TEST:

therascreen® EGFR RGQ PCR Kit

Our study highlights that NGS is able to identify a much higher number of actionable EGFR mutations than RT-qPCR approaches, thereby providing many more patients the opportunity to receive targeted EGFR treatments.

Of 85 patients with NSCLC with disease progression after TKI treatment, T790M mutations were detected during digital PCR in 30 of 85 patients, which is 35.2% of the sample, and with traditional real-time PCR, positive mutations came out only in 3 out of 85 patients.

P2; Trial completion date: Sep 2024 --> Dec 2025 | Trial primary completion date: Sep 2024 --> Dec 2025

This improved method demonstrates that cytology smears can be used as a test material for the detection of EGFR mutations in patients with NSCLC with sparse cells.

Further study is suggested to review other parameters in characterizing the insufficient sample.

"Our results evidenced an equivalent detection ability between PCR-based techniques for circulating EGFR mutations. The NGS assay allowed detection of a wider range of EGFR mutations but showed a poor ability to detect T790M."

P2; Trial completion date: Mar 2024 --> Sep 2024 | Trial primary completion date: Mar 2024 --> Sep 2024

P2; Trial completion date: Mar 2023 --> Mar 2024 | Trial primary completion date: Mar 2023 --> Mar 2024

P2; Recruiting --> Active, not recruiting | N=80 --> 116

P2; N=36 --> 0 | Trial completion date: Dec 2024 --> Feb 2023 | Not yet recruiting --> Withdrawn | Trial primary completion date: Dec 2024 --> Feb 2023

The frequency of mutated cases is slightly higher than the frequency already described in a previous study by our team (11.5%) but close to the frequency of mutated cases in the Cauca-sian population (16%). The most frequent activating mutations are deletions of exon 19 and the L858R mutation of exon 21, the two representing 90% of activating mutations.

P2; Initiation date: Feb 2022 --> Feb 2023

"This study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs."

"Five of the 7 ex20ins were found in advanced stage patients, two of them received first line chemotherapy and two an EGFR TKI (afatinib or osimertinib), with short responses. Conclusion Implementation of NGS in routine clinical practice allows identifying rare ex20ins in EGFR, including new variants, which would be missed by other methodologies. New therapeutic approaches are needed for ex20ins p, which respond poorly to current treatments."

P2; Trial primary completion date: Mar 2022 --> Mar 2023

Notably, the LAMP assay was more time-saving, cost-effective, and straightforward. However, further investigation is required to develop a more sensitive assay.

The ex19del activating mutation of the EGFR gene frequency in adenocarcinoma samples was 9.7%. A more frequent EGFR alteration was detected when assessing the locus copy number in the tumor relative to the non-tumor tissue: changes were recorded for 30% of adenocarcinoma cases and 69.1% of squamous cell lung cancer.

The overall agreement between results obtained with the Idylla™ EGFR Mutation Test and Qiagen Therascreen® EGFR RGQ PCR was 100% Our results showed that EGFR mutation is a rare mutation type in a consecutive series of de novo SCLC. Furthermore, the performance of Idylla™ EGFR Mutation Test and Qiagen Therascreen® EGFR RGQ PCR on archived paraffin sections of limited quantities is available with the high agreement of results.

"Our results evidenced an equivalent ability in detecting EGFR T790M mutations between the PCR-based techniques (Therascreen® and Idylla™) investigated. The NGS assay allowed the detection of a wider range of EGFR and additional gene mutations, but showed a significantly poor ability to detect T790M. Table 1."

"RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA."

"Three differences of prevalence are observed in our study for mexican population with lung AC: EGFR 32.7%, 13.9% KRAS and ALK 6.7%. According to recent NGS studies in Mexico, EGFR has a prevalence of 36.7% and 20% for KRAS. In Mexico and Latin America (LA) the mutational frequency is inverse compared to Caucasian population, with KRAS (20-25%) being more frequent than EGFR (10-15%) and ALK (4.5-5%)."

P3; In NSCLC SCC, a combination index of EGFR FISH plus EGFR IHC results was associated with improved OS when cetuximab was added to CT, representing a potential predictive molecular paradigm for patients suitable for EGFR-antibody therapy.

Receiver operating characteristic analysis showed an area under the curve of 0.977 for Cobas and 0.846 for ddPCR. In line with the FLAURA trial of osimertinib making its way to first-line and given the IASLC recommendations, it is important to understand the attributes of these tests to initiate appropriate treatment.

"EBUS-TBNA combined with ROSE has a high yield in the diagnosis of both neoplastic and non-neoplastic diseases with mediastinal involvement. In younger patients, the former may present with nonspe- cific symptoms and often have advanced disease at the time of diagnosis. Molecular testing in lung cancer is mandatory, especially in young pa- tients."

This is the first report of LAMP liquid biopsy detecting oncogene in a plasma sample. The EGFR-LAMP assay similar performance to the NGS assay in terms of detecting EGFR mutation in NSCLC tumors. The LAMP assay has advantage of time-saving, cost-effective, and simple test compared to the NGS assay.

Cobas was superior to therascreen for detection of plasma EGFR mutations in advanced NSCLC. Plasma cfDNA EGFR mutation analysis is complex; therefore, the diagnostic accuracy of commercially available assays should be validated.

For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen® EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice.

PCR-based assays are a fast and convenient test, allowing the detection of primary and secondary EGFR mutations from plasma. Cobas proved to be a reliable test, whereas digital PCR produced too many inconclusive results to be currently recommended as a principal testing device.

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.

The Z statistics score was 25.4 and Cronbachs alpha was 0.973 for cobas compared to tissue, and they were 6.91 and 0.8008 for ddPCR respectively.Conclusions From this first experience from India, the advantages of upfront liquid biopsy are evident, and adopting the same, may prove promising in a clinician's armamentarium. Cobas has advantages of broader mutation spectrum compared to ddPCR, however is limited by sensitivity, whereas ddPCR is limited by specificity although can provide absolute quantification

"Switching CDx tests from target polymerase chain reaction (PCR)- to next-generation sequencing (NGS)-based methods may lead to obvious changes in clinical practice. When the CDx test is required to change, the investigation of this influence is warranted in future studies."

"We demonstrated a potentially preferable way to profile treatment-naïve patients with NSCLC by NGS and from our early experience in EGFR mutant cases, the advantages of NGS over single gene testing is clearly evident."

Conclusion The sensitivity, specificity, and accuracy of detecting EGFR mutations in LAMP method were nearly equivalent to that in Therascreen method. This method may be a valuable alternative for the identification of EGFR mutations in pulmonary adenocarcinoma.

Reliance on commercially available PCR kit methods may provide insufficient information to support appropriate decision-making for emergent Exon20ins-directed therapies. The large number of insertion variants suggests that NGS platforms, academic or commercially available, would also improve their detection rate by capturing the full breadth of variants that have been identified.

"Our results indicated that, although precise, the molecular diagnosis by NGS of patients with advanced stage NSCLC adenocarcinoma histology was not cost-effective in terms of quality-adjusted life years from the perspective of the Brazilian supplementary health system."

Legal entity responsible for the study: Hinde El Fatemi. Funding: Has not received any funding.