Long-term therapy with temozolomide is a feasible option for newly diagnosed glioblastoma: a single-institution experience with as many as 101 temozolomide cycles.

OBJECT: The objective of this study was to report the authors' experience with the long-term administration of temozolomide (TMZ; > 6 cycles, up to 101) in patients with newly diagnosed glioblastoma and to analyze its feasibility and safety as well as its impact on survival. The authors also compared data obtained from the group of patients undergoing long-term TMZ treatment with data from patients treated with a standard TMZ protocol. METHODS: A retrospective analysis was conducted of 37 patients who underwent operations for glioblastoma between 2004 and 2012. Volumetric analysis of postoperative Gd-enhanced MR images, obtained within 48 hours, confirmed tumor gross-total resection (GTR) in all but 2 patients. All patients received the first cycle of TMZ at a dosage of 150 mg/m(2) starting on the second or third postsurgical day. Afterward, patients received concomitant radiochemotherapy according to the Stupp protocol. With regard to adjuvant TMZ therapy, the 19 patients in Group A, aged 30-72 years (mean 56.1 years), received 150 mg/m(2) for 5 days every 28 days for more than 6 cycles (range 7-101 cycles). The 18 patients in Group B, aged 46-82 years (mean 64.8 years), received the same dose, but for no more than 6 cycles. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was analyzed for both groups and correlated with overall survival (OS) and progression-free survival (PFS). The impact of age, sex, Karnofsky Performance Scale score, and Ki 67 staining were also considered. RESULTS: All patients but 1 in Group A survived at least 18 months (range 18-101 months), and patients in Group B survived no more than 17 months (range 2-17 months). The long-term survivors (Group A), defined as patients who survived at least 12 months after diagnosis, were 51.3% of the total (19/37). Kaplan-Meier curve analysis showed that patients treated with more than 6 TMZ cycles had OS and PFS that was significantly longer than patients receiving standard treatment (median OS 28 months vs 8 months, respectively; p = 0.0001; median PFS 20 months vs 4 months, respectively; p = 0.0002). By univariate and multivariate Cox proportional hazard regression analysis, MGMT methylation status and number of TMZ cycles appeared to be survival prognostic factors in patients with glioblastoma. After controlling for MGMT status, highly significant differences related to OS and PFS between patients with standard and long-term TMZ treatment were still detected. Furthermore, in Group A and B, the statistical correlation of MGMT status to the number of TMZ cycles showed a significant difference only in Group A patients, suggesting that MGMT promoter methylation was predictive of response for long-term TMZ treatment. Prolonged therapy did not confer hematological toxicity or opportunistic infections in either patient group. CONCLUSIONS: This study describes the longest experience so far reported with TMZ in patients with newly diagnosed glioblastomas, with as many as 101 cycles, who were treated using GTR. Statistically significant data confirm that median survival correlates with MGMT promoter methylation status as well as with the number of TMZ cycles administered. Long-term TMZ therapy appears feasible and safe.

Bioinformatic analysis of the LCN2-SLC22A17-MMP9 network in cancer: The role of DNA methylation in the modulation of tumor microenvironment.

Several features of cancer cells such as proliferation, invasion, metastatic spreading, and drug resistance are affected by their interaction with several tumor microenvironment (TME) components, including neutrophil gelatinase-associated lipocalin (NGAL), solute carrier family 22 member 17 (SLC22A17), and matrix metallopeptidase 9 (MMP9). These molecules play a key role in tumor growth, invasion, and iron-dependent metabolism of cancer cells. However, the precise epigenetic mechanisms underlying the gene regulation of Lipocalin 2 (LCN2), SLC22A17, and MMP9 in cancer still remain unclear. To this purpose, computational analysis was performed on TCGA and GTEx datasets to evaluate the expression and DNA methylation status of LCN2, SLC22A17, and MMP9 genes in different tumor types. Correlation analysis between gene/isoforms expression and DNA methylation levels of LCN2, SLC22A17, and MMP9 was performed to investigate the role of DNA methylation in the modulation of these genes. Protein network analysis was carried out using reverse phase protein arrays (RPPA) data to identify protein-protein interactions of the LCN2-SLC22A17-MMP9 network. Furthermore, survival analysis was performed according to gene expression and DNA methylation levels. Our results demonstrated that LCN2 and MMP9 were mainly upregulated in most tumor types, whereas SLC22A17 was largely downregulated, representing a specific hallmark signature for all gastrointestinal tumors. Notably, the expression of LCN2, SLC22A17, and MMP9 genes was negatively affected by promoter methylation. Conversely, intragenic hypermethylation was associated with the overexpression of SLC22A17 and MMP9 genes. Protein network analysis highlighted the role of the LCN2-SLC22A17-MMP9 network in TME by the interaction with fibronectin 1 and claudin 7, especially in rectal tumors. Moreover, the impact of expression and methylation status of LCN2, SLC22A17, and MMP9 on overall survival and progression free interval was tumor type-dependent. Overall, our analyses provide a detailed overview of the expression and methylation status of LCN2, SLC22A17, and MMP9 in all TCGA tumors, indicating that the LCN2-SLC22A17-MMP9 network was strictly regulated by DNA methylation within TME. Our findings pave the way for the identification of novel DNA methylation hotspots with diagnostic and prognostic values and suitable for epi-drug targeting.

Liquid Biopsy and Circulating Biomarkers for the Diagnosis of Precancerous and Cancerous Oral Lesions.

Oral cancer is one of the most common malignancies worldwide, accounting for 2% of all cases annually and 1.8% of all cancer deaths. To date, tissue biopsy and histopathological analyses are the gold standard methods for the diagnosis of oral cancers. However, oral cancer is generally diagnosed at advanced stages with a consequent poor 5-year survival (~50%) due to limited screening programs and inefficient physical examination strategies. To address these limitations, liquid biopsy is recently emerging as a novel minimally invasive tool for the early identification of tumors as well as for the evaluation of tumor heterogeneity and prognosis of patients. Several studies have demonstrated that liquid biopsy in oral cancer could be useful for the detection of circulating biomarkers including circulating tumor DNA (ctDNA), microRNAs (miRNAs), proteins, and exosomes, thus improving diagnostic strategies and paving the way to personalized medicine. However, the application of liquid biopsy in oral cancer is still limited and further studies are needed to better clarify its clinical impact. The present manuscript aims to provide an updated overview of the potential use of liquid biopsy as an additional tool for the management of oral lesions by describing the available methodologies and the most promising biomarkers.

Excision Repair Cross Complementation Group 1 Single Nucleotide Polymorphisms and Nivolumab in Advanced Non-Small Cell Lung Cancer.

Background: We hypothesized that non-small cell lung cancer (NSCLC) patients with a tumor positive for single nucleotide polymorphisms (SNPs) of the Excision Repair Cross Complementation Group 1 (ERCC-1) gene could be more genetically instable and consequently more responsive to a programmed cell death-1 (PD-1) blockade. Methods: We evaluated the T19007C and C8092A ERCC-1 SNPs by pyrosequencing assay, on tumor specimens from two independent cohorts of patients who relapsed after one or more prior systemic treatments for advanced NSCLC and who received nivolumab (3 mg/kg intravenously every 2 weeks) as part of the Italian Expanded Access Program. We aimed to assess the outcome of enrolled subjects according to the ERCC-1 SNPs status, to evaluate the role of these polymorphisms as putative biomarkers associated with a response/clinical benefit to anti-PD-1 therapies. Results: Of the 45 patients included in the final analysis, 21 (47%) and 16 (36%) were positive for the T19007C and C8092A polymorphic genotype (PG), respectively. In univariate analyses, overall survival (OS) and progression free survival (PFS) were shorter in patients with the T19007C PG, but neither difference achieved statistical significance (P = 0.131 and P = 0.717, respectively). The presence of the C8092A PG was associated with a longer OS and PFS, although statistical significance was only reached for PFS (P = 0.112 and P = 0.025, respectively). These results were confirmed by multivariate analyses. The response rate was only significantly higher in patients with the C8092A PG vs. wild type ERCC-1 (62 vs. 7%, P < 0.001). Conclusions: Results from this hypothesis generating pilot study, provided suggestive evidence that a subgroup of NSCLC patients could benefit differently from nivolumab according to the C8092A ERCC-1 SNP status. However, these data warrant further investigation.

Genes and pathways differentially expressed in the brains of Fxr2 knockout mice.

Fragile X syndrome is a common inherited form of mental retardation and originates from the absence of expression of the FMR1 gene. This gene and its two homologues, FXR1 and FXR2, encode for a family of fragile X related (FXR) proteins with similar tissue distribution, together with sequence and functional homology. Based on these characteristics, it has been suggested that these proteins might partly complement one another. To unravel the function of Fxr2 protein, the expression pattern of 12,588 genes was studied in the brains of wild-type and Fxr2 knockout mice, an animal model which shows behavioral abnormalities partly similar to those observed in Fmr1-knockout mice. By genome expression profiling and stringent significance tests we identify genes and gene groups de-regulated in the brains of Fxr2 knockout mice. Differential expression of candidate genes was validated by real-time PCR, in situ hybridization, immunohistochemistry and western blot analysis. A number of differentially expressed genes associated with the Fxr2 phenotype have been previously involved in other memory or cognitive disorders.

Portable Intraoperative Computed Tomography Scan in Image-Guided Surgery for Brain High-grade Gliomas: Analysis of Technical Feasibility and Impact on Extent of Tumor Resection.

BACKGROUND: Intraoperative magnetic resonance imaging is the gold standard among image-guided techniques for glioma surgery. Scant data are available on the role of intraoperative computed tomography (i-CT) in high-grade glioma (HGG) surgery. OBJECTIVE: To verify the technical feasibility and usefulness of portable i-CT in image-guided surgical resection of HGGs. METHODS: This is a retrospective series control analysis of prospectively collected data. Twenty-five patients (Group A) with HGGs underwent surgery using i-CT and 5-aminolevulinic acid (5-ALA) fluorescence. A second cohort of 25 patients (Group B) underwent 5-ALA fluorescence-guided surgery but without i-CT. We used a portable 8-slice CT scanner and, in both groups, neuronavigation. Extent of tumor resection (ETOR) and pre- and postoperative Karnofsky performance status (KPS) scores were measured; the impact of i-CT on overall survival (OS) and progression-free survival (PFS) was also analyzed. RESULTS: In 8 patients (32%) in Group A, i-CT revealed residual tumor, and in 4 of them it helped to also resect pathological tissue detached from the main tumor. EOTR in these 8 patients was 97.3% (96%-98.6%). In Group B, residual tumor was found in 6 patients, whose tumor's mean resection was 98% (93.5-99.7). The Student t test did not show statistically significant differences in EOTR in the 2 groups. The KPS score decreased from 67 to 69 after surgery in Group A and from 74 to 77 in Group B (P = .07 according to the Student t test). Groups A and B did not show statistically significant differences in OS and PFS (P = .61 and .46, respectively, by the log-rank test). CONCLUSION: No statistically significant differences in EOTR, KPS, PFS, and OS were observed in the 2 groups. However, i-CT helped to verify EOTR and to update the neuronavigator with real-time images, as well as to identify and resect pathological tissue in multifocal tumors. i-CT is a feasible and effective alternative to intraoperative magnetic resonance imaging. Portable i-CT can provide useful real-time information during brain surgery and can be easily introduced in neurosurgical theaters in daily practice.

Identification of pharmacological targets in amyotrophic lateral sclerosis through genomic analysis of deregulated genes and pathways.

Amyotrophic Lateral Sclerosis (ALS) is a progressive and disabling neurodegenerative disorder characterized by upper and lower motor neuron loss, leading to respiratory insufficiency and death after 3-5 years. Riluzole is currently the only FDA approved drug for ALS, but it has only modest effects on survival. The majority of ALS cases are sporadic and probably associated to a multifactorial etiology. With the completion of genome sequencing in humans and model organisms, together with the advent of DNA microarray technology, the transcriptional cascades and networks underlying neurodegeneration in ALS are being elucidated providing new potential pharmacological targets. The main challenge now is the effective screening of the myriad of targets to identify those with the most therapeutic utility. The present review will illustrate how the identification, prioritization and validation of preclinical therapeutics can be achieved through genomic analysis of critical pathways and networks deregulated in ALS pathology.

Genomic analysis: toward a new approach in breast cancer management.

Breast cancer is a clinically heterogeneous and complex disease that can affect differently individuals with seemingly identical clinicopathologic parameters. This heterogeneity is strictly linked to individuals and tumors genetic variability. Currently, the development of high-throughput technologies are proving novel tools to tackle this complexity. By DNA microarray technology, genomic analysis has been used successfully for breast carcinomas stratification into molecular subgroups with relevant implications for clinical outcomes, and detection of prognostic/treatment predictive signatures. Indeed, DNA microarray has rapidly improved becoming a powerful diagnostic tool. Information derived from these assays allows clinicians to estimate the risk for distant recurrence, and predict accurately which patients are likely to benefit from adjuvant therapy. This review will describe the state-of-the-art of genomic analysis in breast cancer and introduce the clinicians to a genomic approach to cancer management, illustrating how it can help in defying a better diagnosis, prognosis and therapeutic treatment.

Systems biology of apoptosis and survival: implications for drug development.

The advent of "systems biology" has highlighted that any function of a biological system is rarely attributable to a single molecule or a single process. Hence, complex processes, such as apoptosis and survival, depend on the activity of an integrated network of genes and their encoded proteins, which almost never work alone but interact with each other in highly structured and incredibly complex ways. With the completion of genome sequencing in humans and model organisms, and the advent of DNA microarray technology, the transcriptional cascades and gene networks regulating neuronal apoptosis and survival are being elucidated providing new potential pharmacological targets. The emerging challenge is the effective selection of the myriad of targets to identify those with the most therapeutic utility. The present review will illustrate how the identification, prioritization and validation of preclinical therapeutics can be achieved through genomic analysis of critical pathways and networks in neuronal apoptosis and survival.

Gastric inhibitory polypeptide and its receptor are expressed in the central nervous system and support neuronal survival.

The development of neuronal apoptosis depends on an intrinsic transcriptional program. By DNA microarray technology, we have previously implicated a number of genes in different paradigms of neuronal apoptosis. In the present study, we investigated the spatiotemporal pattern of expression of two of these genes, gastric inhibitory polypeptide (Gip) and its receptor (Gipr) in the rat central nervous system. The levels of their transcripts were measured with real-time quantitative polymerase chain reaction and in situ-hybridization. Widespread expression of Gip and Gipr was found in adult rat brain, whereas during postnatal cerebellum development, they were highly expressed in the external and internal granule layer, and in Purkinje cells. To investigate the possible biological function of Gip we examined its effects in vitro. Addition of Gip to cultured cerebellar granule neurons reduced the extent of apoptotic death induced by switching the growing medium from 25 to 5 mM K+. This neurotrophic effect was mimicked by that of PACAP38 and IGF1. We conclude that Gip acts as an endogenous neurotrophic factor and supports neuronal survival.

CXCR4 and CXCL12 immunoreactivities differentiate primary non-small-cell lung cancer with or without brain metastases.

Synchronous or metachronous brain metastases (BMs) occur in about 33% of patients affected by non-small-cell lung cancer (NSCLC). To date, no reliable biological marker is able to identify patients who will develop BMs. In the present study, using a quantitative double-labeling immunofluorescence analysis, we evaluated the expression of chemokine CXCL12 and its receptor, CXCR4, in primary NSCLC histological specimens of patients with and without BMs. The immunoreactivity of CXCL12 and CXCR4 was significantly higher in NSCLC samples of patients with BMs. We performed Receiver Operating Characteristics (ROC) analysis in order to define optimal cut-off values for CXCL12 and CXCR4 immunoreactivity that could discriminate between NSCLC patients without and with BMs. ROC curves showed a good diagnostic accuracy and adequate predictive power for both CXCL12 and CXCR4. These findings suggest a possible role for the CXCL12/CXCR4 axis in the metastatic evolution of NSCLC, and its potential use as prognostic markers and drug targets.

Cutting edge: MHC class I-Ly49 interaction regulates neuronal function.

MHC class I molecules (MHC-I) have been implicated in nervous system development in the mouse. In this study we present evidence for the interaction of MHC-I with the NK cell receptor Ly49 in primary cortical neuronal cultures. We show that MHC-I and Ly49 are expressed on neuronal soma and axon surfaces, with Ly49 also present on dendrites. Anti-MHC-I Abs reduce synapsin-I expression and enhance neurite outgrowth and neuronal death. Conversely, anti-Ly49 mAbs increase synapsin-I expression, reduce neurite outgrowth, and promote neuron viability. Because we show that Ly49 genes are selectively expressed in the adult brain, these findings suggest an unsuspected role for the MHC-I-Ly49 interaction in the development and function of the brain.

A natural antisense transcript against Rad18, specifically expressed in neurons and upregulated during beta-amyloid-induced apoptosis.

Apoptosis, the main form of programmed cell death, is associated to a complex and dynamic transcriptional and post-transcriptional programme. By microarray analysis, we have previously implicated 241 genes differentially expressed in rat cortical neurons exposed to beta-amyloid (Abeta) protein, the major constituent of amyloid plaques in Alzheimer's disease. A large number of identified genes have no name or known function. In the present study, we have investigated one of these genes that encodes for a natural antisense transcript against Rad18 (NAT-Rad18). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) confirmed differential expression of this transcript in cortical neurons exposed to Abeta. In situ hybridization, qRT-PCR and immunohistochemistry were used to assess the regional and cellular distribution of NAT-Rad18 in adult rat brain. These experiments showed a widespread distribution of NAT-Rad18, with the highest levels in the cerebellum, brainstem and cortex, where it was specifically expressed by neurons. NAT-Rad18 was also strongly expressed in epithelial cells of choroid plexus and cerebral vessels. At the cellular level, expression of Rad18 was counterbalanced by that of its natural antisense transcript, as shown by both in situ hybridization and immunohistochemistry. These experiments suggest the existence of a NAT that exerts a post-transcriptional control over Rad18.

Genomic profiling of cortical neurons following exposure to beta-amyloid.

In vitro and in vivo studies have shown that beta-amyloid peptide induces neuronal cell death. To explore the molecular basis underlying beta-amyloid-induced toxicity, we analyzed gene expression profiles of cultured rat cortical neurons treated for 24 and 48 h with synthetic beta-amyloid peptide. From the 8740 genes interrogated by oligonucleotide microarray analysis, 241 genes were found to be differentially expressed and segregated into distinct clusters. Functional clustering based on gene ontologies showed coordinated expression of genes with common biological functions and metabolic pathways. The comparison with genes differentially expressed in cerebellar granule neurons following serum and potassium deprivation indicates the existence of common regulatory mechanisms underlying neuronal cell death. Our results offer a genomic view of the changes that accompany beta-amyloid-induced neurodegeneration.