Luca Morandi

Recurrence Pattern After Temozolomide Concomitant With and Adjuvant to Radiotherapy in Newly Diagnosed Patients With Glioblastoma: Correlation With MGMT Promoter Methylation Status

PURPOSE The aim of the present study was to evaluate factors predicting the recurrence pattern after the administration of temozolomide (TMZ), initially concurrent with radiotherapy (RT) and subsequently as maintenance therapy, which has become standard treatment for patients with newly diagnosed glioblastoma (GBM). PATIENTS AND METHODS Ninety-five patients with newly diagnosed GBM were treated with RT plus TMZ (75 mg/m(2)/d) followed by maintenance TMZ cycles (150 to 200 mg/m(2) for 5 days every 28 days). Assessable MGMT methylation status and magnetic resonance imaging follow-up were mandatory in all cases. RESULTS After a median follow-up of 18.9 months (range, 6.6 to 44.8 months), 79 patients (83%) had recurrence: inside the RT field in 57 patients (72.2%), outside in 17 patients (21.5%), and at RT margin in five patients (6.3%). MGMT status was correlated with the site of recurrence, which occurred inside, or at the margin of, the RT field in 51 patients (85%) with MGMT unmethylated status and in 11 patients (57.9%) with MGMT methylated status (P = .01). Recurrences outside the RT field occurred after a longer time interval than those inside the RT field (14.9 v 9.2 months, P = .02). CONCLUSION After the administration of TMZ concomitant with and adjuvant to RT in patients with GBM, the pattern of, and time to, recurrence are strictly correlated with MGMT methylation status.

O6-methylguanine DNA-methyltransferase methylation status can change between first surgery for newly diagnosed glioblastoma and second surgery for recurrence: clinical implications

O(6)-methylguanine DNA-methyltransferase (MGMT) promoter methylation status is a prognostic factor in newly diagnosed glioblastoma patients. However, it is not yet clear whether, and if so how, MGMT methylation status may change. Moreover, it is unknown whether the prognostic role of this epigenetic feature is retained during the disease course. A retrospective analysis was made using a database of 614 glioblastoma patients treated prospectively from January 2000 to August 2008. We evaluated only patients who met the following inclusion criteria: age > or = 18 years; performance status 0-2; histological diagnosis of glioblastoma at both first and second surgery for recurrence; postoperative treatment consisting of: (i) radiotherapy (RT) followed by adjuvant temozolomide (TMZ) until 2005 and (ii) TMZ concurrent with and adjuvant to RT after 2005; a time interval > or = 3 months between first and second surgery. MGMT status was evaluated at first and second surgery in all 44 patients (M:F 32:12, median age: 49 years, range: 27-67 years). In 38 patients (86.4%), MGMT promoter status was assessable at both first and second surgery. MGMT methylation status, changed in 14 patients (37%) of second surgery samples and more frequently in methylated than in unmethylated patients (61.5% vs 24%, P = .03). The median survival was significantly influenced only by MGMT methylation status determined at first surgery (P = .04). Significant changes in MGMT methylation status during the course of GBM occur more frequently in MGMT methylated than unmethylated cases. MGMT methylation status determined at first surgery appears to be of prognostic value; however, it is not predictive of outcome following second surgery.

Genetic similarities and differences between lobular in situ neoplasia (LN) and invasive lobular carcinoma of the breast

One of the most controversial issues in breast pathology is whether lobular neoplasia (LN) is a risk factor or a precursor lesion of invasive lobular carcinoma (ILC). This is consequent to the fact that no conclusive data on the biology of LN exist. Molecular studies of LN and ILC are scanty, variable, and not consistent. Clonality of 12 cases of LN and ILC present simultaneously in the same block has been studied. Cells from both lesions were obtained by microdissection and were studied for mitochondrial DNA (mtDNA), D-loop sequencing, and neighbor-joining trees. Eight of the same cases were studied with comparative genomic hybridization (CGH) array to have additional data consistent with mtDNA. In all cases, loss of heterozygosity was studied for D16S496,locus 16q22.1 related to e-cadherin. It appears that no fewer than eight cases were genetically very similar (clonal) with mtDNA. Seven of these cases appeared also clonal with CGH array. It is concluded that in the present series, LN and ILC are genetically related lesions in the majority of cases and that LN might be the precursor of ILC.

Expression of p63 is the sole independent marker of aggressiveness in localised (stage I–II) Merkel cell carcinomas

Merkel cell carcinoma of the skin is a malignant neuroendocrine tumour, whose prognostic criteria are a matter of dispute. Specifically, no predictor is presently available in stage I–II tumours. We collected clinical and follow-up data from 70 Merkel cell carcinomas of the skin. The same cases were studied for p63 expression by immunohistochemistry, by reverse-transcription PCR (RT-PCR) and TP63 gene status by FISH and for presence of Merkel cell polyomavirus by PCR. Stage emerged as a significant prognostic parameter (P=0.008). p63 expression, detected in 61% (43/70) of cases by immunohistochemistry, was associated with both decreased overall survival (P<0.0001) and disease-free survival (P<0.0001). Variable expression patterns of the different p63 isoforms were found only in cases immunoreactive for p63. In these latter lesions, at least one of the N-terminal p63 isoforms was detected and TAp63α was the most frequently expressed isoform. TP63 gene amplification was observed by FISH in only one case. Presence of Merkel cell polyomavirus DNA sequences was detected in 86% (60/70) of Merkel cell carcinomas and did not emerge as a significant prognostic parameter. Merkel cell carcinoma cases at low stage (stage I-II) represented over half (40/70 cases, 57%) of cases, and the clinical course was uneventful in 25 of 40 cases while 15 cases died of tumour (10/40 cases) within 34 months or were alive with disease (5/40 cases) within 20 months. Interestingly, a very strict correlation was found between evolution and p63 expression (P<0.0001). The present data indicate that p63 expression is associated with a worse prognosis in patients with Merkel cell carcinoma, and in localised tumours it represents the single independent predictor of clinical evolution.

Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.

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.

Molecular Diagnosis in Ewing Family Tumors: The Rizzoli Experience—222 Consecutive Cases in Four Years

The Ewings family of tumors (EFTs) are characterized by chimeric transcripts generated by specific chromosomal rearrangements. The most common fusions are between the EWSR1 gene on chromosome 22 and the ETS family of transcription factors; rarely, FUS (on chromosome 16) substitutes for EWSR1. The detection of specific translocations using molecular analysis is now a routine part of the pathological examination of EFT. Here, we report our experience with molecular diagnosis of EFT during the 4 years (2006-2009) at the Rizzoli Institute. We analyzed 222 consecutive tumors with a presumptive diagnosis of EFT using molecular techniques and IHC. We found five distinct types of EWSR1-FLI1 fusion transcripts resulting from translocation t(11;22), three types of EWSR1-ERG transcripts resulting from t(21;22), and one type of t(2;22) resulting in EWSR1-FEV fusion. Molecular investigation validated 92% of cases ultimately diagnosed as EFT; IHC validated 76% of the cases. Thus, despite the difficulties and limitations associated with both molecular and IHC analysis on fresh and formalin-fixed, paraffin-embedded tissue, a combination of these techniques is the best approach to enhancing the accuracy of EFT diagnosis. We also present our method for choosing which molecular techniques to apply. Finally, we collected the most prevalent breakpoints reported in the literature, indicating which exons are involved, the sequence breakpoints, and the NCBI reference sequences.

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.

Somatic complex I disruptive mitochondrial DNA mutations are modifiers of tumorigenesis that correlate with low genomic instability in pituitary adenomas

Mitochondrial DNA (mtDNA) mutations leading to the disruption of respiratory complex I (CI) have been shown to exhibit anti-tumorigenic effects, at variance with those impairing only the function but not the assembly of the complex, which appear to contribute positively to cancer development. Owing to the challenges in the analysis of the multi-copy mitochondrial genome, it is yet to be determined whether tumour-associated mtDNA lesions occur as somatic modifying factors or as germ-line predisposing elements. Here we investigated the whole mitochondrial genome sequence of 20 pituitary adenomas with oncocytic phenotype and identified pathogenic and/or novel mtDNA mutations in 60% of the cases. Using highly sensitive techniques, namely fluorescent PCR and allele-specific locked nucleic acid quantitative PCR, we identified the most likely somatic nature of these mutations in our sample set, since none of the mutations was detected in the corresponding blood tissue of the patients analysed. Furthermore, we have subjected a series of 48 pituitary adenomas to a high-resolution array comparative genomic hybridization analysis, which revealed that CI disruptive mutations, and the oncocytic phenotype, significantly correlate with low number of chromosomal aberrations in the nuclear genome. We conclude that CI disruptive mutations in pituitary adenomas are somatic modifiers of tumorigenesis most likely contributing not only to the development of oncocytic change, but also to a less aggressive tumour phenotype, as indicated by a stable karyotype.

N − 3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure

In cardiac cells the effects of n − 3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n − 3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n − 3 PUFAs is related to a direct modulation of genes in cardiac cells.