% and 92%]), a metastatic rate slightly better than enucleation reports, and a survival rate of almost 90% at 5 years. Our preliminary results show a tumor response in almost all cases, with no major acute or subacute side effects. We thus plan to continue with our treatment procedures and our dose prescription.

Locally advanced rectal cancer: Qualitative and quantitative evaluation of diffusion-weighted MR imaging in the response assessment after neoadjuvant chemo-radiotherapy

Purpose to investigate the added value of qualitative and quantitative evaluation of diffusion weighted (DW) magnetic resonance (MR) imaging in response assessment after neoadjuvant chemo-radiotherapy (CRT) in patients with locally advanced rectal cancer (LARC). Methods 31 patients with LARC (stage ≥ T3) were enrolled in the study. All patients underwent conventional MRI and DWI before starting therapy and after neoadjuvant CRT. All patients underwent surgery; pathologic staging represented the reference standard. For qualitative analysis, two radiologists retrospectively reviewed conventional MR images and the combined set of conventional and DW MR images and recorded their confidence level with respect to complete response (ypCR). For quantitative analysis, tumor’s apparent diffusion coefficient (ADC) values were measured at each examination. ADC pre-CRT, ADC post-CRT and Δ ADC post−ADC pre of the three groups of response (ypCR, partial response ypPR, stable disease ypSD) were compared. Receiver-operating characteristics (ROC) curve analysis was employed to investigate the discriminatory capability for ypCR, responders (ypCR, ypPR) and ypSD of each measure. Results addition of DWI to conventional T2-weighted sequences improved diagnostic performance of MRI in the evaluation of ypCR. A low tumor ADC value in the pre-CRT examination, a high ADC value in the post-CRT examination, a high Δ ADC post−ADC pre [>0.3 (×10−3 mm2/s)] were predictive of ypCR. Conclusions DW sequences improve MR capability to evaluate tumor response to CRT. Nevertheless, no functional MR technique alone seems accurate enough to safely select patients with ypCR.