Luca Bertero

Seizure control following radiotherapy in patients with diffuse gliomas: a retrospective study

BACKGROUND Little information is available regarding the effect of conventional radiotherapy on glioma-related seizures. METHODS In this retrospective study, we analyzed the seizure response and outcome following conventional radiotherapy in a cohort of 43 patients with glioma (33 grade II, 10 grade III) and medically intractable epilepsy. RESULTS At 3 months after radiotherapy, seizure reduction was significant (≥ 50% reduction of frequency compared with baseline) in 31/43 patients (72%) of the whole series and in 25/33 patients (76%) with grade II gliomas, whereas at 12 months seizure reduction was significant in 26/34 (76%) and in 19/25 (76%) patients, respectively. Seizure reduction was observed more often among patients displaying an objective tumor response on MRI, but patients with no change on MRI also had a significant seizure reduction. Seizure freedom (Engel class I) was achieved at 12 months in 32% of all patients and in 38% of patients with grade II tumors. Timing of radiotherapy and duration of seizures prior to radiotherapy were significantly associated with seizure reduction. CONCLUSIONS This study showed that a high proportion of patients with medically intractable epilepsy from diffuse gliomas derive a significant and durable benefit from radiotherapy in terms of epilepsy control and that this positive effect is not strictly associated with tumor shrinkage as shown on MRI. Radiotherapy at tumor progression seems as effective as early radiotherapy after surgery. Prospective studies must confirm and better characterize the response to radiotherapy.

Antiangiogenic therapy of brain tumors: the role of bevacizumab

Abstract Angiogenesis is one of the hallmarks of cancer, including brain tumors. Malignant gliomas have the highest degree of vascular proliferation among solid tumors; thus, angiogenic pathways represent an attractive target to interfere with tumor growth. Up to date VEGF pathway targeting with specific drugs has yielded interesting therapeutics results. In particular bevacizumab, a monoclonal antibody against VEGF-A, has shown clinical activity in malignant gliomas, especially glioblastomas, in terms of a high response rate on MRI and a significant increase in progression-free survival.

Anti-Angiogenic Approaches to Malignant Gliomas

Despite advances in multidisciplinary approaches, the prognosis for most patients with malignant gliomas is poor. Malignant gliomas are highly vascularized tumors with elevated expression of vascular endothelial growth factor (VEGF), an important mediator of angiogenesis. Recent studies of bevacizumab, an anti-VEGF monoclonal antibody, alone or associated with chemotherapy, have demonstrated high response rates and prolongation of median and 6-month progression-free survival. Clinical evaluation of several multitarget small molecule tyrosine kinase inhibitors is ongoing. Other promising antiangiogenic compounds are cilengitide and continuous temozolomide. Toxicity is acceptable. Open issues are represented by patterns of tumor progression, resistance mechanisms and biomarkers.

STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis

The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors.Reactive astrocytes expressing STAT3 support the metastatic growth of tumor cells colonizing the brain and can be pharmacologically targeted to improve the clinical management of patients with secondary brain tumors.

The role of the AR/ER ratio in ER-positive breast cancer patients

The significance of androgen receptor (AR) in breast cancer (BC) management is not fully defined, and it is still ambiguous how the level of AR expression influences oestrogen receptor-positive (ER+) tumours. The aim of the present study was to analyse the prognostic impact of AR/ER ratio, evaluated by immunohistochemistry (IHC), correlating this value with clinical, pathological and molecular characteristics. We retrospectively selected a cohort of 402 ER+BC patients. On each tumour, IHC analyses for AR, ER, PgR, HER2 and Ki67 were performed and AR+ cases were used to calculate the AR/ER value. A cut-off of ≥2 was selected using receiver-operating characteristic (ROC) curve analyses. RNA from 19 cases with AR/ER≥2 was extracted and used for Prosigna-PAM50 assays. Tumours with AR/ER≥2 (6%) showed more frequent metastatic lymph nodes, larger size, higher histological grade and lower PgR levels than cases with AR/ER<2. Multivariate analysis confirmed that patients with AR/ER≥2 had worse disease-free interval (DFI) and disease-specific survival (DSS) (hazard ratios (HR) = 4.96 for DFI and HR = 8.69 for DSS, both P ≤ 0.004). According to the Prosigna-PAM50 assay, 63% (12/19) of these cases resulted in intermediate or high risk of recurrence categories. Additionally, although all samples were positive for ER assessed by IHC, the molecular test assigned 47.4% (9/19) of BCs to intrinsic non-luminal subtypes. In conclusion, the AR/ER ratio ≥2 identifies a subgroup of patients with aggressive biological features and may represent an additional independent marker of worse BC prognosis. Moreover, the Prosigna-PAM50 results indicate that a significant number of cases with AR/ER≥2 could be non-luminal tumours.

Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene

Background:Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies.Methods:We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs.Results:By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12).Conclusions:mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.

Caveolin 1 expression favors tumor growth and is associated with poor survival in primary lung adenocarcinomas

Despite the consolidated clinico-pathological correlates of Caveolin 1 expression in non–small cell lung cancer, the available data on the role of Caveolin 1 in relation to proliferation, migration, and metastasis in lung adenocarcinoma cells is still scant. Here, we aimed to confirm whether Caveolin 1 may act as a promoter of cell growth in human lung adenocarcinoma using in vitro and in vivo models, supported by a survival analysis of Caveolin 1 expression in a series of 116 primary lung adenocarcinomas. The silencing of endogenous Caveolin 1 expression in H522 lung adenocarcinoma cells through stable shRNA transfection significantly inhibited cellular proliferation in vitro and in vivo, in a lung adenocarcinoma xenograft mouse model. The bioluminescence imaging analysis revealed that tumors derived from Caveolin 1 shRNA-transfected cells grew slower than control xenografts. However, this difference progressively diminished over time and was definitively lost after 21 days. This was consistent with a progressive Caveolin 1 re-expression, which started at day 7. The association between the restored expression of Caveolin 1 and the restart of tumor growth in vivo supports the booster role of Caveolin 1 in lung adenocarcinoma progression. To further confirm this role, Caveolin 1 expression was assessed by immunohistochemistry in a series of 116 human lung adenocarcinomas. Positive Caveolin 1 tumors accounted for 20% of cases and were associated with a significantly worse overall survival compared to Caveolin 1-negative cancers. Taken together, these data highlight that Caveolin 1 expression confers a proliferative advantage in lung adenocarcinoma cells, thus fostering increased tumor aggressiveness.

Ki-67 proliferation index but not mitotic thresholds integrates the molecular prognostic stratification of lower grade gliomas

Despite several molecular signatures for “lower grade diffuse gliomas” (LGG) have been identified, WHO grade still remains a cornerstone of treatment guidelines. Mitotic count bears a crucial role in its definition, although limited by the poor reproducibility of standard Hematoxylin & Eosin (H&E) evaluation. Phospho-histone-H3 (PHH3) and Ki-67 have been proposed as alternative assays of cellular proliferation. Therefore in the present series of 141 LGG, the molecular characterization (namely IDH status, 1p/19q co-deletion and MGMT promoter methylation) was integrated with the tumor “proliferative trait” (conventional H&E or PHH3-guided mitotic count and Ki-67 index) in term of prognosis definition. Exclusively high PHH3 and Ki-67 values were predictor of poor prognosis (log rank test, P = 0.0281 for PHH3 and P = 0.032 for Ki-67), unlike standard mitotic count. Based on Cox proportional hazard regression analyses, among all clinical (age), pathological (PHH3 and Ki-67) and molecular variables (IDH, 1p/19q codeletion and MGMT methylation) with a prognostic relevance at univariate survival analysis, only IDH expression (P = 0.001) and Ki-67 proliferation index (P = 0.027) proved to be independent prognostic factors. In addition, stratifying by IDH expression status, high Ki-67 retained its prognostic relevance uniquely in the IDH negative patient (P = 0.029) doubling their risk of death (hazard ratio = 2.27). Overall, PHH3 immunostaining is the sole reliable method with a prognostic value to highlight mitotic figures in LGG. Ki-67 proliferation index exceeds PHH3 mitotic count as a predictor of patients prognosis, and should be integrated with molecular markers in a comprehensive grading system for LGG.

Pharmacologic Therapies for Malignant Glioma: A Guide for Clinicians

This review covers the medical options for malignant gliomas based on the results of recent clinical trials and updated information on molecular markers of prognostic and predictive value. In addition to alkylating agents, the antiangiogenic drug bevacizumab is increasingly used, particularly in cases of recurrence. Supportive care, including antiedema agents, antiepileptic drugs and anticoagulants, represent complementary treatment approaches of the utmost clinical importance.

Molecular alterations of neuroendocrine tumours of the lung

Neuroendocrine tumours of the lung comprise low [typical carcinoid (TC)], intermediate [atypical carcinoid (AC)] and high‐grade [small‐cell lung cancer (SCLC) and large‐cell neuroendocrine carcinoma (LCNEC)] malignancies, while a pre‐invasive lesion [diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH)] may generate a subset of peripheral carcinoid tumours. These neoplasms are differentiated conventionally based on mitotic rate, presence of necrosis and cytological details, according to the 2015 World Health Organisation (WHO) classification. Clinical data and molecular alterations distinguish carcinoids and high‐grade carcinomas into two separate categories. Previous studies have demonstrated a significantly higher rate of chromosomal aberrations in carcinomas (e.g. 3p and 17p deletions), but restriction of multiple endocrine neoplasia type 1 (MEN1) mutations to carcinoids. High‐grade carcinomas are also characterised by TP53 and RB1 gene inactivation. In this review, a critical analysis of the diagnostic and prognostic role of Ki67 labelling index and a concise discussion of the most relevant findings regarding molecular characterisation of lung neuroendocrine neoplasms are reported. In addition, we illustrate how the development of promising therapeutic strategies based on the identification of molecular targets (mTOR inhibitors in carcinoids and targeting of the Notch ligand DLL3 in SCLC) may require the assessment of predictive biomarkers, even in the group of neuroendocrine tumours of the lung.