CD20(+) T cells have a predominantly Tc1 effector memory phenotype and are expanded in the ascites of patients with ovarian cancer.

Copyright © 2015 Taylor & Francis Group, LLCRecently, a small subset of T cells that expresses the B cell marker CD20 has been identified in healthy volunteers and in patients with rheumatoid arthritis and multiple sclerosis. The origin of these CD20-positive T cells as well as their relevance in human disease remains unclear. Here, we identified that after functional B cell/T cell interaction CD20 molecules are transferred to the cell surface of T cells by trogocytosis together with the established trogocytosis marker HLA-DR. Further, the presence of CD20 on isolated CD20(+) T cells remained stable for up to 48h of ex vivo culture. These CD20(+) T cells almost exclusively produced IFNγ (∼70% vs. ∼20% in the CD20(-) T cell population) and were predominantly (CD8(+)) effector memory T cells (∼60-70%). This IFNγ producing and effector memory phenotype was also determined for CD20(+) T cells as detected in the peripheral blood and ascitic fluids of ovarian cancer (OC) patients. In the latter, the percentage of CD20(+) T cells was further strongly increased (from ∼6% in peripheral blood to 23% in ascitic fluid). Taken together, the data presented here indicate that CD20 is transferred to T cells upon intimate T cell/B cell interaction. Further, CD20(+) T cells are of memory and IFNγ producing phenotype and are present in increased amounts in ascitic fluid of OC patients.Netherlands Organization for Scientific ResearchDutch Cancer SocietyAlexander von Humboldt Foundatio

Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3

Background: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. Results: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p <= 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. Conclusions: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts

Detecting cervical cancer by quantitative promoter hypermethylation assay on cervical scrapings: A feasibility study

Current morphology-based cervical cancer screening is associated with significant false-positive and false-negative results. Tumor suppressor gene hypermethylation is frequently present in cervical cancer. It is unknown whether a cervical scraping reflects the methylation status of the underlying epithelium, and it is therefore unclear whether quantitative hypermethylation specific PCR (QMSP) on cervical scrapings could be used as a future screening method augmenting the current approach. Cervical scrapings and paired fresh frozen cervical tissue samples were obtained from 53 cervical cancer patients and 45 controls. All scrapings were morphologically scored and analyzed with QMSP for the genes APC, DAPK, MGMT, and GSTP1. To adjust for DNA input, hypermethylation ratios were calculated against DNA levels of a reference gene. Hypermethylation ratios of paired fresh frozen tissue samples and scrapings of cervical cancer patients and controls were strongly related (Spearman correlation coefficient, 0.80 for APC, 0.98 for DAPK, and 0.83 for MGMT; P <0.001). More cervical cancer patients than controls were DAPK positive (P <0.001). When cutoff levels for ratios were defined to be above the highest ratio observed in controls, QMSP in cervical scrapings identified 32 (67%) of 48 cervical cancer patients. This feasibility study demonstrates that QMSP on cervical scrapings holds promise as a new diagnostic tool for cervical cancer. The addition of more genes specifically methylated in cervical cancer will further improve the assay

Detecting cervical cancer by quantitative promoter hypermethylation assay on cervical scrapings:A feasibility study

Current morphology-based cervical cancer screening is associated with significant false-positive and false-negative results. Tumor suppressor gene hypermethylation is frequently present in cervical cancer. It is unknown whether a cervical scraping reflects the methylation status of the underlying epithelium, and it is therefore unclear whether quantitative hypermethylation specific PCR (QMSP) on cervical scrapings could be used as a future screening method augmenting the current approach. Cervical scrapings and paired fresh frozen cervical tissue samples were obtained from 53 cervical cancer patients and 45 controls. All scrapings were morphologically scored and analyzed with QMSP for the genes APC, DAPK, MGMT, and GSTP1. To adjust for DNA input, hypermethylation ratios were calculated against DNA levels of a reference gene. Hypermethylation ratios of paired fresh frozen tissue samples and scrapings of cervical cancer patients and controls were strongly related (Spearman correlation coefficient, 0.80 for APC, 0.98 for DAPK, and 0.83 for MGMT; P <0.001). More cervical cancer patients than controls were DAPK positive (P <0.001). When cutoff levels for ratios were defined to be above the highest ratio observed in controls, QMSP in cervical scrapings identified 32 (67%) of 48 cervical cancer patients. This feasibility study demonstrates that QMSP on cervical scrapings holds promise as a new diagnostic tool for cervical cancer. The addition of more genes specifically methylated in cervical cancer will further improve the assay

Ovarian stimulation for IVF: mild approaches

Item does not contain fulltextIn contrast to current approaches, the aim of mild stimulation is to develop safer and more patient-friendly protocols in which the risks of the treatment as a whole are minimized. Mild stimulation is defined as the method when exogenous gonadotropins are administered at lower doses, and/or for a shorter duration in GnRH antagonist co-treated cycles, or when oral compounds (antiestrogens, aromatase inhibitors) are used for ovarian stimulation for IVF, with the aim of limiting the number of oocytes obtained to fewer than eight. In this chapter we discuss the relevant physiology of follicle development, the development of milder stimulation protocols, the implications of mild stimulation, the current state of affairs, and future developments