Aris Spathis

Hypoxia-inducible factors in mantle cell lymphoma: implication for an activated mTORC1→HIF-1α pathway

Aberrant activation of phosphoinositide-3 kinase/Akt (PI3K/Akt) and mammalian target of rapamycin (mTOR) signaling is implicated in the pathogenesis of mantle cell lymphoma (MCL). We previously showed oncogenic activation of PI3K/Akt pathway in a subset of MCL patients. In this study, we investigated downstream the immunohistochemical expression of Ser2448pmTOR [indicative of mTOR complex 1 (mTORC1) activation status] as well as of hypoxia-inducible factor 1 alpha (HIF-1α), hypoxia-inducible factor 2 alpha (HIF-2α), p53, and p21 in the same series of MCL patients. Additionally, correlation of these proteins with activated Akt (Ser473pAkt) and established histological prognostic factors was examined. Thirty-five tissue samples (28 classical type and seven blastoid variant) were included. The neoplastic cells expressed Ser2448pmTOR in 61.7%, HIF-1α in 73.5%, HIF-2α in 23.5%, and p53 in 18.2% of patients, while p21 was negative in all examined samples. In addition, 72% of patients who expressed HIF-1α had also Ser2448pmTOR expression (p = 0.041). HIF-1α expression was also correlated to an elevated (≥30%) Ki-67 (p = 0.031) and blastoid variant of disease (p = 0.017). In conclusion, we report for the first time common expression of HIF-alphas, especially HIF-1α, in MCL patients. Furthermore, an overall activation of mTORC1→HIF-1α axis and a potential role of Ser2448pmTOR in the regulation of HIF-1α in MCL patients are suggested. Finally, HIF-1α appears to be associated with more aggressive disease. A pathogenetic role for both mTORC1 and HIF-1α in MCL is implied, which will possibly lead to more efficient target therapies.

Differential effects of stent-graft fabrics on arterial stiffness in patients undergoing endovascular aneurysm repair.

Purpose: To evaluate the differential effects of endograft fabric types on pulse wave velocity (PWV), a valid, clinically feasible marker of arterial stiffness, in patients undergoing endovascular repair (EVAR) of abdominal aortic aneurysms (AAAs). Methods: As part of a registered study (ClinicalTrials.gov identifier NCT00636766), 118 consecutive men (mean age 71±8 years) with AAA undergoing elective EVAR were divided into groups according to the type of fabric in the implanted endografts: 46 had polytetrafluoroethylene (PTFE)-covered stent-grafts and 72 received stent-grafts covered with polyester fabric. Patients with Marfan syndrome, collagen-related disorders, and end-stage renal failure were excluded. PWV, clinical characteristics (comorbidities, body mass index, blood pressure, lipids, and glucose profile), novel biomarkers [interleukin (IL)-6, IL-8, IL-10, and osteoprotegerin (OPG)] were determined at baseline and prospectively after 12 months. Results: At baseline, standard multiple regression analysis revealed the independent association of mean blood pressure, OPG, and AAA diameter with PWV after adjustment for age (R2=0.729, p=0.036). At follow-up, serum levels of both IL-8 and IL-10 were significantly increased, while OPG decreased in both groups (p<0.05). However, between-group comparison revealed a more adverse effect of polyester covered endografts on serum IL-8 (p<0.001) and OPG (p=0.048) levels. At the same time, PWV was considerably increased in both polyester (+4.12±0.33 m/s, p<0.001) and PTFE (+2.82=0.25 m/s, p=0.003) groups; the effect was more pronounced in the former group (p=0.033). In multivariate analysis, the polyester endograft type emerged as an independent determinant of PWV change after EVAR (R2=0.460, p=0.040). Conclusion: Both endograft types increased PWV, while recipients of polyester covered stent-grafts showed greater PWV elevation. Further investigation will clarify the inflammatory response and the differential clinical impact of endograft types.

An Intelligent Clinical Decision Support System for Patient-Specific Predictions to Improve Cervical Intraepithelial Neoplasia Detection

Nowadays, there are molecular biology techniques providing information related to cervical cancer and its cause: the human Papillomavirus (HPV), including DNA microarrays identifying HPV subtypes, mRNA techniques such as nucleic acid based amplification or flow cytometry identifying E6/E7 oncogenes, and immunocytochemistry techniques such as overexpression of p16. Each one of these techniques has its own performance, limitations and advantages, thus a combinatorial approach via computational intelligence methods could exploit the benefits of each method and produce more accurate results. In this article we propose a clinical decision support system (CDSS), composed by artificial neural networks, intelligently combining the results of classic and ancillary techniques for diagnostic accuracy improvement. We evaluated this method on 740 cases with complete series of cytological assessment, molecular tests, and colposcopy examination. The CDSS demonstrated high sensitivity (89.4%), high specificity (97.1%), high positive predictive value (89.4%), and high negative predictive value (97.1%), for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+). In comparison to the tests involved in this study and their combinations, the CDSS produced the most balanced results in terms of sensitivity, specificity, PPV, and NPV. The proposed system may reduce the referral rate for colposcopy and guide personalised management and therapeutic interventions.

The diagnostic accuracy of two methods for E6&7 mRNA detection in women with minor cytological abnormalities

Objective. To examine the diagnostic accuracy of nucleic acid sequence based amplification (NASBA) and flow cytometry for E6&7 human papillomavirus (HPV) mRNA detection in the triage of minor cytological abnormalities. Design. Prospective diagnostic accuracy study. Setting. Gynecology outpatient clinics of a university hospital. Population. 472 women with low‐grade squamous intraepithelial lesion (LSIL) or atypical squamous cells of undetermined significance (ASCUS). Methods. Residual material of the liquid‐based smears was tested by NASBA and by flow cytometry for E6&E7 mRNA expression. Histological diagnosis was used as reference standard. Main outcome measures. Accuracy indices of the two techniques and of type 16‐specific NASBA for the detection of cervical intraepithelial neoplasia (CIN) 2+ and CIN3+, accuracy indices at age >35 years, correlation between NASBA and flow, comparison between integrated and episomal high‐risk HPV infection for risk of CIN2+. Results. Both tests showed increased positivity rates with increasing severity of the lesion (p < 0.05, chi‐squared test for trend). There was a positive correlation between NASBA and flow results (phi coefficient = 0.325). NASBA‐positive cases were more likely to have CIN2+ than were NASBA‐negative/DNA‐positive for types 16, 18, 31, 33, 45 (25/73 vs. 4/52, p= 0.0004; Fisher‘s exact test). In the LSIL group the NASBA accuracy indices for CIN3+ were: sensitivity 75%, specificity 78.7% and positivity rate 20.8%, and for flow 77.8%, 64.5% and 35.9%, respectively. Conclusions. NASBA has favorable specificity and positivity rates for triaging LSIL prior to colposcopy. A relatively low sensitivity warrants cytological surveillance of the NASBA‐negative LSILs. Flow cytometry does not perform as well overall.

Identification of Women for Referral to Colposcopy by Neural Networks: A Preliminary Study Based on LBC and Molecular Biomarkers

Objective of this study is to investigate the potential of the learning vector quantizer neural network (LVQ-NN) classifier on various diagnostic variables used in the modern cytopathology laboratory and to build an algorithm that may facilitate the classification of individual cases. From all women included in the study, a liquid-based cytology sample was obtained; this was tested via HPV DNA test, E6/E7 HPV mRNA test, and p16 immunostaining. The data were classified by the LVQ-NN into two groups: CIN-2 or worse and CIN-1 or less. Half of the cases were used to train the LVQ-NN; the remaining cases (test set) were used for validation. Out of the 1258 cases, cytology identified correctly 72.90% of the CIN-2 or worst cases and 97.37% of the CIN-1 or less cases, with overall accuracy 94.36%. The application of the LVQ-NN on the test set allowed correct classification for 84.62% of the cases with CIN-2 or worse and 97.64% of the cases with CIN-1 or less, with overall accuracy of 96.03%. The use of the LVQ-NN with cytology and the proposed biomarkers improves significantly the correct classification of cervical precancerous lesions and/or cancer and may facilitate diagnosis and patient management.

Genetic Variability and Phylogeny of High Risk HPV Type 16, 18, 31, 33 and 45 L1 Gene in Greek Women

The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single infections of HPV type 16 (n = 104), HPV 31 (n = 40), HPV 33 (n = 7), HPV 18 (n = 5), and HPV 45 (n = 4) were typed by microarrays method, amplified by PCR then sequenced and phylogenetically analyzed. For HPV 16, 9 variants with nucleotide variations were included into the study. For HPV 31, 33, 18 and 45, nucleotide variations were identified in 6, 4, 2 and 3 variants, respectively. The Bayesian inference and Maximum Parsimony methods were used in order to construct the phylogenetic trees. When types were analyzed independently HPV 16 (European and non-European) and HPV 18 (African and non-African) formed distinct clades. The genomic characterization of HPV variants will be important for illuminating the geographical relatedness and biological differences and for the determination of their risk.

Dasatinib inhibits proliferation and induces apoptosis in the KASUMI-1 cell line bearing the t(8;21)(q22;q22) and the N822K c-kit mutation

Activating mutations of the c-kit gene are frequently found in CBF (core binding factor) leukemias. We evaluated the effect of tyrosine kinase inhibitor dasatinib in leukemic cell lines bearing or not c-kit mutations. Our data demonstrate that in the AML Kasumi-1 cell line, bearing the N822K c-kit mutation, dasatinib is a potent suppressor of c-kit and Src kinase activity and inhibits the phosphorylation of their downstream target AKT, possibly through the Src-mediated VEGF/VEGFR receptor type 2 pathway. Dasatinib also effectively blocks proliferation and induces apoptosis through caspase-3 activation in Kasumi-1 cells. These data further encourage the integration of dasatinib in the treatment of CBF AML with c-kit mutations in the context of clinical trials, which are eagerly anticipated.

mRNA and DNA Detection of Human Papillomaviruses in Women of All Ages Attending Two Colposcopy Clinics

Objective HPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals. Methods 1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results. Results HPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests’ positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used. Conclusions Overall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results.

Promoter Methylation of p16INK4A, hMLH1, and MGMT in Liquid-Based Cervical Cytology Samples Compared with Clinicopathological Findings and HPV Presence

Cervical cancer is a common cancer inflicting women worldwide. Even though, persistent infection with oncogenic Human Papillomavirus (HPV) types is considered the most important risk factor for cervical cancer development, less than 5% of women with HPV will eventually develop cervical cancer supporting that other molecular events, like methylation-dependent inactivation of tumor suppressor genes, may cocontribute in cervical carcinogenesis. We analyzed promoter methylation of three candidate genes (p16, MGMT, and hMLH1) in 403 liquid-based cytology samples. Methylation was commonly identified in both benign and pathologic samples and correlated with higher lesion grade determined by cytological, colposcopical, or histological findings, with HPV DNA and mRNA positivity of specific HPV types and p16INK4A protein expression. Overall accuracy of methylation is much lower than traditional diagnostic tests ranking it as an ancillary technique with more data needed to identify the exact value of methylation status in cervical carcinogenesis.

Molecular epidemiology of HPV infection using a clinical array methodology in 2952 women in Greece

The molecular epidemiology of human papillomavirus (HPV) infection in a sample of Greek women (n = 2952, mean age 42.2 ± 13.3 years) was examined. HPV prevalence was 50.7% (95% confidence interval, 48.8-52.6). The most frequent HPV types were HPV 53, 51 and 66 (10.2%, 9.4% and 9.3%, respectively). HPV positivity was associated with age, age of sexual debut, number of sexual partners and duration of sexual relationship, while marriage or multiparity protected against infection (all p <0.001). Follow-up of this cohort will assist in predicting the effect of vaccination with the new HPV vaccines on future screening with HPV-based tests for cervical cancer.