Elisavet Georgiou

Aberrant p16 promoter methylation among Greek lung cancer patients and smokers: correlation with smoking.

Genetic and environmental factors (dietary and smoking) influence lung cancer epidemiology and induce epigenetic modifications that should be assessed in individual populations. We analyzed p16 methylation among Greek non-small cell lung carcinoma patients and smokers using two-stage methylation-specific polymerase chain reaction. One hundred and fifty specimens from cancerous and adjacent non-cancerous tissue, bronchial washings and sputum from patients and 48 specimens, mostly sputum, from disease-free smokers were included. p16 methylation was very frequent in biopsies (82.85%) and bronchial washings (non-small cell lung carcinoma, 80.35%; small cell lung carcinoma, 16.66%) from patients, but also in adjacent non-cancerous tissue (45.71%). Concordance of p16 methylation and positivity by cytological examination was 51.78%. Methylation was also observed in sputum from asymptomatic cytology-negative smokers (22.5%) and chronic obstructive pulmonary disease patients (three of eight). Among disease-free individuals, methylation correlated only with heavy smoking (>50 pack-years, P<0.001) and differed among male and female disease-free smokers. In summary, p16 methylation is very frequent among non-small cell lung carcinoma patients, and correlates with heavy cigarette consumption only in disease-free smokers.

p16 promoter methylation in Pb2+-exposed individuals

Background. One of the principle symptoms of lead poisoning is the development of neurological disorders. Neuronal response is closely related to DNA methylation changes. Aim. In this study, we estimated p16 methylation in nine individuals exposed to lead using methylation-specific polymerase chain reaction followed by analysis of the methylated cytosine content of the product by thermal denaturation. Results. We found that, based on lead blood concentration, lead-exposed individuals were divided into two groups. Among highly exposed individuals (blood Pb2+ concentration = 51–100 μg/dL), we observed complete CpG methylation, whereas for low Pb2+ concentrations (blood Pb2+ concentration = 6–11 μg/dL), we observed partial methylation. Conclusion. Our results show that among lead-overexposed individuals, p16 methylation is frequent and extensive, and suggest that DNA methylation could be involved in the mechanism by which lead induces neurotoxicity.

Frequent presence of incomplete HPV16 E7 ORFs in lung carcinomas: memories of viral infection.

BACKGROUND HPV16 E6/E7 oncoproteins are critical for cervical carcinogenesis. The corresponding oncogenes are also detected in head and neck cancer, but in lung cancer their presence is strongly debated. PCR-based detection protocols amplify different target sequences. OBJECTIVES To examine the frequency of different length HPV16 E7 segments in lung carcinomas. STUDY DESIGN We designed four different amplification schemes for the detection of overlapping segments of the HPV16 E7 ORF, all suitable for specific HPV detection in cervical carcinoma. In two schemes, the entire E7 ORF was targeted while in the remaining schemes internal, smaller sequences were targeted. In total, 76 specimens were used; 29 lung carcinoma specimens, 16 non-cancerous lung tissue specimens from the same patients and 31 bronchial washings from different lung cancer patients. RESULTS Amplification of the entire HPV16 E7 ORF, using two protocols, demonstrated the absence of the specific HPV16 E7 sequences (74 samples either tested negative by the first PCR protocol or false positive by the second, based on sequencing or AvaII or PvuII digestion). However, both schemes targeting smaller E7 segments revealed the frequent presence of HPV16 E7 sequences in lung carcinoma specimens (14/23 positive by either scheme). CONCLUSIONS HPV16 E7 sequences are frequently observed in lung carcinomas. Decreasing the size of PCR-target sequences increases the detection frequency, possibly indicating the presence of incomplete viral ORFs. Restriction endonuclease analysis is critical for verifying the reliability of the detection of these sequences.

Ear lobe involvement in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia worldwide. Although indolent, it is refractory to treatment with only allogeneic stem cell transplantation a curative option for selected patients. CLL involves the skin usually in its advanced stages [1]. We present an uncommon cutaneous presentation of CLL and review the literature on all similar cases. A 67-year-old man presented with multiple, asymptomatic, erythematous papules with mild induration, symmetrically distributed on both ear lobes (Figure 1a). The lesions had been present for about four months and disturbed him cosmetically. The patient had a history of chronic lymphocytic leukemia, fi rst diagnosed three years previously, for which he had failed to follow-up and had not been treated. His absolute lymphocyte count at the time of presentation was 90000/μl. Biopsy from an indurated papule revealed a diffuse cutaneous infi ltration by small neoplastic lymphoid cells with scant cytoplasm and clumped chromatin (Figure 1b). Interspersed among them there were larger lymphoid cells with more conspicuous nucleoli. On immunohistochemical analysis the following immunophenotype was observed: CD20+ (Figure 1c), CD45RA+, PAX5+, BCL2+, CD5+ (Figure 1d), CD23++/(Figure 1e), CD45RO-, CD3-, CD43-, ZAP70-, cyclinD1-, CD10-, CD138-, κ-, λ-. The Ki-67/MIB-1 labeling index was approximately 15 %. These fi ndings were consistent with the diagnosis of cutaneous involvement by his known CLL. Clinical and computed thoracoabdominal tomography fi ndings revealed Binet stage B (or Rai stage II) disease. Systemic chemotherapy started with rituximab – fl udarabine – cyclophosphamide and after the fi rst cycle the absolute lymphocyte count was 15000/μl. In addition, the skin lesions regressed during the fi rst treatment cycles. Skin infi ltration by malignant B-lymphocytes in CLL occurs in 4–20 % of cases. [2] It manifests as solitary, grouped, or generalized papules, plaques, nodules, or large tumors, generalized eruption or even erythroderma. There is a spectrum of atypical cutaneous manifestations, associated with infi ltrations by leukemic cells at site of infections (Borrelia burgdorferi, herpes viruses) or surrounding epithelial neoplasms, probably as part of a specifi c malignant lymphocyte response to the cutaneous infection or tumor. [3] This infi ltration is considered reactive and not metastatic but it may lead to the initial diagnosis of CLL when it is histologically observed. Clinical Letters

Epigenetically - Targeted Therapies for the Treatment of Hematological Malignancies

Epigenetic modifications, which are heritable changes in gene expression not involving DNA sequence alterations, are important early events in the multi -step process of tumorigenesis. Among them, DNA methylation and histone acetylation are the most extensively studied. Although they are, by definition, somatically heritable, epigenetic modifications of DNA and histones are also reversible. This characteristic difference from genetic alterations makes them interesting targets for therapeutic intervention. The huge amount of knowledge gathered in the field of epigenetics the last decade, was followed by the development of novel therapies: old drugs finding new identity and new targets and an increasing list of novel compounds for the treatment of malignant diseases. Hematological malignancies offer a broad spectrum of diseases where epigenetic therapies are shown to be active, providing encouraging results. Some of the more recent reports on this field of therapeutic interventions are reviewed below.

Association of two synonymous splicing-associated CpG single nucleotide polymorphisms in calpain 10 and solute carrier family 2 member 2 with type 2 diabetes

Coding synonymous single nucleotide polymorphisms (SNPs) have attracted little attention until recently. However, such SNPs located in epigenetic, CpG sites modifying exonic splicing enhancers (ESEs) can be informative with regards to the recently verified association of intragenic methylation and splicing. The present study describes the association of type 2 diabetes (T2D) with the exonic, synonymous, epigenetic SNPs, rs3749166 in calpain 10 (CAPN10) glucose transporter (GLUT4) translocator and rs5404 in solute carrier family 2, member 2 (SLC2A2), also termed GLUT2, which, according to prior bioinformatic analysis, strongly modify the splicing potential of glucose transport-associated genes. Previous association studies reveal that only rs5404 exhibits a strong negative T2D association, while data on the CAPN10 polymorphism are contradictory. In the present study DNA from blood samples of 99 Greek non-diabetic control subjects and 71 T2D patients was analyzed. In addition, relevant publicly available cases (40) resulting from examination of 110 Personal Genome Project data files were analyzed. The frequency of the rs3749166 A allele, was similar in the patients and non-diabetic control subjects. However, AG heterozygotes were more frequent among patients (73.24% for Greek patients and 54.55% for corresponding non-diabetic control subjects; P=0.0262; total cases, 52.99 and 75.00%, respectively; P=0.0039). The rs5404 T allele was only observed in CT heterozygotes (Greek non-diabetic control subjects, 39.39% and Greek patients, 22.54%; P=0.0205; total cases, 34.69 and 21.28%, respectively; P=0.0258). Notably, only one genotype, heterozygous AG/CC, was T2D-associated (Greek non-diabetic control subjects, 29.29% and Greek patients, 56.33%; P=0.004; total cases, 32.84 and 56.58%, respectively; P=0.0008). Furthermore, AG/CC was strongly associated with very high (≥8.5%) glycosylated plasma hemoglobin levels among patients (P=0.0002 for all cases). These results reveal the complex heterozygotic SNP association with T2D, and indicate possible synergies of these epigenetic, splicing-regulatory, synonymous SNPs, which modify the splicing potential of two alternative glucose transport-associated genes.

Bilateral Aleukemic Myeloid Sarcoma of the Eyelids With Indolent Course

Abstract:Leukemic infiltrates may be seen in the skin in the absence of detectable bone marrow involvement. Leukemia cutis may exceptionally occupy the eyelids. An unusual case of a 58-year-old man presenting bilateral erythematous eyelid lesions, proven to be aleukemic leukemia cutis, is reported. Biopsy was conducted and hematoxylin/eosin stained sections were histologically evaluated. Immunohistochemistry was also performed.Light microscopy revealed cutaneous infiltration by a neoplastic population consisting of medium-sized cells. These cells infiltrated the overlying epidermis leading to focal microulcerations. The morphological and immunohistochemical characteristics of the neoplastic population were compatible with myeloid leukemia cutis. The bone marrow biopsy was normocellular for the patients age. Although chemotherapy was advised, the patient refused any treatment. He remains free of leukemia or evolution of eyelid lesions approximately 1 year after diagnosis. Leukemia cutis of the eyelids is a rare manifestation of acute leukemia and may remain aleukemic in adults for an indefinite period of time.

Mycosis fungoides in patients with psoriasis: an ongoing issue

235 axilla [3, 7]. The tumour in our case also appeared along the milk lines, which might explain why it appeared on the trunk and why the major histopathological components showed apocrine differentiation. When dermatologists encounter a well-demarcated cystic nodule on the trunk, a clinical diagnosis of other tumours, such as epidermoid cyst and steatocystoma, is usually made, without detailed histopathological examination. However, as suggested by the present case, dermatologists should be aware that AMT can, although rarely, appear on the trunk. Further studies should address the pathogenesis of AMT in association with the mammary glands.

Age-dependent methylation in epigenetic clock CpGs is associated with G-quadruplex, co-transcriptionally formed RNA structures and tentative splice sites

ABSTRACT Horvath’s epigenetic clock consists of 353 CpGs whose methylation levels can accurately predict the age of individuals. Using bioinformatics analysis, we investigated the conformation, energy characteristics and presence of tentative splice sites of the sequences surrounding the epigenetic clock CpGs, in relation to the median methylation changes in different ages, the presence of CpG islands and their position in genes. Common characteristics in the 100 nt sequences surrounding the epigenetic clock CpGs are G-quadruplexes and/or tentative splice site motifs. Median methylation increases significantly in sequences which adopt less stable structures during transcription. Methylation is higher when CpGs overlap with G-quadruplexes than when they precede them. Median methylation in epigenetic clock CpGs is higher in sequences expressed as single products rather than in multiple products and those containing single donors and multiple acceptors. Age-related methylation variation is significant in sequences without G-quadruplexes, particularly those producing low stability nascent RNA and those with splice sites. CpGs in sequences close to transcription start sites and those which are possibly never expressed (hypothetical proteins) undergo similar extent of age-related median methylation decrease and increase. Preservation of methylation is observed in CpG islands without G-quadruplexes, contrary to CpGs far from CpG islands (open sea). Sequences containing G-quadruplexes and RNA pseudoknots, determining the recognition by H3K27 histone methyltransferase, are hypomethylated. The presented structural DNA and co-transcriptional RNA analysis of epigenetic clock sequences, foreshadows the association of age-related methylation changes with the principle biological processes of DNA and histone methylation, splicing and chromatin silencing.

The cellular microenvironment and neoplastic population in mycosis fungoides skin lesions: a clinicopathological correlation

BackgroundThe cellular microenvironment has been proven to play a crucial role in solid tumours and seems to be important in haematologic malignancies, however, it has not been adequately investigated in primary cutaneous T cell lymphomas.ObjectivesThe aim of this study was to register the composition of the cellular microenvironment in mycosis fungoides skin lesions and correlate the composing parameters with the clinical data and follow-up results.Materials and methodsThe presence of eosinophilic polymorphonuclear leukocytes, B lymphocytes, CD68+ macrophages, and CD1a+ epidermal Langerhans and antigen-presenting dermal dendritic cells, as well as their relation to clinicopathological parameters, were studied in 16 mycosis fungoides cases of different disease stages. The presence and nature of the participating T cell populations was also investigated.ResultsCD8+ tumour infiltrating T cells and CD56+ cells were found among neoplastic CD4+ T cells in the lesions. Generally, eosinophils and B lymphocytes were absent or in low numbers, regardless of clinical presentation, contrary to tumourous lesions. Macrophages and CD1a+ cells were constantly present, even in early-stage mycosis fungoides. The reduced presence of the CD1a+ population was associated with resistance to therapy (x2; p = 0.012).ConclusionThere is a striking difference in cellular microenvironment composition between early and advanced mycosis fungoides lesions.