Annalisa Pession

MGMT Promoter Methylation Status Can Predict the Incidence and Outcome of Pseudoprogression After Concomitant Radiochemotherapy in Newly Diagnosed Glioblastoma Patients

PURPOSE Standard therapy for glioblastoma (GBM) is temozolomide (TMZ) administration, initially concurrent with radiotherapy (RT), and subsequently as maintenance therapy. The radiologic images obtained in this setting can be difficult to interpret since they may show radiation-induced pseudoprogression (psPD) rather than disease progression. METHODS Patients with histologically confirmed GBM underwent radiotherapy plus continuous daily temozolomide (75 mg/m(2)/d), followed by 12 maintenance temozolomide cycles (150 to 200 mg/m(2) for 5 days every 28 days) if magnetic resonance imaging (MRI) showed no enhancement suggesting a tumor; otherwise, chemotherapy was delivered until complete response or unequivocal progression. The first MRI scan was performed 1 month after completing combined chemoradiotherapy. RESULTS In 103 patients (mean age, 52 years [range 20 to 73 years]), total resection, subtotal resection, and biopsy were obtained in 51, 51, and 1 cases, respectively. MGMT promoter was methylated in 36 patients (35%) and unmethylated in 67 patients (65%). Lesion enlargement, evidenced at the first MRI scan in 50 of 103 patients, was subsequently classified as psPD in 32 patients and early disease progression in 18 patients. PsPD was recorded in 21 (91%) of 23 methylated MGMT promoter and 11 (41%) of 27 unmethylated MGMT promoter (P = .0002) patients. MGMT status (P = .001) and psPD detection (P = .045) significantly influenced survival. CONCLUSION PsPD has a clinical impact on chemotherapy-treated GBM, as it may express the glioma killing effects of treatment and is significantly correlated with MGMT status. Improvement in the early recognition of psPD patterns and knowledge of mechanisms underlying this phenomenon are crucial to eliminating biases in evaluating the results of clinical trials and guaranteeing effective treatment.

Gefitinib in patients with progressive high-grade gliomas: a multicentre phase II study by Gruppo Italiano Cooperativo di Neuro-Oncologia (GICNO)

To investigate the role of gefitinib in patients with high-grade gliomas (HGGs), a phase II trial (1839IL/0116) was conducted in patients with disease recurrence following surgery plus radiotherapy and first-line chemotherapy. Adult patients with histologically confirmed recurrent HGGs following surgery, radiotherapy and first-line chemotherapy, were considered eligible. Patients were treated with gefitinib (250 mgday−1) continuously until disease progression. The primary end point was progression-free survival at 6 months progression-free survival at 6 months (PFS-6). Tissue biomarkers (epidermal growth factor receptor (EGFR) gene status and expression, phosphorylated Akt (p-Akt) expression) were assessed. Twenty-eight patients (median age, 55 years; median ECOG performance status, 1) were enrolled; all were evaluable for drug activity and safety. Sixteen patients had glioblastoma, three patients had anaplastic oligodendrogliomas and nine patients had anaplastic astrocytoma. Five patients (17.9%, 95% CI 6.1–36.9%) showed disease stabilisation. The overall median time to progression was 8.4 (range 2–104+) weeks and PFS-6 was 14.3% (95% CI 4.0–32.7%). The median overall survival was 24.6 weeks (range 4–104+). No grade 3–4 gefitinib-related toxicity was found. Gefitinib showed limited activity in patients affected by HGGs. Epidermal growth factor receptor expression or gene status, and p-Akt expression do not seem to predict activity of this drug.

dMyc Functions Downstream of Yorkie to Promote the Supercompetitive Behavior of Hippo Pathway Mutant Cells

Genetic analyses in Drosophila epithelia have suggested that the phenomenon of “cell competition” could participate in organ homeostasis. It has been speculated that competition between different cell populations within a growing organ might play a role as either tumor promoter or tumor suppressor, depending on the cellular context. The evolutionarily conserved Hippo (Hpo) signaling pathway regulates organ size and prevents hyperplastic disease from flies to humans by restricting the activity of the transcriptional cofactor Yorkie (yki). Recent data indicate also that mutations in several Hpo pathway members provide cells with a competitive advantage by unknown mechanisms. Here we provide insight into the mechanism by which the Hpo pathway is linked to cell competition, by identifying dMyc as a target gene of the Hpo pathway, transcriptionally upregulated by the activity of Yki with different binding partners. We show that the cell-autonomous upregulation of dMyc is required for the supercompetitive behavior of Yki-expressing cells and Hpo pathway mutant cells, whereas the relative levels of dMyc between Hpo pathway mutant cells and wild-type neighboring cells are critical for determining whether cell competition promotes a tumor-suppressing or tumor-inducing behavior. All together, these data provide a paradigmatic example of cooperation between tumor suppressor genes and oncogenes in tumorigenesis and suggest a dual role for cell competition during tumor progression depending on the output of the genetic interactions occurring between confronted cells.

The lethal giant larvae tumour suppressor mutation requires dMyc oncoprotein to promote clonal malignancy

BackgroundNeoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues.ResultsWe observed that lgl mutant cells growing in wild-type imaginal wing discs show poor viability and are eliminated by Jun N-terminal Kinase (JNK)-dependent cell death. Furthermore, they express very low levels of dMyc oncoprotein compared with those found in the surrounding normal tissue. Evidence that this is a cause of lgl mutant cells elimination was obtained by increasing dMyc levels in lgl mutant clones: their overgrowth potential was indeed re-established, with mutant cells overwhelming the neighbouring tissue and forming tumourous masses displaying several cancer hallmarks. Moreover, when lgl mutant clones were induced in backgrounds of slow-dividing cells, they upregulated dMyc, lost apical-basal cell polarity and were able to overgrow. Those phenotypes were abolished by reducing dMyc levels in the mutant clones, thereby confirming its key role in lgl-induced tumourigenesis. Furthermore, we show that the eiger-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating lgl mutant cells in the wing pouch; lgl-/- clonal death in this region is instead driven mainly by dMyc-induced Cell Competition.ConclusionsOur results provide the first evidence that dMyc oncoprotein is required in lgl tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside versus outside the mutant clones plays a key role in driving neoplastic overgrowth.

Gene expression profiling in glioblastoma and immunohistochemical evaluation of IGFBP-2 and CDC20.

Thirty-nine glial tumours (28 glioblastomas (GB) and 11 low-grade gliomas) were investigated with DNA microarrays to reveal a possible specific gene expression profile. Unsupervised classification through hierarchical cluster analysis identified two groups of tumours, the first composed of low-grade gliomas and the second mainly composed of GB. Nine genes were identified as most informative: seven were over-expressed in low-grade gliomas and under-expressed in GB; on the contrary, two genes, insulin-like growth factor binding protein 2 (IGFBP-2) and cell division cycle 20 homologue (CDC20), were over-expressed in GB and under-expressed in low-grade tumours. This same genetic profile was confirmed by reverse transcriptase polymerase chain reaction. Immunohistochemistry for IGFBP-2 was positive in 88.8% of the cases of GB and in only one low-grade glioma, whilst CDC20 immunostained 74.1% of the cases of GB and none low-grade glioma. This was confirmed in an additional series of cases studied with immunohistochemistry only. In conclusion, over-expression of mRNA levels of IGFBP-2 and CDC20 is highly related to GB, IGFBP-2 and CDC-20 gene and protein expressions are strongly correlated, and IGFBP-2 and CDC20 immunopositivity can be useful for the identification of GB in small biopsies.

Promoter methylation analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR using an imprinted gene ( SNURF ) as a reference

BackgroundEpigenetic silencing of the MGMT gene by promoter methylation is associated with loss of MGMT expression, diminished DNA-repair activity and longer overall survival in patients with glioblastoma who, in addition to radiotherapy, received alkylating chemotherapy with carmustine or temozolomide. We describe and validate a rapid methylation sensitive quantitative PCR assay (MS-qLNAPCR) using Locked Nucleic Acid (LNA) modified primers and an imprinted gene as a reference.MethodsAn analysis was made of a database of 159 GBM patients followed between April 2004 and October 2008. After bisulfite treatment, methylated and unmethylated CpGs were recognized by LNA primers and molecular beacon probes. The SNURF promoter of an imprinted gene mapped on 15q12, was used as a reference. This approach was used because imprinted genes have a balanced copy number of methylated and unmethylated alleles, and this feature allows an easy and a precise normalization.ResultsConcordance between already described nested MS-PCR and MS-qLNAPCR was found in 158 of 159 samples (99.4%). The MS-qLNAPCR assay showed a PCR efficiency of 102% and a sensitivity of 0.01% for LNA modified primers, while unmodified primers revealed lower efficiency (69%) and lower sensitivity (0.1%). MGMT promoter was found to be methylated using MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival was of 24 months, being 20 months and 36 months, in patients with MGMT unmethylated and methylated, respectively. Considering MGMT methylation data provided by MS-qLNAPCR as a binary variable, overall survival was different between patients with GBM samples harboring MGMT promoter unmethylated and other patients with any percentage of MGMT methylation (p = 0.003). This difference was retained using other cut off values for MGMT methylation rate (i.e. 10% and 20% of methylated allele), while the difference was lost when 50% of MGMT methylated allele was used as cut-off.ConclusionsWe report and clinically validate an accurate, robust, and cost effective MS-qLNAPCR protocol for the detection and quantification of methylated MGMT alleles in GBM samples. Using MS-qLNAPCR we demonstrate that even low levels of MGMT promoter methylation have to be taken into account to predict response to temozolomide-chemotherapy.

Design and synthesis of novel 3,4-disubstituted pyrazoles for nanomedicine applications against malignant gliomas

A series of novel 3,4-disubstituted pyrazoles were synthesized. The cytotoxicity against U87MG glioma cell line have been investigated in vitro and three of these compounds showed promising inhibitory activity on cell growth with an IC50 lower than 90 microM. AutoDock molecular docking into type I TGF-beta receptor (TGF-beta-RI; PDB: 1py5) has been done for lead optimization of the mentioned compounds as potential TGF-beta-RI1 inhibitors. In particular, 3-aryl-4-amido pyrazole containing long omega-amino-aliphatic chain emerged as a good candidate for further optimization. Entrapment into targetable PEG-based micelles improved growth inhibition IC50 values up to 100 nM and this could lead to a novel drug delivery strategy for treating glioblastoma.

Drosophila Lethal Giant Larvae Neoplastic Mutant as a Genetic Tool for Cancer Modeling

Drosophila lethal giant larvae (lgl) is a tumour suppressor gene whose function in establishing apical-basal cell polarity as well as in exerting proliferation control in epithelial tissues is conserved between flies and mammals. Individuals bearing lgl null mutations show a gradual loss of tissue architecture and an extended larval life in which cell proliferation never ceases and no differentiation occurs, resulting in prepupal lethality. When tissues from those individuals are transplanted into adult normal recipients, a subset of cells, possibly the cancer stem units, are again able to proliferate and give rise to metastases which migrate to distant sites killing the host. This phenotype closely resembles that of mammalian epithelial cancers, in which loss of cell polarity is one of the hallmarks of a malignant, metastatic behaviour associated with poor prognosis. Lgl protein shares with its human counterpart Human giant larvae-1 (Hugl-1) significant stretches of sequence similarity that we demonstrated to translate into a complete functional conservation, pointing out a role in cell proliferation control and tumorigenesis also for the human homologue. The functional conservation and the power of fly genetics, that allows the researcher to manipulate the fly genome at a level of precision that exceeds that of any other multicellular genetic system, make this Drosophila mutant a very suitable model in which to investigate the mechanisms underlying epithelial tumour formation, progression and metastatisation. In this review, we will summarise the results obtained in these later years using this model for the study of cancer biology. Moreover, we will discuss how recent advances in developmental genetics techniques have succeeded in enhancing the similarities between fly and human tumorigenesis, giving Drosophila a pivotal role in the study of such a complex genetic disease.

Amyotrophic lateral sclerosis with mutation of the Cu/Zn superoxide dismutase gene (SOD1) in a patient with Down syndrome

Familial amyotrophic lateral sclerosis can be related to mutations in the Cu/Zn superoxide dismutase gene (SOD1) located on chromosome 21q22.1. This is the first report of a SOD1 mutation in a patient with Down syndrome. A 34-year-old woman with Down syndrome developed a lower motor neuron disease that led to death in two years. Autopsy findings confirmed the diagnosis. DNA examination identified a missense mutation at nucleotide 134 of exon 5 of the SOD1 gene resulting in the aminoacid substitution serine-134-asparagine (S134N). A real time PCR detected the mutation in two out of three alleles. The 70-year-old mother of the patient carries the same mutation but has not yet developed the disease. The missense mutation of SOD1 gene in two of the three alleles could have increased its toxic effects in the Down syndrome patient leading to an earlier onset and rapid progression of the disease.

A novel T137A SOD1 mutation in an Italian family with two subjects affected by amyotrophic lateral sclerosis

Abstract Mutations in the superoxide dismutase-1 (SOD1) gene occur in some forms of familial amyotrophic lateral sclerosis (ALS). To date about 150 mutations are known to involve this gene. Here we describe a novel missense mutation in exon 5 of the SOD1 gene in an Italian family with two members affected by ALS. Sequencing of the SOD1 gene was performed on 11 members of the family and 75 healthy controls. Electron microscopy was also performed on one ALS patient. We identified a heterozygous mutation in codon 137 leading to substitution of threonine by alanine. Further studies are needed to clarify the role of this alteration in ALS aetiopathogensis; nevertheless, T137A seems to represent a new missense mutation of the SOD1 gene in ALS patients.