Paolo Tini

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.

β-catenin and Gli1 are prognostic markers in glioblastoma

Glioblastomas (GBMs), the most common primary malignancies of the central nervous system, are highly aggressive and heterogeneous, and remain a dramatic therapeutic challenge. Markers mirroring the complex molecular profile of GBMs that are predictive of patient outcomes are needed to define novel multi-targeted treatment strategies. Resistance to current GBM therapies is partly due to a subpopulation of stem-like and other self-renewing cells (hereafter called glioma stem-like cancer cells, GSCC), which are therefore of key interest as therapeutic entry points. Wnt and Hedgehog are among the main pathways involved in GSCC renewal. β-catenin and Gli1 are markers of Wnt and Hedgehog activation respectively and both pathways are known to be altered in gliomas. To date, there are no investigations of Gli1 protein expression in GBM tissue, and recently a high expression of β-catenin has been found to have a poor prognostic impact in GBM patients in a study. We have therefore quantified the positivity for β-catenin, Gli1, as well as Ki-67, p53, and EGFR proteins on immunohistochemically-stained GBM sections from 106 patients in an investigation for potential predictive biomarkers. Correlation between these markers and survival was evaluated by pair-wise Pearson correlation coefficient and by bi-dimensional hierarchical clustering, followed by survival estimations using linear regression models and classification trees. We demonstrated that both β-catenin and, for the first time, Gli1 proteins are highly predictive markers of short survival, being found in 75 and 90% of the highly predictive trees, respectively, whereas Ki-67, p53 and EGFR were under 30% and thus, not considered as predictive. Our results indicate a role of β-catenin and Gli1 in GBM malignant behaviour, and suggest that inhibiting members of Wnt and Hedgehog pathways could be a valuable therapeutic strategy for GBM patients. See commentary: A magnifying glass on glioblastoma stem cell signaling pathways

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.

Combined EGFR and autophagy modulation impairs cell migration and enhances radiosensitivity in human glioblastoma cells

Glioblastoma (GBM) remains the most aggressive and lethal brain tumor due to its molecular heterogeneity and high motility and invasion capabilities of its cells, resulting in high resistance to current standard treatments (surgery, followed by ionizing radiation combined with Temozolomide chemotherapy administration). Locus amplification, gene overexpression, and genetic mutations of epidermal growth factor receptor (EGFR) are hallmarks of GBM that can ectopically activate downstream signaling oncogenic cascades such as PI3K/Akt/mTOR pathway. Importantly, alteration of this pathway, involved also in the regulation of autophagy process, can improve radioresistance in GBM cells, thus promoting the aggressive phenotype of this tumor. In this work, the endogenous EGFR expression profile and autophagy were modulated to increase radiosensitivity behavior of human T98G and U373MG GBM cells. Our results primarily indicated that EGFR interfering induced radiosensitivity according to a decrease of the clonogenic capability of the investigated cells, and an effective reduction of the in vitro migratory features. Moreover, EGFR interfering resulted in an increase of Temozolomide (TMZ) cytotoxicity in T98G TMZ‐resistant cells. In order to elucidate the involvement of the autophagy process as pro‐death or pro‐survival role in cells subjected to EGFR interfering, the key autophagic gene ATG7 was silenced, thereby producing a transient block of the autophagy process. This autophagy inhibition rescued clonogenic capability of irradiated and EGFR‐silenced T98G cells, suggesting a pro‐death autophagy contribution. To further confirm the functional interplay between EGFR and autophagy pathways, Rapamycin‐mediated autophagy induction during EGFR modulation promoted further impairment of irradiated cells, in terms of clonogenic and migration capabilities. Taken together, these results might suggest a novel combined EGFR‐autophagy modulation strategy, to overcome intrinsic GBM radioresistance, thus improving the efficacy of standard treatments. J. Cell. Physiol. 229: 1863–1873, 2014.

The combined EGFR protein expression analysis refines the prognostic value of the MGMT promoter methylation status in glioblastoma

BACKGROUND/AIMS To investigate the combined prognostic value of the EGFR expression level and the MGMT promoter methylation status in Glioblastoma (GB). METHODS We assessed the EGFR protein expression level by immune-histochemical (IHC) evaluation and the MGMT promoter methylation status by Polymerase Chain Reaction (PCR) in 169 patients affected by GB. We assessed the prognostic significance of combined MGMT methylation status and EGFR expression level in terms of Overall Survival (OS) with univariate and multivariate analysis, and validated this finding using an external data set of GB patient. RESULTS Clustering survival analysis for the methylation status of MGMT (methMGMT/unmethMGMT) and EGFR expression (High EGFR: H-EGFR; Low EGFR: L-EGFR), identified three different prognostic groups (p=0.001), as follows. Patients with unmethMGMT/H-EGFR had the shortest survival time (median OS: 5 months) and patients co-expressing methMGMT/L-EGFR had the best prognosis (median OS: 35 months), as compared to the other two sub-groups (methMGMT/H-EGFR; unmethMGMT/L-EGFR), which had respectively median OSs of 11 and 12 months. The combined MGMT methylation and EGFR amplification status analysis showed a similar prognostic impact in an independent series, which we used for validation (p=0.001). CONCLUSIONS The EGFR expression evaluation refines the prognostic value of MGMT methylation status in GBs.

Combined Epidermal Growth Factor Receptor and Beclin1 Autophagic Protein Expression Analysis Identifies Different Clinical Presentations, Responses to Chemo- and Radiotherapy, and Prognosis in Glioblastoma

Dysregulated EGFR in glioblastoma may inactivate the key autophagy protein Beclin1. Each of high EGFR and low Beclin1 protein expression, independently, has been associated with tumor progression and poor prognosis. High (H) compared to low (L) expression of EGFR and Beclin1 is here correlated with main clinical data in 117 patients after chemo- and radiotherapy. H-EGFR correlated with low Karnofsky performance and worse neurological performance status, higher incidence of synchronous multifocality, poor radiological evidence of response, shorter progression disease-free (PDFS), and overall survival (OS). H-Beclin1 cases showed better Karnofsky performance status, higher incidence of objective response, longer PDFS, and OS. A mutual strengthening effect emerges in correlative power of stratified L-EGFR and H-Beclin1 expression with incidence of radiological response after treatment, unifocal disease, and better prognosis, thus identifying an even longer OS group (30 months median OS compared to 18 months in L-EGFR, 15 months in H-Beclin1, and 11 months in all GBs) (P = 0.0001). Combined L-EGFR + H-Beclin1 expression may represent a biomarker in identifying relatively favorable clinical presentations and prognosis, thus envisaging possible EGFR/Beclin1-targeted therapies.

Diffusion-weighted MR Volumetry for Assessing the Response of Rectal Cancer to Combined Radiation Therapy with Chemotherapy

Editor: In the study by Curvo-Semedo and colleagues (1), which appeared in the September 2011 issue of Radiology, the utility of tumor volumetry derived from diffusion-weighted (DW) magnetic resonance (MR) images was evaluated in the assessment of patients with rectal cancer after preoperative combined radiation therapy with chemotherapy (CRT). They showed that post-CRT DW volumetry and volume shrinkage at both T2-weighted and DW MR imaging were highly accurate in the determination of complete response (areas under the receiver operating characteristic curve [AUC] of 0.93, 0.84, and 0.92, respectively). The apparent diffusion coefficient (ADC) and ADC modification rate were not accurate (AUC, 0.54 and 0.51, respectively). The intraclass correlation coefficient (ICC) between T2-weighted and DW MR volumetry was good for pre-CRT measurements but unsatisfactory for post-CRT measurements. This last observation might be attributable to post-CRT fibrosis, which affects the reliability of segmentation of tumor volume on T2-weighted images with respect to post-CRT DW volumetry based on ADC maps (1). In their study, Curvo-Semedo and colleagues defined complete response as tumor regression grade 1 (2) at pathologic examination or a relapsefree follow-up of 17 months. The preliminary results of our study (3), performed with DW imaging using a b value of 800 sec/mm2, are consistent with those reported by Curvo-Semedo and colleagues. In our study of 21 patients who underwent surgical resection after CRT, the interobserver agreement was good (ICC = 0.948 for T2-weighted MR volumetry and 0.916 for DW MR volumetry), whereas the preand post-CRT DW and T2-weighted volumetry concordance consisted of ICC values of 0.828 and 0.151, respectively. The volume segmented from the DW data set on the basis of hyperintensity of hypercellular tissue on DW images showed a good correlation with tumor regression grade (r = 0.556, P , .05). However, DW MR volumetry has some drawbacks, including the difficulty in detecting small neoplastic residual tumors on the post-CRT ADC maps and T2 shinethrough artifacts, owing to necrosis or mucin, which can produce overestimation of residual tumor at the only qualitative eval uation of DW images. These limitations, in our experience, make it difficult to differentiate between patients with tumor regression grades of 1 or 2 (Mandard five-point scale). The relationship to DW MR volumetry of the nodal stage evaluation could increase its prognostic relevance because good prognosis has been demonstrated for low tumor regression grades ypN2 with respect to ypN+ (4). DW MR volumetry seems to be a promising tool for assessing response after preoperative CRT and deserves further study.

Three-dimensional conformal radiotherapy, temozolomide chemotherapy, and high-dose fractionated stereotactic boost in a protocol-driven, postoperative treatment schedule for high-grade gliomas

AIMS AND BACKGROUND No available scientific report deals with high-dose (> or = 70 Gy) radiotherapy plus temozolomide chemotherapy (TMZ CHT) in high-grade gliomas. The survival results of a protocol-driven, postoperative treatment schedule are reported here to contribute to the discussion on this issue. METHODS AND STUDY DESIGN Uniform criteria were prospectively adopted for case selection during the period 1993-2006 in the management of 123 patients, and we progressively introduced three-dimensional conformal radiotherapy (3D-CRT, 60 Gy), TMZ CHT and a high-dose (70 Gy) stereotactic boost (HDSRT) in the treatment schedule. Palliative radiotherapy was delivered by whole brain irradiation (WBI, 50 Gy) for bulky tumors, whereas radical irradiation was performed with 3D-CRT throughout the study period. Two periods of accrual are considered: 36 patients were treated before 31 December 1999 (29.25%) and 87 (70.75%) after 1 January 2000. This subdivision was due to the implementation of HDSRT hardware and TMZ CHT from January 2000. RESULTS The median overall survival was 13 months and the 1-, 2- and 3-year survival rates were 53%, 19.5% and 11.6%, respectively. The differences in survival related to the treatment variables were highly significant, both in univariate and multivariate analysis. The median survival and 1-, 2- and 3-year survival rates in the palliative WBI group were 9.75 months and 37%, 2%, and 0%, respectively; in the 3D-CRT group 17.25 months and 64%, 34%, and 15%, respectively; in the TMZ CHT concomitant with radiotherapy group 20 months and 61%, 39%, and 21%, respectively; in the TMZ CHT concomitant with and sequential to radiotherapy group 25.75 months and 84%, 54%, and 26%, respectively, and in the HDSRT group 22 months and 72%, 48%, and 37%, respectively. No symptomatic radiation necrosis occurred in any of the groups. CONCLUSIONS The results reported here are generally better than those reported in the literature. The selection of patients on the basis of favorable prognostic factors and suitability to the currently available, aggressive postoperative treatment resources can be the mainstay for improving therapeutic results. In particular, the new treatment option reported here (HDSRT in association with TMZ CHT) proved to be safe and effective in obtaining a relatively favorable outcome.

Diffusion-Weighted Magnetic Resonance Diagnosis of Local Recurrences of Prostate Cancer after Radical Prostatectomy: Preliminary Evaluation on Twenty-Seven Cases

Objectives. To assess the diagnostic performance of diffusion-weighted MR imaging (DWI) in patients affected by prostatic fossa (PF) relapse after radical prostatectomy (RP) for prostatic carcinoma (PC). Methods. Twenty-seven patients showing a nodular lesion in the PF at T2-weighted MR imaging after RP, with diagnosis of PC relapse established by biopsy or PSA determinations, were investigated by DWI. Two readers evaluated the DWI results in consensus and the apparent diffusion coefficient (ADC) of the nodules, separately; a mean value was obtained (ADCm). Results. Relapses did not significantly differ in size in respect of postsurgical benign nodules. The DWI qualitative evaluation showed sensitivity, specificity, accuracy, ppv, and npv values, respectively, of 83.3%, 88.9%, 85.2%, 93.7%, and 72.7% (100%, 87.5%, 95.6%, 93.7%, and 100%, for nodules >6 mm). The intraclass correlation coefficient (ICC) for ADC evaluation between the two readers was 0.852 (95% CI 0.661–0.935; P = 0.0001). The ADCm values for relapses and benign nodules were, respectively, 0.98 ± 0.21 × 10−3 mm2/sec and 1.24 ± 0.32 × 10−3 mm2/sec (P = 0.006). Sensitivity, specificity, accuracy, ppv and npv of ADCm were, respectively, 77.8%, 88.9%, 81.8%, 93.3%, and 66.7% (93.3%, 87.5%, 85.4%, 93.3%, and 87.5% for nodules >6 mm). Conclusions. Diffusion-weighted MR imaging is a promising tool in the management of a hyperintense nodule detected by T2-weighted sequences. This might have a relevant importance in contouring radiotherapy treatment volumes.

The effects of radiotherapy on the survival of patients with unresectable non-small cell lung cancer

ABSTRACT Introduction: Lung cancer represents the leading cause of cancer mortality across the worlds. At present, less than 30% of the patients can undergo curative surgery, while the majority of them (65%) are diagnosed with metastatic disease and directed to systemic treatments. In this context there is a subset of patients (25%) with locally advanced stage disease whose outcome might be improved by using combined strategies of treatment including chemotherapy, radiotherapy and surgery. Areas covered: Here we reviewed possible combination strategies aimed to improve the outcome of lung cancer patients, focusing on the role of radiotherapy both in the adjuvant and oligo-metastatic setting and in synergy with immunotherapy, and finally, we afforded the new challenges concerning the advanced RT and precision oncology. We carried out a focused analysis concerning the key clinical management weaknesses as well as the potential that current research holds. Expert commentary: We believe that the most promising clinical trials in this specific patient subset will build their rationale on the results of well-designed translational models aimed to test the combination of cytotoxic drugs, radiobiology, and immune-pharmacology. In this context, remarkable investigational fields are focused on the attempt to combine radiotherapy with chemo-immunological strategies and precision medicine protocols.