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1.
Cancers (Basel) ; 13(6)2021 Mar 12.
Article in English | MEDLINE | ID: mdl-33809329

ABSTRACT

Microsatellite instability (MSI), a common alteration in endometrial cancers (EC) is known as a biomarker for immune checkpoint therapy response alongside screening for Lynch Syndrome (LS). However, former studies described challenging MSI profiles in EC hindering analysis by using MSI testing methods intensively validated for colorectal cancer (CRC) only. In order to reduce false negatives, this study examined four different PCR-based approaches for MSI testing using 25 EC samples already tested for mismatch repair deficiency (dMMR). In a follow up validation set of 75 EC samples previously tested both for MMR and MSI, the efficiency of a seven-marker system corresponding to the Idylla system was further analyzed. Both Bethesda and Promega marker panels require trained operators to overcome interpretation complexities caused by either hardly visible additional peaks of one and two nucleotides, or small shifts in microsatellite repeat length. Using parallel sequencing adjustment of bioinformatics is needed. Applying the Idylla MSI assay, an evaluation of input material is more crucial for reliable results and is indispensable. Following MMR deficiency testing as a first-line screening procedure, additional testing with a PCR-based method is necessary if inconclusive staining of immunohistochemistry (IHC) must be clarified.

2.
Sci Rep ; 10(1): 11387, 2020 07 09.
Article in English | MEDLINE | ID: mdl-32647293

ABSTRACT

Outcome of immune checkpoint inhibition in cancer can be predicted by measuring PDL1 expression of tumor cells. Search for additional biomarkers led to tumor mutational burden (TMB) as surrogate marker for neoantigens presented. While TMB was previously determined via whole exome sequencing (WES), there have been approaches with comprehensive gene panels as well. We sequenced samples derived from formalin-fixed tumors, a POLE mutated cell line and standard DNA by WES and five different panels. If available, normal tissue was also exome sequenced. Sequencing data was analyzed by commercial software solutions and an in-house pipeline. A robust Pearson correlation (R = 0.9801 ± 0.0167; mean ± sd; N = 7) was determined for the different panels in a tumor paired normal setting for WES. Expanded analysis on tumor only exome sequenced samples yielded similar correlation (R = 0.9439 ± 0.0632; mean ± sd; N = 14). Remaining germline variants increased TMB in WES by 5.761 ± 1.953 (mean ± sd.; N = 7) variants per megabase (v/mb) for samples including synonymous variants and 3.883 ± 1.38 v/mb for samples without synonymous variants compared to tumor-normal paired calling results. Due to limited sample numbers in this study, additional replication is suggested for a clinical setting. Remaining germline variants in a tumor-only setting and artifacts caused by different library chemistries construction might affect the results.


Subject(s)
Antigens, Neoplasm/genetics , Biomarkers, Tumor/genetics , DNA Mutational Analysis/methods , Neoplasms/diagnosis , Algorithms , Antigens, Neoplasm/immunology , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Biomarkers, Tumor/analysis , Cell Line, Tumor , Computational Biology , Drug Resistance, Neoplasm/genetics , Germ-Line Mutation , High-Throughput Nucleotide Sequencing , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/immunology , Exome Sequencing
3.
Int J Cancer ; 138(4): 927-38, 2016 Feb 15.
Article in English | MEDLINE | ID: mdl-26340530

ABSTRACT

Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionally, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.


Subject(s)
Carcinoma, Neuroendocrine/genetics , Carcinoma, Neuroendocrine/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Signal Transduction , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA Mutational Analysis , Flow Cytometry , Fluorescent Antibody Technique , High-Throughput Nucleotide Sequencing , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Receptors, Notch/genetics , Receptors, Notch/metabolism , Retinoblastoma Protein/genetics , Transfection , Tumor Suppressor Protein p53/genetics
4.
J Cancer Res Clin Oncol ; 135(5): 723-30, 2009 May.
Article in English | MEDLINE | ID: mdl-19002495

ABSTRACT

PURPOSE: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) has been linked to activating mutations in the EGFR gene. So far these mutations have been extensively characterized in established cell lines. The aim of this study was to determine the effects of EGFR mutations on downstream signaling in human tumor specimens. METHODS: We have looked for mutations of the EGFR gene in specimens of 67 patients with NSCLC and correlated these with EGFR phosphorylation and the activity of its three main downstream signaling cascades Akt, MAPK and Stat3 by immunohistochemistry. RESULTS: We show that the phosphorylation of tyrosine residues 922 and 1173, but not 1068, are primarily affected by the activating EGFR mutations. Akt activity was significantly higher in patients with EGFR mutations but we found no difference in Stat3 or MAPK phosphorylation. Our results suggest that EGFR mutations not only increase receptor activity, but also alter responses of downstream signaling cascades in human NSCLCs and that these finding differ from results obtained in cell lines.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , ErbB Receptors/genetics , Lung Neoplasms/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , STAT3 Transcription Factor/metabolism , Adult , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , DNA, Neoplasm/genetics , DNA, Neoplasm/isolation & purification , ErbB Receptors/metabolism , Exons/genetics , Female , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Male , Middle Aged , Mutation , Phosphorylation , Polymerase Chain Reaction , Sequence Deletion , Signal Transduction/genetics
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