ABSTRACT
CXCR4 mutations impact disease presentation and treatment outcomes in Waldenström macroglobulinemia. Current techniques used for CXCR4 mutation detection have a number of limitations. The aim of the present study was to develop and analytically validate a novel droplet digital PCR (ddPCR) assay for the simultaneous detection of five of the most common CXCR4 mutations in bone marrow (BM). In silico novel primers and probes designed for simultaneous detection of five hotspot mutations of CXCR4 were first performed. Experimental conditions were optimized, and the assay was analytically validated. The developed assay was further applied in 95 BM samples from patients with IgM gammopathy, 7 BM samples from patients with non-IgM gammopathy and 12 PBMCs from healthy donors, whereas a direct comparison study of Sanger sequencing and allele-specific PCR was performed by using 95 and 39 identical patient tumor DNA samples, respectively. The drop-off ddPCR assay is a robust, cost-effective, highly sensitive, and highly specific screening tool for CXCR4 mutations. Of 95 patients with IgM gammopathy samples, 27 had at least one CXCR4 mutation in their BM samples. With Sanger sequencing, 12 of the 95 samples tested positive, whereas the direct comparison of the developed assay with allele-specific PCR revealed substantial agreement. The clinical performance of the developed assay will be prospectively evaluated in a large number of patients, and the applicability of this assay will be further evaluated.
Subject(s)
Lymphoma, B-Cell , Waldenstrom Macroglobulinemia , Humans , Waldenstrom Macroglobulinemia/diagnosis , Waldenstrom Macroglobulinemia/genetics , Mutation , Polymerase Chain Reaction/methods , Alleles , Lymphoma, B-Cell/genetics , Receptors, CXCR4/geneticsABSTRACT
The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons-intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.
ABSTRACT
In March 2020 the World Health Organization announced a pandemic outbreak. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen for the coronavirus disease-19 (COVID-19) pandemic. The authorities worldwide use clinical science to identify infected people, but this approach is not able to track all symptomatic and asymptomatic cases due to limited sampling capacity of the testing laboratories. This drawback is eliminated by the Wastewater-Based Epidemiology (WBE) approach. In this review, we summarized the peer-reviewed published literature (available as of September 28, 2020), in the field of WBE. The commonly used steps (sampling, storage, concentration, isolation, detection) of the analytical protocols were identified. The potential limitations of each stage of the protocols and good practices were discussed. Finally, new methods for the efficient detection of SARS-CoV-2 were proposed.
ABSTRACT
Nowadays, therapeutic strategies in cancer are subsequently defined according to the molecular profile of the tissue [...].
