Performance of a DNA methylation marker panel using liquid-based cervical scrapes to detect cervical cancer and its precancerous stages

Abstract Background A change of cervical cancer screening algorithms to an HPV-based screening setting is discussed in many countries, due to higher sensitivity of HPV testing compared to cytology. Reliable triage methods are, however, an essential prerequisite in such a setting to avoid overtreatment and higher screening costs. Results In this study, a series of cervical scrapes collected in PreservCyt liquid-based cytology (LBC) medium from women with cervical cancer (n = 5), cervical intraepithelial neoplasia grade 1–3 (n = 74), and normal cytology (n = 201; further n = 352 collected in SureThin®) were assessed for methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 using the GynTect assay and compared to cobas® HPV and CINtec Plus® biomarker results. All samples from women with cervical cancer, 61.2% of CIN3, 44.4% of CIN2 and 20.0% of CIN1 cases were scored positive for the GynTect methylation assay. In contrast, all CIN, irrespective of severity grade, and carcinomas were positive by both, CINtec Plus and cobas HPV. The specificity of GynTect for CIN3+ was 94.6% compared to 69.9% for CINtec Plus and 82.6% for cobas HPV (all HPV types) and 90.6% for cobas HPV 16/18. DNA methylation analysis of this methylation marker panel (GynTect assay) in cervical scrapes consistently detects cervical cancer and the majority of CIN3 as well as a subset of CIN1/2 lesions. The detection rate among cytologically normal samples is extraordinarily low (1.5%). Conclusion GynTect shows excellent performance when using cervical scrape material collected in liquid-based cytology media, a prerequisite for employing such a test as a triage in screening programs. Compared to the other test systems used in this work, GynTect showed higher specificity while still detecting all cancer cases

New Model for Gastroenteropancreatic Large-Cell Neuroendocrine Carcinoma: Establishment of Two Clinically Relevant Cell Lines

<div><p>Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. <i>In vitro</i> and <i>in vivo</i> tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. <i>In vitro</i> and <i>in vivo</i> experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.</p></div

NEC cell lines express general and specific neuroendocrine markers, somatostatin receptors and transcription factors.

<p>RNA from cultured NEC cell lines was isolated and RT-PCR analyses performed for (<b>A</b>) general neuroendocrine markers, (<b>B</b>) somatostatin receptors, (<b>C</b>) specific neuroendocrine markers and (<b>D</b>) transcription factors as indicated. The colon cancer cell line HCT116 served as control cell line.</p

Immunohistochemical expression analyses of the cell lines NEC-DUE1 and NEC-DUE2.

<p>NSE neuron-specific enolase, VMAT vesicular monoamine transporter, CD56 cluster of differentiation 56, NCAM Neural Cell Adhesion Molecule, SSTR somatostatin receptor, CK cytokeratin, CEA carcinoembryonic antigen, Ca 19.9 carbohydrate antigen 19-9, TTF1 thyroid transcription factor 1, CDX2 caudal type homeobox 2.</p

Cytogenetic changes in large-cell NEC cell lines.

<p>DNA was isolated from the cell lines, primary tumors (PT) and hepatic or lymphatic metastases (M) and genetic aberrations were analyzed by aCGH analysis. Amplitudes over the midline reflect chromosomal gains, amplitudes under the midline losses. M I and M II represent the atypically resected liver metastases of the gastroesophageal NEC.</p

Sensitivity of NEC cell lines to conventional chemotherapeutics.

<p>NEC-DUE1 and –DUE2 were treated for 24 hours with etoposide (<b>A</b>), cisplatin (<b>B</b>), 5-FU (<b>C</b>) or oxaliplatin (<b>D</b>). Cell viability was measured using the MTS assay as described in materials and methods. Values represent the mean absorbance at 490 nm ± SD of triplicates.</p

Electron microscopy of large-cell NEC cell lines.

<p>Electron microscopy revealed electron-dense large dense core neurosecretory granules in both NEC cell lines (inset demonstrates magnified electron dense granules). HCT116 served as negative control cell line.</p

NEC-DUE1 and NEC-DUE2 retain the expression of neuroendocrine marker with increasing numbers of passage.

<p>RNA from cultured NEC cell lines NEC-DUE1 (<b>A</b>) and NEC-DUE2 (<b>B</b>) at indicated culture passages was isolated and RT-PCR analyses performed for neuroendocrine markers. Immunocytochemical staining of selected neuroendocrine markers was performed in NEC-DUE1 (<b>C</b>) and NEC-DUE2 (<b>D</b>) cells grown on cover slips at different numbers of culture passage. NC = negative control, p = number of passage.</p

Morphological and immunohistochemical characterization of primary NECs.

<p>Primary tumors from which the cell lines NEC-DUE1 (<b>A</b>) and NEC-DUE2 (<b>B</b>) derived stained with hematoxylin-eosin (HE) showing morphologically large cell neuroendocrine cytology, i.e. large-sized cells with large atypical nuclei revealing a “salt and pepper” chromatin. Synaptophysin (SYN), chromogranin A (CGA), vesicular monoamine transporters (VMAT1, VMAT2), somatostatin receptor (SSTR2A), thyroid transcription factor 1 (TTF1), caudal type homeobox 2 (CDX2), cluster of differentiation 56 (CD56) and cytokeratins (CK) as well as epithelial markers (CEA, Ca19.9) were immunohistochemically evaluated as indicated and proliferation index is demonstrated by staining with MIB-1 antibody.</p