The ability of early (first weeks of treatment) ctDNA kinetics to identify primary resistance to anti-PD1 immunotherapies was evaluated with a validation cohort of 49 patients treated with anti-PD1 for metastatic BRAF or NRAS-mutated melanoma, alone and pooled with the 53 patients from a previously described derivation cohort. BRAF or NRAS mutations were quantified on plasma DNA by digital PCR at baseline and after two or four weeks of treatment. ctDNA kinetics were interpreted according to pre-established biological response criteria. A biological progression (bP, i.e., a significant increase in ctDNA levels) at week two or week four was associated with a lack of benefit from anti-PD1 (4-month PFS = 0%; 1-year OS = 13%; n = 12/102). Patients without initial bP had significantly better PFS and OS (4-month PFS = 78%; 1-year OS = 73%; n = 26/102), as did patients whose ctDNA kinetics were not evaluable, due to low/undetectable baseline ctDNA (4-month PFS = 80%; 1-year OS = 81%; n = 64/102). ctDNA detection at first-line anti-PD1 initiation was an independent prognostic factor for OS and PFS in multivariate analysis. Overall, early ctDNA quantitative monitoring may allow the detection of primary resistances of metastatic melanoma to anti-PD1 immunotherapies.

Circulating tumour DNA (ctDNA) can be used to identify gene alterations. The purpose of this study was to determine whether the detection of ctDNA, based on the identification of BRAF and NRAS mutations before systemic treatment initiation, was associated with the prognosis of metastatic melanoma. In total, 68 BRAF or NRAS-mutated stage IV or unresectable stage III metastatic cutaneous melanoma patients were included and tested for the presence of BRAF and NRAS mutations in circulating DNA before treatment initiation, using the Cobas BRAF/NRAS Mutation Test (Roche). The expected mutation was detected in the plasma of 34/68 patients (50% sensitivity). ctDNA detection was associated with AJCC stage, along with the number and nature of metastases. ctDNA was less frequently detected in NRAS-mutated than in BRAF-mutated melanoma (36% and 66%, respectively). At initiation of first-line treatment, ctDNA detection was associated with poor prognosis in Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis (log-rank: p = 0.002 and p < 0.0001, respectively). In multivariate analysis, ctDNA detection was an independent factor of poor prognosis in OS, after adjustment for AJCC stage, number and nature of metastases and gender (HR = 4.384; 95% CI: (1.308; 14.699); p = 0.017).

Purpose: MEM is an NGS algorithm that uses Expectation-Maximisation to detect the presence of unstable alleles from the NGS sequences of five microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). The purpose of this study was to compare the MEM algorithm with a reference PCR method (MSI-PCR) and MisMatch Repair protein immunohistochemistry (MMR-IHC). Methods: FFPE colorectal cancer samples from 146 patients were analysed in parallel by MSI-PCR and NGS using the MEM algorithm. MMR-IHC results were available for 133 samples. Serial dilutions of an MSI positive control were performed to estimate the limit of detection. Results: the MEM algorithm was able to detect unstable alleles of each microsatellite with up to a 5% allelic fraction. Of the 146 samples, 28 (19.2%) were MSI in MSI-PCR. MEM algorithm results were in perfect agreement with those of MSI-PCR, at both MSI status and individual microsatellite level (Cohen’s kappa = 1). A high level of agreement was noted between MSI-PCR/MEM algorithm results and MMR-IHC results (Cohen’s kappa = 0.931). Conclusion: the MEM algorithm can determine the MSI status of colorectal cancer samples on a small NGS panel, using only five microsatellites approved by international guidelines, and can be combined with screening for targetable mutations.

Fixed tissues are the standard samples used in routine practice for molecular testing. But sometimes tissues are lacking or difficult to obtain. In these cases, circulating tumor DNA released from tumor cells can be used as an alternative source of tumor DNA. We present the case of a 63-year-old Caucasian woman with a metastatic melanoma and a very poor performance status. A plasma sample was tested and the BRAF p.V600E mutation was detected. Based on this result, a treatment combining a BRAF inhibitor and a MEK inhibitor was immediately started. This patient achieved a complete response. In addition, by repeating the plasma test, we could obtain a precise kinetic of release of mutated BRAF DNA in plasma. We report here for the first time the efficient treatment of a metastatic melanoma patient on the basis of circulating tumor DNA analysis. This urgent treatment provided a dramatic response in a patient with a very poor initial condition. The kinetic data most likely reflect treatment efficacy.

Background/Objectives: Circulating tumor DNA (ctDNA) analysis is a powerful tool for non-invasive monitoring of tumor burden and treatment response. Reliable quantification methods are critical for the effective use of ctDNA as a tumor biomarker. Digital PCR (dPCR) offers high sensitivity and quantification, but requires the prior knowledge of tumor-specific genomic alterations. Next-generation sequencing (NGS) provides a more comprehensive approach but is semi-quantitative, relying on variant allelic fraction (VAF), which can be influenced by non-tumor cell-free DNA. Methods: We developed a novel quantitative NGS (qNGS) method for absolute quantification of nucleotide variants, utilizing unique molecular identifiers (UMIs) and of quantification standards (QSs), short synthetic DNA sequences modified to include characteristic mutations for unique identification in sequencing data. We evaluated the performance of this method using plasma samples spiked with mutated DNA and plasma pools from cancer patients. We further applied our technique to plasma samples from four non-small cell lung cancer (NSCLC) patients enrolled in the ELUCID trial. Results: Our qNGS approach demonstrated robust linearity and correlation with dPCR in both spiked and patient-derived plasma samples. Notably, the analysis of clinical samples from the ELUCID trial revealed the ability of our method to simultaneously quantify multiple variants in a single plasma sample. Significant differences in ctDNA levels were observed between baseline and post-treatment samples collected after three weeks of front-line therapy. Conclusions: We introduce a novel qNGS method that enables the absolute quantification of ctDNA, independent of non-tumor circulating DNA variations. This technique was applied for the first time to serial samples from NSCLC patients, demonstrating its ability to simultaneously monitor multiple variants, making it a robust and versatile tool for precision oncology.

Background and objective: Detection of genomic rearrangements like ALK fusions are mandatory in non-small cell lung cancer (NSCLC) as those alterations can be targeted by an increasing number of drugs. Fluorescence in-situ hybridization (FISH) is the gold standard but multiplexed technologies allow the analysis of several targets simultaneously. We have evaluated a novel RT-PCR based assay for the detection of ALK, ROS1 and RET rearrangement in NSCLC patients in this research study. Materials and Methods: FFPE tissue sections from 309 patients with late stage NSCLC were screened for ALK and ROS1 status using immunohistochemistry (IHC) and confirmed with FISH. Total nucleic acids were extracted from tissue sections using the Maxwell RSC RNA FFPE kit (Promega). Fusions were detected by RT-PCR using a prototype ALK/RET/ROS1 Fusion Panel assay (Roche). A subset of discordant cases were further analyzed by RNA sequencing using the QIAseq Targeted RNAscan Lung Cancer Panel (Qiagen). Results: All 309 samples were tested by RT-PCR with a 0% invalid rate. ALK fusions were detected in 39 samples: 29 were ALK FISH+, 5 were ALK FISH not contributive, 3 were ALK FISH- (less than 15% tumor cells presenting a gene fusion), and 2 were ALK IHC- and not tested by FISH. ROS1 fusions were detected in 12 samples: 9 were ROS1 FISH+, 1 was ROS1 FISH-, and 2 were ROS1 IHC- and not tested by FISH. RET fusions were detected in 3 samples. The remaining 255 samples were RT-PCR-: 5 were ALK FISH+ containing fusions not covered by the RT-PCR assay, 1 was ALK FISH+ containing ALK polysomy, 1 was ROS1 FISH+ containing a fusion not covered by the RT-PCR assay, 1 was ROS1 FISH+ containing ROS1 amplification, 1 was ROS1 FISH+ but ROS1 RNAseq-, and 246 samples were IHC- or IHC+/FISH-. The overall concordance rate between RT-PCR and FISH for ALK and ROS1 gene rearrangements was 91.9%. Although the RT-PCR assay did not detect 5 ALK and 1 ROS1 fusions due to design limitations, it did identify the presence of fusions in 9 tumors that were undetected by FISH (8 ALK and 1 ROS1). Conclusion: Using limited amount of biological material, RT-PCR was able to detect a substantial majority of ALK and ROS1 fusions identified by FISH, as well as fusions that were missed by the initial IHC/FISH screening. This study demonstrates RT-PCR as a feasible approach to detecting fusions in NSCLC. Citation Format: Marc G. Denis, Audrey Vallée, Christine Sagan, Guillaume Herbreteau, Sandrine Charpentier, Jaya Rajamani, Michael Lee, Ellen Ordinario. Detection of ALK, ROS1 and RET fusion transcripts in FFPE samples of non-small cell lung cancer patients using a novel RT-PCR based assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1271.