Fluctuation of Acquired Resistance Mutations and Re-Challenge with EGFR TKI in Metastatic NSCLC: A Case Report.

Osimertinib has become the preferred first-line therapy for epidermal growth factor receptor ( mutation-positive metastatic non-small cell lung cancer (NSCLC) in recent years. Originally, it was approved for second-line treatment after epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) of the first and second generations had failed and T790M had emerged as a mode of resistance. Osimertinib itself provokes a wide array of on- and off-target molecular alterations that can limit therapeutic success.

Generation of Antibodies Selectively Recognizing Epitopes in a Formaldehyde-Fixed Cell-Surface Antigen Using Virus-like Particle Display and Hybridoma Technology.

Efficient induction of target-specific antibodies can be elicited upon immunization with highly immunogenic virus-like particles (VLPs) decorated with desired membrane-anchored target antigens (Ags). However, for example, for diagnostic purposes, monoclonal antibodies (mAbs) are required to enable the histological examination of formaldehyde-fixed paraffin-embedded (FFPE) biopsy tissue samples. Aiming at the generation of FFPE-antigen-specific mAbs and as a proof of concept (POC), we first established a simplified protocol using only formaldehyde and 90 °C heat fixation (FF90) of cells expressing the target Ag nerve growth factor receptor (NGFR).

Development of the NOGGO GIS v1 Assay, a Comprehensive Hybrid-Capture-Based NGS Assay for Therapeutic Stratification of Homologous Repair Deficiency Driven Tumors and Clinical Validation.

The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA).

Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.

Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein.

Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors.

To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 10 transducing units/mL (TU/mL).

Response of an HER2-Mutated NSCLC Patient to Trastuzumab Deruxtecan and Monitoring of Plasma ctDNA Levels by Liquid Biopsy.

HER2-targeted therapy is currently the subject of several studies in lung cancer and other solid tumors using either tyrosine kinase inhibitors (TKI) or targeted-antibody-drug conjugates. We describe a 61-year-old female patient with HER2 mutated adenocarcinoma of the lungs who received chemo-immunotherapy, followed by trastuzumab deruxtecan (T-DXd) and third-line Ramucirumab/Docetaxel at disease progression. Plasma ctDNA monitoring was obtained at 12 timepoints during therapy and revealed HER2 mutation allele frequencies that corresponded to the clinical course of disease.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a 'pull-down assay' used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs.

Outcome of Ordinary Polymorphous Adenocarcinomas of the Salivary Glands in Comparison With Papillary and Cribriform Subtypes.

Background/aim: Polymorphous adenocarcinoma (PAC) is a low-grade salivary gland malignancy in contrast to variants with papillary (PAP) or cribriform (CASG) architecture and confers the second most common malignancy of minor salivary glands. Our study aimed to identify prognostic factors and to evaluate histomorphological and molecular diagnostic criteria of PACs.

Patients And Methods: A series of 155 PACs, including 10 PAPs and 12 CASGs from the population-based Cancer Registry of North Rhine-Westphalia (LKR-NRW) and the Hamburg Salivary Gland Reference Centre (HRC) were analyzed.

Infectious RNA: Human Immunodeficiency Virus (HIV) Biology, Therapeutic Intervention, and the Quest for a Vaccine.

Different mechanisms mediate the toxicity of RNA. Genomic retroviral mRNA hijacks infected host cell factors to enable virus replication. The viral genomic RNA of the human immunodeficiency virus (HIV) encompasses nine genes encoding in less than 10 kb all proteins needed for replication in susceptible host cells.

Components of a HIV-1 vaccine mediate virus-like particle (VLP)-formation and display of envelope proteins exposing broadly neutralizing epitopes.

The sequence diversity of HIV-1 is the biggest hurdle for the design of a prophylactic vaccine. Mosaic (Mos) antigens consisting of synthetically shuffled epitopes from various HIV-1 strains are currently tested in the clinical vaccine trial Mosaico (NCT03964415). Besides adenovirus vectors encoding variants of Mos.

Comparison of Resistance Spectra after First and Second Line Osimertinib Treatment Detected by Liquid Biopsy.

Since 2009, several first, second, and third generation tyrosine kinase inhibitors (TKI) have been approved for targeted treatment of mutated metastatic non-small lung cancer (NSCLC). A vast majority of patients is improving quickly on treatment; however, resistance is inevitable and typically occurs after one year for TKI of the first and second generation. Osimertinib, a third generation TKI, has recently been approved for first line treatment in the palliative setting and is expected to become approved for the adjuvant setting as well.

Integration of Tumor Mutation Burden and PD-L1 Testing in Routine Laboratory Diagnostics in Non-Small Cell Lung Cancer.

In recent years, Non-small cell lung cancer (NSCLC) has evolved into a prime example for precision oncology with multiple FDA-approved "precision" drugs. For the majority of NSCLC lacking targetable genetic alterations, immune checkpoint inhibition (ICI) has become standard of care in first-line treatment or beyond. PD-L1 tumor expression represents the only approved predictive biomarker for PD-L1/PD-1 checkpoint inhibition by therapeutic antibodies.

Determination of PD-L1 Expression in Circulating Tumor Cells of NSCLC Patients and Correlation with Response to PD-1/PD-L1 Inhibitors.

Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch System. We tested an epitope-independent method (Parsortix system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients.

Nucleocytosolic depletion of the energy metabolite acetyl-coenzyme a stimulates autophagy and prolongs lifespan.

Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast.