Non-invasive detection of EGFR mutations by cell-free loop-mediated isothermal amplification (CF-LAMP)

Arjuna, Srividya; Venkataram, Rajesh; Dechamma, Pandyanda Nanjappa; Chakraborty, Gunimala; Babu, Nishith; D'Cruz, Audrey Madonna; Hosmane, Giridhar Belur; Chakraborty, Anirban

doi:10.1038/s41598-020-74689-3

Cited by 7 publications

(4 citation statements)

References 22 publications

(28 reference statements)

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“…A second report from the same research group demonstrated a cost effective and sensitive LAMP for detecting therapeutically-relevant EGFR hotspot mutations in a non-invasive manner. The results consistency when compared to those obtained by ultrasensitive droplet digital PCR (ddPCR) assay indicates the robustness of the developed method 33 .…”

Section: Discussionsupporting

confidence: 53%

Fluorescent and colorimetric RT-LAMP as a rapid and specific qualitative method for chronic myeloid leukemia diagnosis

Aoki

Marin

Zanette

et al. 2022

Analytical Biochemistry

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The detection of BCR-ABL1 mRNA transcripts is essential to molecular chronic myeloid leukemia (CML) diagnosis. In most cases, the RT-qPCR technique is performed as the gold standard diagnosis tool for clinical cases. However, this method requires expensive reagents and equipment, such as a real-time thermal cycler, probes and master mix. Consequently, the development and validation of simple and lowcost methods are essential for a rapid CML diagnosis in less specialized and equipped centers. In this study, we develop and demonstrate an accessible, rapid, and low-cost method using RT-LAMP for BCR-ABL1 detection in both cell lines and CML clinical samples, using colorimetric and uorescent assays. Differently to the Q-LAMP assay described in 2019 by Stella and collaborators, the samples here were analyzed by RT-qPCR and the results were compared to the results obtained by uorescent and colorimetric RT-LAMP. The obtained data indicates that the proposed method here described is a cheaper, robust and speci c approach for CML diagnosis with outstanding performance.

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Section: Discussionsupporting

confidence: 53%

Fluorescent and colorimetric RT-LAMP as a rapid and specific qualitative method for chronic myeloid leukemia diagnosis

Aoki

Marin

Zanette

et al. 2022

Analytical Biochemistry

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“…Here we demonstrate a compact AS-Mini-LAMP + USS mutant speci c reaction that consists of 6-primers (FIP, BIP, F3, B3, USS FB, USS BB) spanning 155 bp, within the typical range of plasma cfDNA. In contrast to alternative mutation-selective LAMP design strategies (PNA-LNA-LAMP 11 and AS-LAMP/CF-LAMP 13,14 ) selectivity is encoded at the terminal 5' position of both the FIP and BIP primers, promoting selective self-primed exponential ampli cation upon self-hybridisation and loop formation from the FIP and BIP initiated reactions. Using synthetic template models, the mutant selective reaction (AS-Mini-LAMP Mut + USS WT ) demonstrated analytical sensitivity of 500 copies of mutant DNA within 25 minutes, > 20 minutes ahead of non-selective WT template.…”

Section: Discussionmentioning

confidence: 99%

“…Arjuna et al, 13 developed allele-speci c LAMP (AS-LAMP), a typical 6-primer LAMP reaction, incorporating the T790M EGFR mutation or wild type counterpart in the backward inner primer (BIP). Subsequent application on cfDNA template material (termed CF-LAMP) included a pre-LAMP conventional PCR using LAMP outer primers (F3 and B3) to enrich for copies of target cfDNA prior to CF-LAMP 14 . Our previous work describes a universal self-stabilising single base-LAMP method (USS-sbLAMP) 5 , a typical 6-primer reaction encoding single base variant selectivity at the 5' end of both the forward and backward inner primers (FIP and BIP), coupled with unmodi ed self-stabilising (USS) competitive primers for enhanced prevention of un-speci c wild type variant base ampli cation.…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Allsopp

Alexandrou

Toumazou

et al. 2022

Preprint

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Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

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“…However, there are no reports of such variations being detected in plasma by droplet digital PCR (ddPCR) technique, the most advanced third generation ultrasensitive PCR technique. We have previously reported that ddPCR can efficiently detect EGFR variations in lung cancer patient-derived plasma [11,12]. Here we are reporting a series of 6 cases of de novo SCLC harbouring common as well as rare activating EGFR variations in the plasma as detected by droplet digital PCR (ddPCR).…”

Section: Introductionmentioning

confidence: 99%

Detection of clinically-relevant <em>EGFR</em> variations in <em>de novo</em> small cell lung carcinoma by droplet digital PCR

Venkataram¹,

Shetty²,

Prasad³

et al. 2022

Monaldi Arch Chest Dis

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Targeted therapy that utilizes tyrosine kinase inhibitors (TKIs), specific to epidermal growth factor receptors (EGFR) has changed the landscape of treatment of non-small cell lung cancer (NSCLC). The success or failure of this approach depends on presence of certain variations in the tyrosine kinase domain of EGFR gene. Generally, patients diagnosed with Small cell lung cancer (SCLC) are considered ineligible for TKI therapy owing to the absence of EGFR variations. . However, there is evidence of these variations being detected in SCLCs, both in de-novo and in transformed SCLCs (TKI-treated adenocarcinomas). Despite the presence of clinically-relevant EGFR variations in SCLCs, the response to TKIs has been inconsistent. Liquid biopsy is a well-established approach in lung cancer management with proven diagnostic, prognostic and predictive applications. It relies on detection of circulating tumor-derived nucleic acids present in plasma of the patient. In this study, a liquid biopsy approach was utilized to screen 118 consecutive lung cancer patients for four clinically-relevant variations in EGFR gene, which included three activating/sensitizing variations (Ex18 G719S, Ex19del E746-A750 and Ex21 L858R) and one acquired/resistance (Ex20 T790M, de novo) variation by droplet digital PCR, the most advanced third generation PCR technique. As expected, clinically-relevant EGFR variations were found in majority of the non-small cell lung cancer cases. However, among the handful of small cell lung cancer samples screened, sensitizing variations (Ex18 G719S and Ex21 L858R) were seen in almost all of them. Interestingly, Ex20 T790M variation was not detected in any of the cases screened. The results of our study indicate that EGFR variations are present in SCLCs and highly sensitive liquid biopsy techniques like ddPCR can be effectively utilized for this purpose of screening EGFR variations in such samples.

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Non-invasive detection of EGFR mutations by cell-free loop-mediated isothermal amplification (CF-LAMP)

Cited by 7 publications

References 22 publications

Fluorescent and colorimetric RT-LAMP as a rapid and specific qualitative method for chronic myeloid leukemia diagnosis

Fluorescent and colorimetric RT-LAMP as a rapid and specific qualitative method for chronic myeloid leukemia diagnosis

Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Detection of clinically-relevant <em>EGFR</em> variations in <em>de novo</em> small cell lung carcinoma by droplet digital PCR

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