Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ CT ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

Hypermethylated RASSF1A as Circulating Tumor DNA Marker for Disease Monitoring in Neuroblastoma.

PURPOSE: Circulating tumor DNA (ctDNA) has been used for disease monitoring in several types of cancer. The aim of our study was to investigate whether ctDNA can be used for response monitoring in neuroblastoma. METHODS: One hundred forty-nine plasma samples from 56 patients were analyzed by quantitative polymerase chain reaction (qPCR) for total cell free DNA (cfDNA; albumin and ß-actin) and ctDNA (hypermethylated RASSF1A). ctDNA results were compared with mRNA-based minimal residual disease (qPCR) in bone marrow (BM) and blood and clinical patient characteristics. RESULTS: ctDNA was detected at diagnosis in all patients with high-risk and stage M neuroblastoma and in 3 of 7 patients with localized disease. The levels of ctDNA were highest at diagnosis, decreased during induction therapy, and not detected before or after autologous stem-cell transplantation. At relapse, the amount of ctDNA was comparable to levels at diagnosis. There was an association between ctDNA and blood or BM mRNA, with concordant results when tumor burden was high or no tumor was detected. The discrepancies indicated either low-level BM infiltration (ctDNA negative/mRNA positive) or primary tumor/soft tissue lesions with no BM involvement (ctDNA positive/mRNA negative). CONCLUSION: ctDNA can be used for monitoring disease in patients with neuroblastoma. In high-risk patients and all patients with stage M at diagnosis, ctDNA is present. Our data indicate that at low tumor load, testing of both ctDNA and mRNA increases the sensitivity of molecular disease monitoring. It is likely that ctDNA can originate from both primary tumor and metastases and may be of special interest for disease monitoring in patients who experience relapse in other organs than BM.

Mesenchymal Neuroblastoma Cells Are Undetected by Current mRNA Marker Panels: The Development of a Specific Neuroblastoma Mesenchymal Minimal Residual Disease Panel.

Patients with neuroblastoma in molecular remission remain at considerable risk for disease recurrence. Studies have found that neuroblastoma tissue contains adrenergic (ADRN) and mesenchymal (MES) cells; the latter express low levels of commonly used markers for minimal residual disease (MRD). We identified MES-specific MRD markers and studied the dynamics of these markers during treatment. PATIENTS AND METHODS: Microarray data were used to identify genes differentially expressed between ADRN and MES cell lines. Candidate genes were then studied using real-time quantitative polymerase chain reaction in cell lines and control bone marrow and peripheral blood samples. After selecting a panel of markers, serial bone marrow, peripheral blood, and peripheral blood stem cell samples were obtained from patients with high-risk neuroblastoma and tested for marker expression; survival analyses were also performed. RESULTS: PRRX1, POSTN, and FMO3 mRNAs were used as a panel for specifically detecting MES mRNA in patient samples. MES mRNA was detected only rarely in peripheral blood; moreover, the presence of MES mRNA in peripheral blood stem cell samples was associated with low event-free survival and overall survival. Of note, during treatment, serial bone marrow samples obtained from 29 patients revealed a difference in dynamics between MES mRNA markers and ADRN mRNA markers. Furthermore, MES mRNA was detected in a higher percentage of patients with recurrent disease than in those who remained disease free (53% v 32%, respectively; P = .03). CONCLUSION: We propose that the markers POSTN and PRRX1, in combination with FMO3, be used for real-time quantitative polymerase chain reaction-based detection of MES neuroblastoma mRNA in patient samples because these markers have a unique pattern during treatment and are more prevalent in patients with poor outcome. Together with existing markers of MRD, these new markers should be investigated further in large prospective studies.