When False-Positives Arise: Troubleshooting a SARS-Coronavirus-2 (SARS-CoV-2) Detection Assay on a Semi-Automated Platform.

BACKGROUND: During the COVID-19 pandemic, many molecular diagnostic laboratories performed high-throughput SARS-CoV-2 testing often with implementation of automated workflows. In parallel, vaccination campaigns resulted increasingly in specimens from fully vaccinated patients, with resultant clinical inquiries regarding positive results in this patient population. This prompted a quality improvement initiative to investigate the semi-automated testing workflow for false-positive results. The troubleshooting workflow is described and procedural improvements are outlined that serve as a resource for other molecular diagnostic laboratories that need to overcome testing anomalies in a semi-automated environment. METHODS: This workflow utilized the MagMax-96 Viral RNA kit and the CDC 2019-nCoV RT-qPCR Panel on the Agilent Bravo Liquid-Handler (Bravo). Screening of the environment, personnel, and the mechanical performance of instrumentation using low Ct checkerboard challenges was executed to identify sources of cross-contamination. Evaluation of the assay and reporting design was conducted. RESULTS: Specimen contamination was observed during the viral extraction process on the Bravo. Changes to the program reduced plate contamination by 50% and importantly revealed consistent hallmarks of contaminated samples. We adjusted the reporting algorithm using these indicators of false positives. False positives that were identified made up 0.11% of the 45 000+ tests conducted over the following 8 months. CONCLUSIONS: These adjustments provided confident and quality results while maintaining turnaround time for patients and pandemic-related public health initiatives. This corrected false-positive rate is concordant with previously published studies from diagnostic laboratories utilizing automated systems and may be considered a laboratory performance standard for this type of testing.

SLAM/SAP signaling regulates discrete γδ T cell developmental checkpoints and shapes the innate-like γδ TCR repertoire.

During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate γδ T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM-SAP signaling pathway regulates the development and function of multiple innate-like γδ T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent γδ TCR repertoire. SAP deficiency resulted in both a significant loss of an immature Gzma + Blk + Etv5 + Tox2 + γδT17 precursor population, and a significant increase in Cd4 + Cd8+ Rorc + Ptcra + Rag1 + thymic γδ T cells. SAP-dependent diversion of embryonic day 17 thymic γδ T cell clonotypes into the αß T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered γδ TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset. Together, the data suggest that SAP-dependent γδ/αß T cell lineage commitment regulates γδ T cell developmental programming and shapes the γδ TCR repertoire.

Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers.

Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.

Small-cell transformation of ALK-rearranged non-small-cell adenocarcinoma of the lung.

Anaplastic lymphoma kinase (ALK) gene rearrangements are present in ∼5% of non-small-cell lung cancers (NSCLCs). These rearrangements occur because of a chromosomal inversion within the short arm of Chromosome 2, which results in the formation of the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion oncogene. Whereas NSCLC transformation to SCLC is a rare phenomenon described in epidermal growth factor receptor (EGFR) mutant cancers primarily after treatment with targeted therapy, it is exceedingly rare in ALK-rearranged adenocarcinomas. It is currently unclear what the therapeutic significance of the rearrangement is in this transformed tumor as there is a paucity of medical literature describing follow-up care and outcomes of patients in this rare scenario. We describe a unique case in which a patient with ALK-rearranged adenocarcinoma underwent small-cell transformation at a metastatic site with retained ALK rearrangement and was provided clinical follow-up after treatment with second-generation tyrosine kinase inhibiter (TKI) therapy.