Loop-Mediated Isothermal Amplification as Point-of-Care Testing for EGFR-Mutated Lung Adenocarcinoma.

Liquid biopsy has been adapted as a diagnostic test for EGFR mutations in patients with advanced or metastatic non-small cell lung cancer (NSCLC). Loop-mediated isothermal amplification (LAMP) has been widely used for the rapid detection of pathogens through DNA amplification. This study investigated the efficacy of an EGFR-LAMP assay using plasma samples of patients with resected NSCLC tumors. The EGFR status was investigated using both LAMP and next-generation sequencing (NGS) assays in cases that met the following criteria: (1) pulmonary adenocarcinoma with EGFR mutation detected by the Therascreen EGFR PCR Kit and (2) preoperative plasma samples contained enough DNA for the LAMP and NGS experiments. Among 51 specimens from patients with EGFR-mutated tumors or metastatic lymph nodes, the LAMP assay detected 1 EGFR mutation that was also detected in the NGS assay. However, a plasma sample that demonstrated EGFR wild type in the LAMP assay showed an EGFR mutant status in NGS. The detection rates (1.9% in LAMP and 3.9% in NGS) were very low in both assays, demonstrating a similar performance in detecting EGFR mutations in NSCLC tumors; therefore, it could be a more suitable test for the advanced stage, not the early stage. Notably, the LAMP assay was more time-saving, cost-effective, and straightforward. However, further investigation is required to develop a more sensitive assay.

Development and clinical validation of loop-mediated isothermal amplification (LAMP) assay to diagnose high HBV DNA levels in resource-limited settings.

OBJECTIVE: A massive scale-up of testing and treatment is indicated to globally eliminate hepatitis B virus (HBV) infection. However, access to a polymerase chain reaction (PCR), a key test to quantify HBV DNA levels and determine treatment eligibility, is limited in resource-limited countries. We have developed and evaluated the loop-mediated isothermal amplification (LAMP) assay to diagnose clinically important HBV DNA thresholds defined by the WHO (≥20 000 and ≥ 200 000 IU/mL). METHODS: Pan-genotypic primer sets were designed on conserved HBV gene regions. Accuracy of LAMP to identify highly viraemic patients was evaluated in 400 and 550 HBV-infected people in France and Senegal, respectively. RESULTS: Our primers successfully detected eight major HBV genotypes/sub-genotypes (A1/2/3/B/C/D/E/F) with a detection limit ranging between 40 and 400 IU/mL. In France, the area under the receiver operating characteristic curve (AUROC), sensitivity and specificity of bead-based extraction and real-time turbidimetric LAMP were 0.95 (95% CI 0.93-0.97), 91.1% and 86.0%, respectively, to diagnose HBV DNA ≥20 000 IU/mL; and 0.98 (0.97-0.99), 98.0% and 94.6% for ≥200 000 IU/mL. The performance did not vary by viral genotypes. In Senegal, using a field-adapted method (reagent-free boil-and-spin extraction and inexpensive end-point fluorescence detection), the AUROC, sensitivity and specificity were 0.95 (0.93-0.97), 98.7% and 91.5%, respectively, to diagnose HBV DNA ≥200 000 IU/mL. The assay was not adapted to discriminate low-level viraemia. DISCUSSION: We have developed a simple, rapid (60 min), and inexpensive (US$8/assay) alternative to PCR to diagnose high viraemia ≥200 000 IU/mL. HBV-LAMP may contribute to eliminating HBV mother-to-child transmission by identifying high-risk pregnant women eligible for antiviral prophylaxis in resource-limited countries.

Comparative study of the loop-mediated isothermal amplification method and the QIAGEN therascreen PCR kit for the detection of EGFR mutations in non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are important biomarkers in the treatment of patients with advanced or metastatic diseases. The therascreen EGFR Rotor-Gene Q (RGQ) PCR Kit® (Qiagen, Inc.) is an approved diagnostic test for EGFR mutations in non-small cell lung cancer (NSCLC). This study aims to investigate the diagnostic capability of a loop-mediated isothermal amplification (LAMP) assay as an accurate, efficient, and cost-effective alternative to the therascreen assay. METHODS: EGFR mutations were investigated by LAMP and therascreen assays using tissue samples that were surgically resected or biopsied from 117 consecutive patients with NSCLC tumors. The EGFR status from the LAMP assay was compared with that of the therascreen assay. Next-generation sequencing (NGS) was performed to confirm EGFR status of tumors that did not match in both assays. To establish an optimal LAMP AUC value, receiver operating characteristics (ROC) curve analysis was performed within tumors with exon 19 deletion or L858R point mutation. RESULTS: Of the 117 tumors assayed, 45 tumors with EGFR mutations and 68 tumors with EGFR wild type were matched in both assays, four tumors having mismatched EGFR statuses. NGS further confirmed that two of the four discordant tumors had the same EGFR status that was determined by the LAMP assay. The AUC values were 0.973 (95% CI: 0.929-1.00) in exon 19 deletion, and 0.952 (95% CI: 0.885-1.00) in L858R point mutation. In exon 19 deletion, sensitivity, specificity, and accuracy were 89.3%, 98.9%, and 96.6%, respectively, and 94.7%, 95.9%, and 95.7%, respectively, in L858R using AUC value of 0.222. CONCLUSIONS: The LAMP assay compared favorably with the therascreen assay and has potential as an effective, simple, rapid, and low-cost diagnostic alternative. Based on these results, a liquid biopsy LAMP system should be developed for point-of-care testing of oncogenes in the near future.

Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification.

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

Mff functions with Pex11pß and DLP1 in peroxisomal fission.

PEROXISOMAL DIVISION COMPRISES THREE STEPS: elongation, constriction, and fission. Translocation of dynamin-like protein 1 (DLP1), a member of the large GTPase family, from the cytosol to peroxisomes is a prerequisite for membrane fission; however, the molecular machinery for peroxisomal targeting of DLP1 remains unclear. This study investigated whether mitochondrial fission factor (Mff), which targets DLP1 to mitochondria, may also recruit DLP1 to peroxisomes. Results show that endogenous Mff is localized to peroxisomes, especially at the membrane-constricted regions of elongated peroxisomes, in addition to mitochondria. Knockdown of MFF abrogates the fission stage of peroxisomal division and is associated with failure to recruit DLP1 to peroxisomes, while ectopic expression of MFF increases the peroxisomal targeting of DLP1. Co-expression of MFF and PEX11ß, the latter being a key player in peroxisomal elongation, increases peroxisome abundance. Overexpression of MFF also increases the interaction between DLP1 and Pex11pß, which knockdown of MFF, but not Fis1, abolishes. Moreover, results show that Pex11pß interacts with Mff in a DLP1-dependent manner. In conclusion, Mff contributes to the peroxisomal targeting of DLP1 and plays a key role in the fission of the peroxisomal membrane by acting in concert with Pex11pß and DLP1.