Identification of an MSI-H Tumor-Specific Cytotoxic T Cell Epitope Generated by the (−1) Frame of U79260(FTO)

Microsatellite instability (MSI-H) induced by defects of the DNA mismatch repair system results in insertion or deletion of single nucleotides at short repetitive DNA sequences. About 15% of sporadic and approximately 90% of hereditary nonpolyposis colorectal cancers display MSI-H. When affecting coding regions, MSI-H results in frameshift mutations and expression of corresponding frameshift peptides (FSPs). Functional tumor promoting relevance has been demonstrated for a growing number of genes frequently hit by MSI-H. Contrary, immune reactions against FSPs are involved in the immune surveillance of MSI-H cancers. Here, we provide conclusive data that the (−1) frame of U79260(FTO) encodes an HLA-A0201-restricted cytotoxic T cell epitope (FSP11; TLSPGWSAV). T cells specific for FSP11 efficiently recognized HLA-A0201(pos) tumor cells harboring the mutated reading frame. Considering the exceptionally high mutation rate of U79260(FTO) in MSI-H colorectal carcinoma (81.8%), this recommends that FSP11 be a component of future vaccines

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

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

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