Regulatory T Cell Counts and Development of Malignancy in Patients with HIV Infection.

BACKGROUND: T-regulatory cells (Tregs) play an important role in maintaining homeostasis by attenuating the cytokine response to T-cell receptor (TCR) stimulation and by suppressing the functioning of neighboring immune cells. In Human Immunodeficiency Virus (HIV) infection, Tregs can be either beneficial, by suppressing generalized T-cell activation, or detrimental, by suppressing protective anti-HIV cell-mediated immunity. An imbalance of Tregs and effector T-cells can blunt immune responses to malignant cells or facilitate inflammation-mediated pathologies. OBJECTIVE: The purpose of our study was to explore the possible correlation between Tregs' concentration and HIV infection's parameters as well as the development of hematological and solid malignancies. METHODS: In a longitudinal prospective study, ex vivo phenotyping of fresh peripheral blood mononuclear cells from patients with primary HIV infection was performed at baseline. All patients were then followed up every 3 months and the development of solid or hematological malignancies was noted. RESULTS: A total of 155 patients were included in the study and the median follow-up period was 64 months. Treg counts were significantly higher among males, patients with high viral load (>350 copies/ml) and patients with virological failure to antiretroviral treatment (ART). Linear regression analysis showed a significant negative correlation between Treg levels and CD4 (+) T-cell counts. Patients with neoplasia had lower levels of Tregs while increasing levels showed a negative correlation with the development of neoplasia. CONCLUSION: In our population of HIV-infected patients, high levels of Tregs were associated with disease progression, and low baseline levels were associated with a higher probability of developing neoplasia.

Colorectal Cancer CTCs Detection Using Two FCM Protocol Approaches.

BACKGROUND: The presence and quantification of circulating tumor cells (CTCs) could minimize the mortality among cancer patients by tailored, personalized, and targeted therapy and could also help in the field of investigation about new therapeutic targets. Identification of CTCs has been performed using molecular techniques and more advanced automated techniques such as CellSearch® and Amnis®. Our aim was to test the possibility of identifying CTCs in colorectal cancer patients using a lower cost and less complex flow cytometry-based method. METHODS: Besides the CellSearch® system for CTCs enumeration and Amnis® Imaging Flow Cytometers, which are both commercially available, we tested and developed two FCM protocol approaches after immunomagnetic enrichment for CTCs enumeration in the blood of colorectal cancer patients. The CTCs numbers were assessed at baseline before anesthesia and the curative surgery and day one after the curative surgery. Blood from healthy donors was used as negative control. The research was performed by the Cyflow® Space cytometer (Partec, Münster, Germany). RESULTS: In the patient group, the enumeration using the direct protocol with a threshold 3 cells/mL, showed a mean of CTCs before surgery of 32, while after surgery it was 20, with sensitivity (SN) of 49.2% and specificity (SP) of 58.3%. On the other hand, using the intracellular protocol with a threshold of 1 cell/mL in patients' group, the mean of CTCs before surgery was 65 and after surgery it was 60, with a sensitivity (SN) of 62.7% and specificity (SP) of 70%. In the intracellular protocol the most significant correlation identified was for the expression of CKs with adenocarcinomas (r = 0.256, p = 0.044), with the existence of lymph node infiltration (r = 0.380, p = 0.008), and with the stage of the disease (r = 0.391, p = 0.003). For the direct protocol, considerable correlation was found with the samples of the right colon (r = 0.369, p = 0.002). CONCLUSIONS: Our findings demonstrate that we established two FCM low cost protocol approaches to detect and enumerate CTCs in colorectal cancer patients. In both FCM protocols (direct and intracellular) we observed a statistically significant increase (p ˂ 0.05) of CTCs number in the patient group. No statistical significance (p ˂ 0.05) of CTCs before and after surgery in patient group in both protocols was observed.

BRAF Mutation Status in Primary, Recurrent, and Metastatic Malignant Melanoma and Its Relation to Histopathological Parameters.

BACKGROUND: BRAF mutations are a common finding in malignant melanoma (MM). Nevertheless, apart from their significance as a therapeutic target in advanced melanoma, their prognostic value is still debated. OBJECTIVE: To assess BRAF mutation status in primary, recurrent, or metastatic MM and its correlations with histopathological findings. METHODS: We analyzed 203 samples from 178 consecutive patients: 129 primary cutaneous MM, 49 metastatic and recurrent MM of unknown primary site, and 25 cases of recurrences or metastases of primary MM. BRAF mutations in exon 15 were identified with real-time polymerase chain reaction and/or direct sequencing or pyrosequencing. Histopathological examination was performed according to standard procedures. RESULTS: We observed a 42.1% prevalence of BRAF mutations at codon 600 among our patients, 84% of whom harbored the V600E mutation. Mutations showed a statistically significant increase in younger patients (P = 0.011), in ulcerated tumors (P = 0.020), and in tumors lacking solar elastosis in adjacent dermis (P = 0.008). Mutations were also more common in male patients, as well as in primary MMs of the torso, and in nonvisceral metastases, however without reaching statistical significance. Logistic regression analysis identified type and ulceration as the only significant predictors of BRAF mutation. The highest frequencies of mutated BRAF were identified in superficial spreading and nodular types, and the lowest in acral lentiginous and lentigo maligna types. In situ MM and primary dermal melanoma displayed intermediate frequencies. CONCLUSION: Frequency of mutated BRAF is type-related and correlated with ulceration, a known adverse prognostic factor.

Neonatal screening for congenital CMV infection stresses the importance of maternal nonprimary infection even in an area where prenatal serology testing is common.

AIM AND METHODS: Dried blood spots from 2149 newborns were examined to diagnose congenital cytomegalovirus (cCMV). RESULTS: Prenatal CMV-IgG antibodies had been measured during prenatal care in 1287 (60.3%) of mothers and 980 (76.1%) of them were found seropositive. cCMV incidence was 0.47%. All newborns were asymptomatic; 9/10 were born post nonprimary maternal infection; two developed sensorineural hearing loss. CONCLUSIONS: In a country where prenatal CMV testing is common and therefore a false sense of control might prevail, nonprimary maternal infection should not be overlooked. Indeed, women of childbearing age should be educated on CMV prevention measures irrespectively to their serostatus.

Concomitant Human Herpes Virus 6 and nivolumab-related pneumonitis: Potential pathogenetic insights.

The development of immune system modulating agents, such as immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment. Nivolumab, a human monoclonal antibody against PD-1, has emerged as an efficient treatment for various malignancies, including non-small cell lung cancer (NSCLC); however, it is associated with important immune related side-effects, attributed to organ-specific inflammation, such as immune-mediated pneumonitis, a relatively uncommon, albeit potentially fatal adverse event. We herein present the unique case of severe interstitial pneumonitis with concomitant detection of Human Herpes Virus 6 (HHV-6) in a nivolumab treated patient with NSCLC. Potential pathogenetic mechanisms are discussed.

The Yokohama system for reporting directly sampled endometrial cytology: The quest to develop a standardized terminology.

BACKGROUND: The main purpose of directly sampled endometrial cytology is to detect invasive endometrial malignancies. With this principle in mind, The Yokohama System (TYS) Working Group, composed of cytopathologists, surgical pathologists, and gynecologic oncologists met at the 2016 International Congress of Cytology, Yokohama, with the aim to publish a standardized reporting system inclusive of specific diagnostic categories and cytomorphologic criteria for uniform and reliable diagnosis of endometrial malignancies on directly sampled endometrial samples. METHODS: The diagnostic cytopathologic criteria previously published in the literature by the Japanese and Greek working group on endometrial cytology (Yanoh et al. [2012] Acta Cytol. 56:233; Margari et al. [2016] Diagn Cytopathol. 44:888-901) were critically reviewed with the aim of correlating the diagnostic classes to well defined risk categories for endometrial carcinoma (EC). Moreover, two classes of "atypical" endometrial cells were correlated respectively to a low- and high risk group. Some methodological suggestions for the application of ancillary special technologies to liquid based samples were also given. RESULTS: The TYS group conceived a new Bethesda-style classification for directly sampled endometrial cytology which correlates the cytologic diagnostic classes with definite risk categories. The cytomorphologic findings have been correlated to the molecular pathology of EC, also through the application of ancillary special techniques to liquid-based samples. CONCLUSIONS: The success of TYS will depend on the acceptance of TYS by all the relevant pathology and gynecologic oncology communities who, by their joint efforts, will adopt, critically evaluate, and optimize this method with the only aim of further improving the impact of endometrial cytology on patients' care.

Application of Immunocytochemistry on Cell Block Sections for the Investigation of Thyroid Lesions.

OBJECTIVE: To investigate the potential of Classification and Regression Trees (CARTs) for the diagnosis of thyroid lesions based on cell block immunocytochemistry and cytological outcome. STUDY DESIGN: A total of 956 histologically confirmed cases (673 benign and 283 malignant) from patients with thyroid nodules were prepared via liquid-based cytology and evaluated; 4 additional slides were stained for cytokeratin 19 (CK-19), galectin 3 (Gal-3), Hector Battifora mesothelial cell 1 (HBME-1), and thyroglobulin. On the basis of immunocytochemistry and the cytological diagnosis, a CART algorithm was constructed and used for evaluation. RESULTS: The major important factors contributing to the diagnostic CART model were: cytological outcome, CK-19, Gal-3, and HBME-1. The sensitivity and specificity of the cytological diagnosis were 96.27% and 88.26%, respectively (cut-off: category 3 of The Bethesda System [TBS-3]). The introduction of immunocytochemistry and the CART model increased the sensitivity and specificity to 98.88% and 99.11%, respectively. CK-19 presented the best performance for discriminating papillary thyroid carcinomas, followed by HBME-1 and Gal-3. In the TBS-2 cases, CK-19 and, subsequently, Gal-3 were important immunocytochemistry markers. Ultimately, CK-19 and HBME-1 on TBS-5 or TBS-6 cases demonstrated the best results. CONCLUSIONS: The hierarchical structure of the CART model provides a diagnostic algorithm linked with the risk of malignancy at every step of the procedure. It also provides guidance on the use of ancillary examinations as it goes by simple, human understandable rules.

High levels of IL-6 and IL-8 characterize early-on idiopathic pulmonary fibrosis acute exacerbations.

INTRODUCTION: Controversy exists about the pathogenesis of idiopathic pulmonary fibrosis acute exacerbations (IPF-AEs). According to one hypothesis IPF-AEs represent the development of any etiology diffuse alveolar damage (DAD) upon usual interstitial pneumonia (UIP), whilst other researchers argue that an accelerated phase of the intrinsic fibrotic process of unknown etiology prevails, leading to ARDS. Different cytokines might be involved in both processes. The aim of this study was to assess pro-inflammatory and pro-fibrotic cytokines in the peripheral blood from stable and exacerbated IPF patients. METHODS: Consecutive IPF patients referred to our department were included. Diagnoses of IPF and IPF-AE were based on international guidelines and consensus criteria. The interleukins (IL)-4, IL-6, IL-8, IL-10, and IL-13 as well asactive transforming growth factor-beta (TGF-ß) were measured in blood from both stable and exacerbated patients on the day of hospital admission for deterioration. Subjects were followed for 12months. Mann-Whitney test as well as Tobit and logistic regression analyses were applied. RESULTS: Among the 41 patients studied, 23 were stable, and 18 under exacerbation; of the latter, 12 patients survived. The IL-6 and IL-8 levels were significantly higher in exacerbated patients (p=0.002 and p=0.046, respectively). An increase in either IL-6 or IL-8 by 1pg/ml increases the odds of death by 5.6% (p=0.021) and 6.7% (p=0.013), respectively, in all patients. No differences were detected for the other cytokines. CONCLUSION: High levels of IL-6 and IL-8 characterize early-on IPF-AEs and an increase in the levels of IL-6 and IL-8 associates with worse outcome in all patients. However, as the most representative pro-fibrotic cytokines, TGF-ß, IL-10, IL-4 and IL-13 were not increased and given the dualistic nature, both pro-inflammatory and pro-fibrotic of IL-6 further studies are necessary to clarify the enigma of IPF-AEs etiopathogenesis.

Evaluation Analysis of miRNAs Overexpression in Liquid-Based Cytology Endometrial Samples.

Background: miRNAs have an important role as their deregulation is linked to endometrial cancer. Methods: A custom miScript® miRNA PCR Array was used to investigate for the first time the expression of eight miRNAs in forty-nine histologically confirmed Liquid Based cytology endometrial samples. The expression profile of the same miRNAs was also examined in sixty formalin-fixed tissue samples. Results: Expression of seven miRNAs was significantly higher in malignant samples with three of them (mir-182, mir-141 and mir-205) performing optimally. Conclusion: These results suggest the potential use of this non-invasive method of sampling for miRNA expression studies. Furthermore miRNA overexpression could serve as an ancillary or reflex test for optimal identification of malignant samples especially in morphologically inadequate samples.

Integrating TYMS, KRAS and BRAF testing in patients with metastatic colorectal cancer.

AIM: To investigate the impact of thymidylate synthase (TYMS), KRAS and BRAF in the survival of metastatic colorectal cancer (mCRC) patients treated with chemotherapy. METHODS: Clinical data were collected retrospectively from records of consecutive patients with mCRC treated with fluoropyrimidine-based chemotherapy from 1/2005 to 1/2007. Formalin-fixed paraffin-embedded tissues were retrieved for analysis. TYMS genotypes were identified with restriction fragment analysis PCR, while KRAS and BRAF mutation status was evaluated using real-time PCR assays. TYMS gene polymorphisms of each of the 3' untranslated region (UTR) and 5'UTR were classified into three groups according to the probability they have for high, medium and low TYMS expression (and similar levels of risk) based on evidence from previous studies. Univariate and multivariate survival analyses were performed. RESULTS: The analysis recovered 89 patients with mCRC (46.1% de novo metastatic disease and 53.9% relapsed). Of these, 46 patients (51.7%) had colon cancer and 43 (48.3%) rectal cancer as primary. All patients were treated with fluoropyrimidine-based chemotherapy (5FU or capecitabine) as single-agent or in combination with irinotecan or/and oxaliplatin or/and bevacizumab. With a median follow-up time of 14.8 mo (range 0-119.8), 85 patients (95.5%) experienced disease progression, and 63 deaths (70.8%) were recorded. The 3-year and 5-year OS rate was 25.4% and 7.7% while the 3-year progression-free survival rate was 7.1%. Multivariate analysis of TYMS polymorphisms, KRAS and BRAF with clinicopathological parameters indicated that TYMS 3'UTR polymorphisms are associated with risk for disease progression and death (P < 0.05 and P < 0.03 respectively). When compared to tumors without any del allele (genotypes ins/ins and ins/loss of heterozygosity (LOH) linked with high TYMS expression) tumors with del/del genotype (low expression group) and tumors with ins/del or del/LOH (intermediate expression group) have lower risk for disease progression (HR = 0.432, 95%CI: 0.198-0.946, P < 0.04 and HR = 0.513, 95%CI: 0.287-0.919, P < 0.03 respectively) and death (HR = 0.366, 95%CI: 0.162-0.827, P < 0.02 and HR = 0.559, 95%CI: 0.309-1.113, P < 0.06 respectively). Additionally, KRAS mutation was associated independently with the risk of disease progression (HR = 1.600, 95%CI: 1.011-2.531, P < 0.05). The addition of irinotecan in 1st line chemotherapy was associated independently with lower risk for disease progression and death (HR = 0.600, 95%CI: 0.372-0.969, P < 0.04 and HR = 0.352, 95%CI: 0.164-0.757, P < 0.01 respectively). CONCLUSION: The TYMS genotypes ins/ins and ins/LOH associate with worst prognosis in mCRC patients under fluoropyrimidine-based chemotherapy. Large prospective studies are needed for validation of our findings.

Association of codon 72 polymorphism of p53 with the severity of cervical dysplasia, E6-T350G and HPV16 variant lineages in HPV16-infected women.

Purpose. Polymorphic variability in the tumour-suppressor protein p53 at codon 72 has a considerable impact on cervical cancer development. The present study clarified the association between p53 codon 72 genotypes and the risk of cervical disease in Greek patients. We also examined whether the presence of specific p53 genotypes in combination with HPV16 variants or E6 T350G sequence variation can modify an individual's susceptibility to cervical disease.Methodology. The analysis of p53 genotypes was performed through PCR-RFLP. Sequence and phylogenetic tree analyses of the HPV16 E6 gene were also performed in order to identify HPV16 variants and T350G sequence variation.Results/Key findings. The outcomes of the present analysis revealed that women who are homozygous for the arg genotype are at a 4.17-fold higher risk of developing HPV16-associated HSIL+ (OR=4.17, 95 % CI:1.48-4.9, P=0.0049). Moreover, p53 arg/arg patients infected by an HPV16 prototype strain were associated with an increased risk of more severe lesions, while a significant relationship between the p53 arg/arg genotype in patients with T350G sequence variation and the risk of high-grade squamous intraepithelial lesions (HSILs) was revealed.Conclusion. The oncogenic potential of the virus is increased by the presence of the p53 arg/arg genotype in the Greek population in such a way that the specific protein interaction E6 (L83V)-p53 (Arg-72) can modify an individual's susceptibility to cervical disease.

Quantitative and qualitative analysis of regulatory T cells in B cell chronic lymphocytic leukemia.

Accumulated data indicate a significant role of T cell dysfunction in the pathogenesis of chronic lymphocytic leukemia. In CLL, regulatory T cells are significantly higher and show lower apoptotic levels compared to healthy donors. We demonstrate that CLL derived CD4+CD25-CD127- and CD4+CD25lowCD127- subpopulations share a common immunophenotypic profile with conventional Tregs and are associated with advanced stage disease. We further provide evidence that the increased number of Tregs contributes indirectly to the proliferation of the CLL clone, by suppressing the proliferation of Teffs which in turn suppress CLL cells. These data are further supported by our observations that CLL derived Tregs appear rather incapable of inducing apoptosis of both normal B cells and CLL cells, in contrast to normal Tregs, suggesting an immunoediting effect of CLL cells on Tregs which negatively affects the functionality of the latter and contributes to the failure of Tregs in CLL to efficiently eliminate the abnormal clone.

Internal quality control in an academic cytopathology laboratory for the introduction of a new reporting system for endometrial cytology.

BACKGROUND: To evaluate reproducibility of a reporting system for endometrial cytology. METHODS: Cytologic slides from 49 patients, prepared via liquid based cytology, were blindly examined by five cytopathologists of various experience levels, applying a recently introduced reporting system as previously reported. The agreement among cytopathologists was evaluated via Kappa (κ) statistics and the Kendall's Coefficient of Variation (W); cytologic results were compared with the relevant histologic report. RESULTS: Substantial agreement among all five raters was found in the benign, ACE-L and malignant categories, fair agreement in inadequate and ACE-H categories, whereas only slight agreement in ACE-U. For the three more experienced cytopathologists, an almost perfect agreement was found in inadequate, benign, and ACE-L categories, substantial agreement in ACE-H and malignant categories and fair agreement in ACE-U category. Overall agreement for all five cytopathologists and for all categories was moderate, whereas it was very high for the three senior raters. Using the Kendall's test, both five cytopathologists (W = 0.81) and the three senior ones (W = 0.93) had very high agreement. Sensitivity: 83.33-92.59%, specificity: 83.33-94.74%, ROC area: 71.72-90.3%. CONCLUSION: Application of appropriate statistical tests shows that integration of a new reporting cytologic system is effective with an overall accuracy around 90%. Both statistical tests applied disclosed lower agreement rates among both all five raters and the three most experienced ones in the intermediate categories constituting the gray zone, thus delineating the need for better training of cytopathologists to correctly identify diagnostic criteria for classification of a given case into these categories.

The Profile of Circulating Matrix Metalloproteinases in Patients Undergoing Lower Limb Endovascular Interventions for Peripheral Arterial Disease.

BACKGROUND: Matrix metalloproteinases (MMPs) play a significant role in the development and progression of atherosclerotic vascular disease. We aimed to document the profile of circulating MMPs in peripheral arterial disease (PAD) patients undergoing lower limb endovascular revascularization. METHODS: A total of 46 patients (37 male; mean age 66 ± 11 years) undergoing elective lower limb percutaneous revascularization (angioplasty/stent) for symptomatic PAD were recruited from 2 vascular centers. Exclusion criteria were: acute limb ischemia, active infection and/or wet gangrene, liver disease, end-stage renal disease, and cancer. Patients having open revascularization or hybrid (open combined with endovascular) procedures were also excluded. Peripheral venous blood samples were taken on admission and 24 hrs after the procedure. Levels of MMP-2, MMP-3, MMP-7, and MMP-9 were measured along with tissue inhibitors of MMPs (TIMPs) 1 and 2. RESULTS: Compared to baseline values, there was a significant elevation in serum MMP-3 (P = 0.014) and MMP-7 (P = 0.008) levels, whereas serum MMP-9 showed a nonsignificant trend to increase (P = 0.169). On the other hand, no significant alterations were found 24 hrs after angioplasty/stenting with regard to the MMP-2 level and TIMP-1 and 2 levels. CONCLUSIONS: This study documented the periprocedural profile of circulating MMPs in patients undergoing angioplasty/stenting for PAD. The implications of increased MMP-3 and MMP-7 activity after peripheral endovascular interventions and their potential clinical relevance require further investigation.

Quantitative Measurement of L1 Human Papillomavirus Type 16 Methylation for the Prediction of Preinvasive and Invasive Cervical Disease.

Background: Methylation of the human papillomavirus (HPV) DNA has been proposed as a novel biomarker. Here, we correlated the mean methylation level of 12 CpG sites within the L1 gene, to the histological grade of cervical precancer and cancer. We assessed whether HPV L1 gene methylation can predict the presence of high-grade disease at histology in women testing positive for HPV16 genotype. Methods: Pyrosequencing was used for DNA methylation quantification and 145 women were recruited. Results: We found that the L1 HPV16 mean methylation (±SD) significantly increased with disease severity (cervical intraepithelial neoplasia [CIN] 3, 17.9% [±7.2] vs CIN2, 11.6% [±6.5], P < .001 or vs CIN1, 9.0% [±3.5], P < .001). Mean methylation was a good predictor of CIN3+ cases; the area under the curve was higher for sites 5611 in the prediction of CIN2+ and higher for position 7145 for CIN3+. The evaluation of different methylation thresholds for the prediction of CIN3+ showed that the optimal balance of sensitivity and specificity (75.7% and 77.5%, respectively) and positive and negative predictive values (74.7% and 78.5%, respectively) was achieved for a methylation of 14.0% with overall accuracy of 76.7%. Conclusions: Elevated methylation level is associated with increased disease severity and has good ability to discriminate HPV16-positive women that have high-grade disease or worse.

Image analysis and multi-layer perceptron artificial neural networks for the discrimination between benign and malignant endometrial lesions.

BACKGROUND: This study aims to investigate the efficacy of an Artificial Neural Network based on Multi-Layer Perceptron (ANN-MPL) to discriminate between benign and malignant endometrial nuclei and lesions in cytological specimens. METHODS: We collected 416 histologically confirmed liquid-based cytological smears from 168 healthy patients, 152 patients with malignancy, 52 with hyperplasia without atypia, 20 with hyperplasia with atypia, and 24 patients with endometrial polyps. The morphometric characteristics of 90 nuclei per case were analyzed using a custom image analysis system; half of them were used to train the MPL-ANN model, which classified each nucleus as benign or malignant. Data from the remaining 50% of cases were used to evaluate the performance and stability of the ANN. The MLP-ANN for the nuclei classification (numeric and percentage classifiers) and the algorithms for the determination of the optimum threshold values were estimated with in-house developed software for the MATLAB v2011b programming environment; the diagnostic accuracy measures were also calculated. RESULTS: The accuracy of the MPL-ANN model for the classification of endometrial nuclei was 81.33%, while specificity was 88.84% and sensitivity 69.38%. For the case classification based on numeric classifier the overall accuracy was 90.87%, the specificity 93.03% and the sensitivity 87.79%; the indices for the percentage classifier were 95.91%, 93.44%, and 99.42%, respectively. CONCLUSION: Computerized systems based on ANNs can aid the cytological classification of endometrial nuclei and lesions with sufficient sensitivity and specificity. Diagn. Cytopathol. 2017;45:202-211. © 2016 Wiley Periodicals, Inc.

Endocervical Carcinogenesis and HPV Vaccination: An Occasional Circumstance or a Gap in the Chain?

As a result of the Human Papillomavirus (HPV) vaccination program, the prevalence of precancerous dysplasia and invasive cervical cancer has substantially decreased. In this brief report, we present a case of a young patient who was diagnosed with in situ adenocarcinoma of the cervix. This 30-year-old female had completed the HPV vaccination after she became sexually active and has been undergoing annual gynecological assessments, including clinical examination and Pap test, all of which had been negative. This year, her Pap test revealed a low grade squamous intraepithelial lesion (LGSIL) and additionally a colposcopy was performed. Given the extent of the lesion and since the colposcopy was inadequate, the patient underwent a type 3 large loop excision of the transformation zone and a curettage of the endocervix under local anesthesia. The pathological diagnosis from cervical biopsy revealed an in situ adenocarcinoma of the endocervix with negative limits. The HPV subtypes 16 and 83 were detected with PCR. After proper consultation she decided to preserve her fertility and to undergo a regular follow-up, postponing hysterectomy after the completion of her family planning. In conclusion, this case report highlights the need for diagnostic surveillance regarding HPV-related cervical cancer even after vaccination.

γH2Ax Expression as a Potential Biomarker Differentiating between Low and High Grade Cervical Squamous Intraepithelial Lesions (SIL) and High Risk HPV Related SIL.

BACKGROUND: γH2AX is a protein biomarker for double-stranded DNA breakage; its expression was studied in cervical squamous intraepithelial lesions and carcinomas. METHODS: Immunostaining for phospho-γH2AX was performed in sections from histologically confirmed cervical SIL and carcinomas, as well as from normal cervices used as controls. In total, 275 cases were included in the study: 112 low grade SIL (LGSIL), 99 high grade SIL (HGSIL), 24 squamous cell carcinoma (SCC), 12 adenocarcinoma and 28 cervical specimens with no essential lesions. Correlation of histological grading, high risk vs. low risk HPV virus presence, activated vs. non-activated status (by high risk HPV mRNA expression) and γH2AX expression in both basal and surface segments of the squamous epithelium was performed. RESULTS: Gradual increase of both basal and surface γH2AX expression was noted up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state (p<0.05). Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL. CONCLUSIONS: γH2AX could serve as a potential biomarker discriminating between LGSIL and HGSIL, as well as between LGSIL harboring high risk HPV at an activated state.

Apoptosis Induction and Gene Expression Profile Alterations of Cutaneous T-Cell Lymphoma Cells following Their Exposure to Bortezomib and Methotrexate.

Mycosis fungoides (MF) and its leukemic variant Sézary syndrome (SS) comprise the majority of CTCL, a heterogenous group of non-Hodgkins lymphomas involving the skin. The CTCL's resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation. With the view of a more targeted therapy, we evaluated in vitro the effectiveness of bortezomib and methotrexate, as well as their combination in CTCL cell lines, regarding apoptosis induction. Our data are of clinical value and indicate that the bortezomib/methotrexate combinational therapy has an inferior impact on the apoptosis of CTCL compared to monotherapy, with bortezomib presenting as the most efficient treatment option for SS and methotrexate for MF. Using PCR arrays technology, we also investigated the alterations in the expression profile of genes related to DNA repair pathways in CTCL cell lines after treatment with bortezomib or methotrexate. We found that both agents, but mostly bortezomib, significantly deregulate a large number of genes in SS and MF cell lines, suggesting another pathway through which these agents could induce apoptosis in CTCL. Finally, we show that SS and MF respond differently to treatment, verifying their distinct nature and further emphasizing the need for discrete treatment approaches.