Evidence for the efficacy of disulfiram and copper combination in glioblastoma multiforme - A propos of a case.

Glioblastoma multiforme (GBM) is the most common and aggressive malignancy of the central nervous system. Treatment usually involves a combination of surgical resection, chemotherapy, and radiotherapy, but ultimately this condition is incurable. Besides the dismal prognosis of GBM, financial factors have also presented challenges for advancing treatments. Taking into consideration the high cost of developing new anticancer drugs as well as the fact that GBM is a rare disease, thus further limiting financial incentive for drug development, it becomes obvious that there has been growing interest for repurposing candidates. One of the most promising drugs to repurpose for treating GBM is disulfiram (DSF). DSF is a relatively nontoxic drug used for more than sixty years in the treatment of chronic alcoholism with the ability to readily cross the blood-brain barrier. Repurposing DSF for use as an anticancer drug in general has recently become of interest because of its preclinically described anticancer effects against various human cancers. Interestingly, a number of these effects were shown to be copper (Cu)-dependent. The purpose of this paper was to review the existing literature surrounding preclinical and clinical data on the effects of DSF -alone or in combination with Cu- in GBM. In addition, we present the first case of a GBM patient safely treated with DSF/Cu combination along with standard therapy exhibiting remarkably increased progression-free (PFS) and overall survival (OS).

The evolution of comprehensive genetic analysis in neurology: Implications for precision medicine.

Technological advancements have facilitated the availability of reliable and thorough genetic analysis in many medical fields, including neurology. In this review, we focus on the importance of selecting the appropriate genetic test to aid in the accurate identification of disease utilizing currently employed technologies for analyzing monogenic neurological disorders. Moreover, the applicability of comprehensive analysis via NGS for various genetically heterogeneous neurological disorders is reviewed, revealing its efficiency in clarifying a frequently cloudy diagnostic picture and delivering a conclusive and solid diagnosis that is essential for the proper management of the patient. The feasibility and effectiveness of medical genetics in neurology require interdisciplinary cooperation among several medical specialties and geneticists, to select and perform the most relevant test according to each patient's medical history, using the most appropriate technological tools. The prerequisites for a comprehensive genetic analysis are discussed, highlighting the utility of appropriate gene selection, variant annotation, and classification. Moreover, genetic counseling and interdisciplinary collaboration could improve diagnostic yield further. Additionally, a sub-analysis is conducted on the 1,502,769 variation records with submitted interpretations in the Clinical Variation (ClinVar) database, with a focus on neurology-related genes, to clarify the value of suitable variant categorization. Finally, we review the current applications of genetic analysis in the diagnosis and personalized management of neurological patients and the advances in the research and scientific knowledge of hereditary neurological disorders that are evolving the utility of genetic analysis towards the individualization of the treatment strategy.

RediScore: Prospective validation of a pipeline for homologous recombination deficiency analysis.

Tumors harboring homologous recombination deficiency (HRD) are considered optimal candidates for poly(ADP-ribose) polymerase 1 (PARP) inhibitor treatment. Such deficiency can be detected by analyzing breast cancer type (BRCA)1/2 gene mutations, as well as mutations in other genes of the homologous recombination pathway. The algorithmic measurement of the HRD effect by identifying genomic instability (GI) has been used as biomarker. As compared with the direct measurement of somatic gene alterations, this approach increases the number of patients who could benefit from PARP inhibitor treatment. In the present study, the performance of the Oncoscan CNV assay, accompanied by appropriate bioinformatic algorithms, was evaluated for its performance in GI calculation and was compared with that of a validated next-generation sequencing (NGS) test (myChoice HRD test). In addition, the clinical utility of the GI score (GIS) and BRCA1/2 tumor analysis were investigated in a cohort of 444 patients with ovarian cancer. For that reason, single nucleotide polymorphism (SNP) arrays and appropriate bioinformatics algorithms were used to calculate GIS in 29 patients with ovarian cancer with known GIS status using a validated NGS test. Furthermore, BRCA1/2 analysis results were compared between the aforementioned assay and the amplicon-based Oncomine™ BRCA Research Assay. BRCA1/2 analysis was performed in 444 patients with ovarian cancer, while GIS was calculated in 175 BRCA1/2-negative cases. The bioinformatics algorithm developed for GIS calculation in combination with NGS BRCA1/2 analysis (RediScore), and the OncoscanR pipeline exhibited a high overall agreement with the validated test (93.1%). In addition, the Oncomine NGS assay had a 100% agreement with the validated test. The BRCA1/2 mutation frequency was 26.5% in the examined patients with ovarian cancer. GIS was positive in 40% of the BRCA1/2-negative cases. The RediScore bioinformatics algorithm developed for GIS calculation in combination with NGS BRCA1/2 analysis is a viable and effective approach for HRD calculation in patients with ovarian cancer, offering a positive prediction for PARP inhibitor responsiveness in 55% of the patients.

Different intrafamilial clinical presentation of FMF mutation carriers.

Familial Mediterranean fever (FMF) is a heterogeneous disorder; at present, it is diagnosed using only genetic methods. In the current study, we performed molecular analysis in two families presenting with FMF. In the first family, we report two brothers with a common genotype (M694V/V726A) but with different clinical presentation. In the second family, we identified the M694V and K695R mutations in a presymptomatic carrier.

Heterogeneous molecular mechanisms underlie attenuated familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis is a phenotypically heterogeneous disease predisposing to colorectal cancer. It is dominantly transmitted, when associated with the APC gene, and recessively inherited, when associated with MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and Greek patients with attenuated polyposis, a phenotypic variant whose genetic cause remains unknown in many cases. METHODS: We studied 26 unrelated patients (and 16 relatives) with multiple colorectal adenomas (3-100, by endoscopic analysis) that had screened APC mutation-negative by protein truncation test. We searched for APC rearrangements by multiplex ligation-dependent probe amplification and for MUTYH mutations by sequencing. We performed a screening of five MUTYH recurrent pathogenic mutations in 501 Italian and 144 Greek controls. RESULTS: One patient proved to carry an APC whole-gene deletion; 4 of 25 (16%) patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1, APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy subjects showed that only Y165C and G382D reach a subpolymorphic frequency. CONCLUSION: Attenuated polyposis patients without "conventional" APC mutations are genetically heterogeneous, and the phenotype is not directly related to the germline defect. Therefore, the families' appropriate management requires an accurate genetic and clinical investigation.

Cell-free DNA and RNA in plasma as a new molecular marker for prostate and breast cancer.

In this study, we examined several molecular markers in prostate and breast cancer patients and in normal individuals. The markers tested were: variations in the quantity of plasma DNA, glutathione-S-transferase P1 gene (GSTP1), Ras association domain family 1A (RASSF1A), and ataxia telangiectasia mutated (ATM) methylation status in plasma, carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSMA) mRNA in peripheral blood mononuclear cells (PBMC) and plasma samples from prostate cancer patients. DNA quantification in plasma was performed using real-time PCR (RT-PCR). We assessed the methylation status of GSTP1 in plasma DNA using methylation-specific PCR (MSP) assay, while the methylation status of RASSF1A and ATM genes was examined by the MethyLight technology. RT-PCR analysis was used for the detection of mRNA, PSMA, and CEA. In 58.3% of newly diagnosed prostate cancer patients and 26.7% of prostate cancer patients under therapy, plasma DNA levels were increased. Additionally, 48.5% of breast cancer patients showed plasma DNA levels above the cutoff limit. GSTP1 Promotor hypermethylation was detectable in 75% of plasma samples obtained from patients with newly diagnosed prostate cancer and in 36.8% of patients under therapy, whereas 26% and 14% of the breast cancer patients tested were positive for RASSF1A and ATM methylation, respectively. The combination of DNA load and promotor methylation status identified 88% of prostate cancer patients and 54% of breast cancer patients. This study shows that free-circulating DNA can be detected in cancer patients compared with disease-free individuals, and suggests a new, noninvasive approach for early detection of cancer.

Molecular and morphological characterization of Aedes albopictus in northwestern Greece and differentiation from Aedes cretinus and Aedes aegypti.

The presence of Aedes albopictus (Skuse) was recently confirmed for the first time in northwestern Greece. This location is within the distribution range of a morphologically similar species, Aedes cretinus Edwards, and is a potentially favorable region for the reintroduction of Aedes aegypti (L.). It was thus compelling to use methods in addition to morphology-based keys to correctly identify specimens badly damaged, rubbed, or otherwise altered in their external characteristics. It was decided to use molecular techniques as a novel and reliable method for differentiating the three Stegomyia species. The nuclear internal transcribed spacer 2 (ITS2) fragments from morphologically identified Ae. albopictus and Ae. cretinus specimens were amplified, and their sequences were compared with those in GenBank for Ae. albopictus, Ae. cretinus, and Ae. aegypti. Also, mitochondrial cytochrome oxidase I (COI) fragments were amplified for Ae. albopictus and Ae. cretinus (so far not available in GenBank) and compared with Ae. aegypti fragments. ITS2 and COI sequences generated in our study were deposited in GenBank and could be useful in future studies of mosquitoes by other research workers.

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients.

BACKGROUND: Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions/deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. METHODS: In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation - dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction). RESULTS: A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. CONCLUSION: Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations.

Efficient testing of the RET gene by DHPLC analysis for MEN 2 syndrome in a cohort of patients.

BACKGROUND: Multiple Endocrine Neoplasia type 2 (MEN 2) is an autosomal dominant inherited syndrome characterized by a strong predisposition for developing endocrine tumors. MEN 2 is caused by germline mutations in the ret proto-oncogene. We investigated the feasibility of using the DHPLC technique in mutation detection of the ret gene in members of MTC families. We compared DHPLC analysis with direct sequencing with regard to sensitivity, reliability, cost and time. MATERIALS AND METHODS: Exons 10 and 11 were amplified with PCR from forty-three samples in seventeen unrelated Greek families and were analyzed for mutations by DHPLC and DNA sequencing. RESULTS: Eight PCR amplicons showed a distinct non-wild-type DHPLC profile. Sequence analysis confirmed different nucleotide variations: six of them were localized in exon 10 and two in exon 11. Mutations were detected in five out of seventeen families tested (29%). CONCLUSION: None of the alterations detected by direct sequencing was missed by DHPLC. We conclude that DHPLC is a fast, sensitive, cost-efficient and reliable method for the scanning of ret germline mutations.

Prevalence of human herpesvirus 8 DNA sequences in human immunodeficiency virus-negative individuals without Kaposi's sarcoma in Greece.

BACKGROUND: It has already been established that the prevalence of human herpes virus 8 (HHV8) in the general population varies among different geographical areas. The objective of this study was to evaluate the prevalence of HHV8 infection in the Greek population. MATERIALS AND METHODS: Eight hundred blood samples were collected consecutively from human immunodeficiency virus (HIV)-negative individuals without evidence of Kaposi's sarcoma (KS). All individuals were Greeks and were classified according to gender, age and geographic origin. HHV8 DNA sequences were detected by nested-PCR. RESULTS: An overall HHV8 positivity rate of 9.6% was found. Analysis of the KS330 region within ORF-26 revealed that the HHV8 strains were distributed to the C1, C3 or A1 subtypes. Logistic regression showed no association between HHV8 presence and geographic regions in Greece. The results indicate that very few individuals (4.3%) were exposed to HHV8 before 15 years of age. The infection rate peaked (16.4%) between the ages of 31 and 40. Females and males showed similar prevalence of HHV8. CONCLUSION: The data suggest that HHV8 is spread in Greece, but to a lesser extent than that observed in other Mediterranean countries. The fact that HHV8 was also found in individuals under 15 years of age indicates that a small percent of HHV8 transmission could occur through non-sexual contacts.

Characterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 predispose to breast and ovarian cancer. A multitude of mutations have been described and are found to be scattered throughout these two large genes. We describe analysis of BRCA1 in 25 individuals from 18 families from a Greek cohort. METHODS: The approach used is based on dHPLC mutation screening of the BRCA1 gene, followed by sequencing of fragments suspected to carry a mutation including intron--exon boundaries. In patients with a strong family history but for whom no mutations were detected, analysis was extended to exons 10 and 11 of the BRCA2 gene, followed by MLPA analysis for screening for large genomic rearrangements. RESULTS: A pathogenic mutation in BRCA1 was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. Single base putative pathogenic mutations were identified by dHPLC and confirmed by sequence analysis in 4 families: 5382insC (in two families), G1738R, and 5586G > A (in one family each). In addition, 18 unclassified variants and silent polymorphisms were detected including a novel silent polymorphism in exon 11 of the BRCA1 gene. Finally, MLPA revealed deletion of exon 20 of the BRCA1 gene in one family, a deletion that encompasses 3.2 kb of the gene starting 21 bases into exon 20 and extending 3.2 kb into intron 20 and leads to skipping of the entire exon 20. The 3' breakpoint lies within an AluSp repeat but there are no recognizable repeat motifs at the 5' breakpoint implicating a mechanism different to Alu-mediated recombination, responsible for the majority of rearrangements in the BRCA1 gene. CONCLUSIONS: We conclude that a combination of techniques capable of detecting both single base mutations and small insertions/deletions and large genomic rearrangements is necessary in order to accurately analyze the BRCA1 gene in patients at high risk of carrying a germline mutation as determined by their family history. Furthermore, our results suggest that in those families with strong evidence of linkage to the BRCA1 locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.

Cell-free DNA and RNA in plasma as a new molecular marker for prostate cancer.

Extracellular nucleic acids could serve as molecular markers in the early detection of cancer and in the prediction of disease outcome. In this study we examined six molecular markers, such as: variations in the quantity of DNA in plasma, glutathione-S-transferase P1 (GSTP1) gene methylation status in plasma, carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSMA) mRNA in peripheral blood mononuclear cells (PBMC), and plasma samples from prostate cancer patients in different stages. The combination of DNA load and GSTP1 promoter methylation status identified 83% (10/12) of the prostate cancer patients before therapy. This study shows that free circulating DNA can be detected in patients with prostate cancer compared with disease-free individuals, and suggests a new, noninvasive approach for early detection of prostate cancer.

The 500-base-pair fragment of the putative gene RvD1-Rv2031c is also present in the genome of Mycobacterium tuberculosis.

BACKGROUND: It has been proposed that differentiation between M. bovis and M. tuberculosis is possible by using a PCR assay for the 500bp fragment present only in the M. bovis genome. MATERIALS AND METHODS: Forty clinical samples and 16 clinical isolates from the Department of Microbiology, as well as 4 clinical isolates obtained from another laboratory, were tested for the purpose of this study. As controls we tested 2 M. bovis (M. bovis BCG Pasteur TMC1011 and M. bovis BCG Copenhagen), 1 H37Rv M. tuberculosis strain, 2 M. avium (ATCC15765 and ATCC1975, respectively) and 1 M. paratuberculosis (ATCC19698) strains. RESULTS: None of the mtp40- negative clinical isolates amplified the 500bp fragment, whereas 4 out of 17 mtp40-positive clinical isolates scored positive for the 500bp fragment. All clinical isolates scored positive for IS6110, mtp40, the pncA and oxyR PCR's. All but one of the clinical isolates amplified the 500bp fragment. Sequence analysis of the pncA and oxyR PCR products revealed the presence of nucleotide C at position 169 and G at position 285 respectively, suggesting M. tuberculosis as the causative agent. CONCLUSION: Our data suggest that the 500bp PCR fragment is present not only in M. bovis but also in M. tuberculosis.

Colorectal cancer and inflammatory bowel disease: epidemiology, risk factors, mechanisms of carcinogenesis and prevention strategies.

Patients with long-standing ulcerative colitis and Crohn's disease have an increased risk of developing colorectal cancer and patients with small intestinal Crohn's disease are at increased risk of small bowel adenocarcinoma. Colorectal cancer appearing on the ground of inflammatory bowel disease is the result of a process which is believed to begin from no dysplasia progressing to indefinite dysplasia, low-grade dysplasia, high-grade dysplasia and finally to invasive adenocarcinoma, although colorectal cancer can arise without proceeding through each of these steps. Ulcerative colitis patients with total proctocolectomy and ileal pouch anal-anastomosis have a rather low risk of dysplasia in the ileal pouch, although the anal transition zone should be monitored periodically, especially if chronic pouchitis is present with associated severe villous atrophy. Concerning the risk factors predisposing to colorectal cancer in the setting of ulcerative colitis or Crohn's disease, it seems that the risk increases with longer duration and greater anatomic extent of colitis, the degree of inflammation, and the presence of primary sclerosing cholangitis and family history of colorectal cancer. Concerning the mechanisms of carcinogenesis, it is now well established that the molecular alterations responsible for sporadic colorectal cancer, namely chromosomal instability, microsatellite instability and hypermethylation, also play a role in colitis-associated colon carcinogenesis. Chemoprevention strategies include the administration of agents such as aminosalicylates, ursodeoxycholic acid, and possibly folic acid and statins, the exact role of which remains to be further elucitated.

Elucidation of the transmission of a novel mutation in BRCA1 (1125delCT) in a family with multiple cases of breast cancer.

In a family with multiple members affected by breast cancer we identified the novel mutation 1125delCT (exon 11) in BRCA1. Three out of three offsprings have the novel mutation while the mother affected by breast cancer does not carry the mutation. Linkage analysis revealed the transmission of the healthy haplotype from the mother to the three offsprings while the children inherited the mutated haplotype from the father. Our data document in an unquestionable way where the mutated haplotype was inherited from. In some families, although the transmission pathway seems obvious, the molecular analysis yields surprising results.

The role of collagen IX tryptophan polymorphisms in symptomatic intervertebral disc disease in Southern European patients.

STUDY DESIGN: We conducted a cross-sectional, genotyping study of intervertebral disc disease patients and controls. OBJECTIVES: To determine the contribution of COL9A2 and COL9A3 Tryptophan polymorphisms to intervertebral disc disease development in a genetically heterogeneous, Southern European population compared to previous Finnish studies. SUMMARY OF BACKGROUND DATA: The COL9A2 and COL9A3 genes encode the alpha2 and alpha3 chains of Collagen IX. Recent Finnish studies suggest that a tryptophan polymorphism in the COL9A2 gene (Trp2) results in hereditary intervertebral disc disease, whereas a similar tryptophan mutation in COL9A3 (Trp3) conveys a 3-fold risk of intervertebral disc disease. METHODS: We studied 105 symptomatic patients with radiographically and/or surgically proven lumbar (98%, n = 103) or cervical (2%, n = 2) intervertebral disc disease and 102 age-matched controls without spinal complaints from hospitals in Athens, Greece. Intervertebral disc disease was defined as significant disc herniation resulting in persistent back or leg pain. We genotyped all patients for COL9A2 and COL9A3 allele variations using a polymerase chain reaction-based technique. RESULTS: None of our patients had the Trp2 allele. Consistent with previous Finnish findings, more Greek intervertebral disc disease cases (8.6%) than controls (4.9%) had at least 1 Trp3 allele, but this difference did not reach statistical significance (P = 0.293). The allele frequency of the Trp3 mutation was significantly higher among previously studied Finnish patients with intervertebral disc disease (12.3%) than among the Southern European patients with intervertebral disc disease in our study (4.3%), P = 0.001. CONCLUSIONS: The differences in Trp allele frequency we found between Greek and Finnish patients with intervertebral disc disease most likely represent true differences in polymorphism prevalence between the respective populations. The 2 previously described Trp alleles in COL9A2 and COL9A3 are likely to be less significant susceptibility factors for intervertebral disc disease development in Southern European populations.