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The management of metastatic cutaneous melanoma is conditioned by the identification of BRAF-activating mutations in tumor DNA. Tumor genotyping is usually performed on DNA extracted from tissue samples. However, these invasive samples are rarely repeated during follow-up, and their analysis requires a sample pre-treatment which may take several weeks. Circulating tumor DNA (ctDNA), released into blood by cancer cells, is a good alternative to tissue sampling. ctDNA is not subject to tumor heterogeneity, and can be analyzed rapidly, making possible the detection of mutations in emergency or in patients whose tumor cannot be sampled. ctDNA can also be analyzed repeatedly during follow-up, for postresection minimal residual disease assessment, for therapeutic response monitoring and for early relapse detection.

Simple Summary Microsatellite instability (MSI) assessment has become a major issue in the management of colorectal cancer, with the recent approval of anti-PD1 immunotherapies in MSI-metastatic colorectal cancer. The reference PCR method (MSI-PCR) can be costly, time and tissue-consuming. However, NGS could facilitate the assessment of MSI status while simultaneously screening for targetable oncogenic mutations (KRAS, NRAS, BRAF) for any colorectal cancer, but the algorithms developed to date use a large number of microsatellites that have not been approved by international guidelines and which are generally incompatible with small NGS panels. We present the MEM algorithm, which mimics the interpretation of MSI-PCR data by a human operator to reliably assess MSI status using only five validated microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). We demonstrated that the MEM algorithm was in perfect agreement with MSI-PCR results, in terms of both MSI status and individual microsatellite status, in a cohort of 146 patients. Abstract 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.

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.

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.

Background: The IFCT-1603 trial evaluated atezolizumab in small cell lung cancer (SCLC). The purpose of the present study was to determine whether circulating tumor DNA (ctDNA), prospectively collected at treatment initiation, was associated with the prognosis of SCLC, and whether it identified patients who benefited from atezolizumab. Methods: 68 patients were included in this study: 46 patients were treated with atezolizumab and 22 with conventional chemotherapy. Circulating DNA was extracted from plasma and NGS (Next Generation Sequencing) looked for mutations in the TP53, RB1, NOTCH1, NOTCH2, and NOTCH3 genes. ctDNA was detectable when at least one somatic mutation was identified, and its relative abundance was quantified by the variant allele fraction (VAF) of the most represented mutation. Results: We found that 49/68 patients (70.6%) had detectable baseline ctDNA. The most frequently identified mutations were TP53 (32/49, 65.3%) and RB1 (25/49, 51.0%). Patients with detectable ctDNA had a significantly lower disease control rate at week 6 compared with patients with no detectable ctDNA, regardless of the nature of the treatment. Detection of ctDNA was associated with a poor OS prognosis. The detection of ctDNA at a relative abundance greater than the median value was significantly associated with poor overall survival (OS) and progression free survival (PFS). Interestingly, the benefit in overall survival (OS) associated with low ctDNA was more pronounced in patients treated with atezolizumab than in patients receiving chemotherapy. Among patients whose relative ctDNA abundance was below the median, those treated with atezolizumab tended to have higher OS than those in the chemotherapy arm. Conclusion: ctDNA is strongly associated with the prognosis of SCLC patients treated with second-line immunotherapy. Its analysis seems justified for future SCLC clinical trials.

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 &lt, 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).

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).

International audience; Multiple myeloma (MM) is still considered incurable and new therapeutic approaches are therefore needed. Deep-sequencing analysis revealed the presence of BRAF mutations in up to 15% of patients. The clinical experience of BRAF-targeted therapy in myeloma patients harboring BRAF mutation is still limited. We here report the case of a patient with penta-refractory (bortezomib, lenalidomide, carfilzomib, pomalidomide, and daratumumab) MM with extramedullary BRAF-mutated disease that achieved clinical response to dual BRAF and MEK inhibition. At the time of disease progression, gene sequencing analysis of the tumor at the time of progression demonstrated a clonal evolution with emergence of aNRAS mutation and persistence of BRAF and TP53 mutations. Backtracking of the NRAS mutation was performed by digital polymerase chain reaction on the baseline biopsy and identified the pre-existence of the NRAS at a subclonal level. This observation is the first report of acquired NRAS mutation leading to resistance to dual BRAF/MEK inhibitors in MM. These data suggest that a systematic search for RAS mutations using highly sensitive techniques should be performed before considering targeted therapy in relapsed myeloma with BRAF mutation.

Major advances in the treatment of non-small cell lung cancer (NSCLC) patients have been obtained during the last decade. Molecular testing of tumor samples is therefore mandatory in routine clinical practice. Tumor DNA is also present as cell-free molecules in blood, which is therefore a very useful and convenient source of tumor DNA. In this review, we discuss pre-analytical and analytical aspects of circulating tumor DNA (ctDNA) analysis. We also describe the use of ctDNA analysis in routine clinical practice, and discuss the potential use of ctDNA monitoring both to identify minimal residual disease and as a potential tool to early identify patients’ response to treatment.

Introduction Le dosage des chaines legeres libres Kappa dans le liquide cerebrospinal (LCS) et le calcul de l’index kappa semble apporter une aide interessante au diagnostic biologique de la sclerose en plaques. Objectifs Les etudes precedemment publiees fournissent des cut-offs variables pour le quotient Kappa et l’index Kappa. Nous presentons ici les resultats de l’application de ces differents cut-offs sur les resultats des mesures obtenues au laboratoire. Patients et methodes Nous avons mesure retrospectivement sur 236 couples serums-LCS les parametres suivants : albumine, IgG, IgA, IgM, chaines legeres libres Kappa et chaines legeres libres lambda. Tous les dosages ont ete realises sur Optilite® (Binding Site, Birmingham, Royaume-Uni) avec des reactifs dedies. Nous avons calcule les differents rapports et index. Parmi les 236 couples serums-LCS analyses, on retrouvait a l’IEF la presence de bandes oligoclonales (BOC) d’IgG dans 91 cas et l’absence de BOC dans 145 cas. Resultats Les resultats des dosages ont ete analyses selon la presence (BOC +) ou l’absence (BOC −) de synthese locale intrathecale d’IgG mise en evidence a l’IEF. On note des concentrations de chaines legeres Kappa, un quotient Kappa et surtout un index Kappa significativement plus eleve (p Discussion En appliquant pour l’index Kappa les differents cut-off de 2,9 ; 4,3 ou 5,8, nous obtenons une sensibilite variant de 100 % a 96 %, une specificite de 54 % a 71 %, une valeur predictive positive (VPP) variant de 86 % a 91 % et une valeur predictive negative (VPN) variant de 100 % a 87 %. Conclusion Dans notre etude le cut-off le plus sensible pour l’index IgG est celui determine par Valencia-Vera, c’est egalement celui qui nous fournit la meilleure VPN.

International audience; Objective: Mucosal associated invariant T cells (MAIT) and innate lymphoid cells (ILCs) have immuno-regulatory functions at mucosal sites and have been involved in various inflammatory and autoimmune diseases. The aim of this study was to assess their frequencies in blood in ANCA-associated vasculitis (AAV).Methods: The frequencies and function of MAIT cells, ILCs, gdT, iNKT, NK cells were analyzed by flow cytometry on PBMC of patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) without any treatment, in acute (AP) and remission phase (RP) and compared with healthy controls (HC).Results: The frequencies of MAIT cells were strongly decreased in GPA and MPA in AP compared to HC, both in never treated and in relapsing patients and independently of patient age. This was associated with an activated phenotype of patient MAIT cells, as shown by increased expression of CD69 and IFNg. MAIT cells remained decreased during RP in AAV patients. The frequencies of iNKT and gdT cells were unaffected compared to HC, whereas those of NK cells were slightly reduced during AP in MPA. We also observed a significant decrease in frequencies of total ILCs with decreased ILC2 and ILC3 and increased ILC1 during AP in both GPA and MPA compared to HC. These frequencies normalized during RP. Interestingly , we observed a significant correlation between the frequency of total ILCs and BVAS.Conclusion: We show for the first time that AAV are associated with a major decrease and an activated phenotype of blood MAIT cell. These features persisted during remission suggesting a role for MAIT cells in the pathogenesis of AAV.

e21695 Background: Detection of genomic rearrangements like ALK and ROS1 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) has been so far the gold standard but multiplexed technologies would allow analysis of many potential targets simultaneously. We have evaluated a novel RT-PCR based assay and a targeted RNA sequencing solution for the detection of ALK and ROS1 rearrangement in NSCLC patients. Methods: 41 patients with late stage NSCLC were included in the study. ALK and ROS1 status were screened on FFPE tissue sections using immunohistochemistry and confirmed with FISH. Total nucleic acids were extracted from tissue sections using the Maxwell RSC RNA FFPE kit (Promega). Detection of rearrangements was performed by RT-PCR using the prototype ALK/RET/ROS1 Fusion Panel assay (Roche), which detects 7 ALK fusion variants, 13 ROS1 variants and 6 RET variants. For RNA sequencing, the libraries were prepared with the QIAseq Targeted RNAscan Lung Cancer Panel (Qiagen). Sequencing was performed on a MiSeq instrument (Illumina). Results: We tested 16 ALK FISH+ tumors, 4 ROS1 FISH+ tumors, and 21 samples that were either IHC- or IHC+/FISH-. The RT-PCR assay detected 15 of the 16 ALK FISH+ tumors, and 3 of the 4 ROS1 FISH+ tumors. In the 21 IHC- or IHC+/FISH- samples, 1 additional ROS1 fusion and 1 RET fusion were detected. The remaining 19 assays samples were negative. 2 fusions were missed by RT-PCR because the assay design does not cover these rearrangements (KLC1-ALK and CTNND1-ROS1). With targeted RNA sequencing, we were able to detect 14 of the 16 ALK FISH+ tumors and all 4 of the ROS1 FISH+ tumors. We also identified 1 additional ROS1 fusion and 1 RET fusion in the 21 IHC- or IHC+/FISH- samples. 17 patients were negative and 2 tests were not contributive. Overall, using FISH as a reference, the sensitivity of both assays was 90% (18/20). Conclusions: Using limited amount of biological material, both RT-PCR and targeted RNA sequencing were able in one assay to detect the overwhelming majority of ALK and ROS1 fusions identified with FISH. These approaches also allowed us to detect gene rearrangements that were missed by the initial IHC/FISH screening. The RT-PCR assay requires less RNA (as little as 3 ng) and has a much faster turn-around time. The RNA sequencing approach allowed us to identify the fusion partners, but it requires much more RNA and is rather difficult to implement in routine practice.

International audience; Immunotherapies have changed the medical management of metastatic melanoma. However, the early detection of patients who do not respond to these treatments is a key issue. We evaluated the quantitative monitoring of circulating tumor DNA (ctDNA) as an early predictor of response to anti-PD1. Patients treated with anti-PD1 for metastatic mutated melanoma were selected. The somatic alteration detected on the tumor tissue was quantified on plasma DNA by digital PCR (dPCR) at treatment initiation, after 2 and 4 weeks of treatment, and then every 4 weeks until progression. The absence of biological response (defined as a significant decrease in the amount of ctDNA relative to the baseline level) after 2 weeks of treatment was associated with a lack of clinical benefit under anti-PD1. In the presence of a biological response at week 2, detection of subsequent biological progression (significant increase in the amount of ctDNA relative to its nadir) was 100% predictive of progressive disease, on average 75 days prior to radiological detection. Patients with a persistent biological response beyond week 16 did not experience any progressive disease and exhibited sustained responses. In conclusion, we show that quantitative monitoring of ctDNA, using criteria accounting for dPCR measurement imprecision, allows the early and specific detection of patients who do not respond to anti-PD1 therapy.

The management of metastatic melanoma has evolved since the onset of treatments with BRAF inhibitors. In order to predict which patients are likely to respond to these treatments, the therapeutic strategy is now conditioned by the search for the activating mutations of the BRAF gene. Tumor genotyping is routinely performed from DNA extracted from tissue or cellular specimens from the primary tumor, metastases, or neoplastic effusions. Due to their invasiveness, these specimens are rarely repeated during the management. In addition, the analysis of the tumor material requires a pretreatment of the sample (formalin fixation, paraffin inclusion, preparation of tissue sections) and may take up to several weeks, making emergency treatment with BRAF inhibitors impossible. Circulating tumor DNA (ctDNA), released by cancer cells in the blood stream, appears as an alternative to tissue sampling. The pre-analytical conditions are now well defined, and several technological approaches can be used to demonstrate the desired molecular alterations. ctDNA is less affected by tumor heterogeneity, can be collected in a minimally invasive manner and analyzed rapidly. Furthermore, ctDNA can be repeatedly analyzed during follow-up, which makes it possible to envisage its use as a specific tumor marker, in order to monitor the response to the treatment and to detect treatment failure.

International audience; Circulating tumor DNA is a promising non-invasive tool for cancer monitoring. The main objective of our work was to investigate the relationship between mutant BRAF DNA in plasma and clinical response. Thirty-eight stage IV patients with a V600 mutated BRAF melanoma were included prior to any treatment. DNA was extracted from plasma and mutant DNA was detected using the amplification-refractory mutation system method. Before the beginning of any treatment, the corresponding BRAF mutation was detected in 29 of the 38 tested plasma samples (76.3% positive per cent agreement). We observed a strong correlation between the presence of circulating mutated DNA and overall survival (OS; P=.02), and with the number of metastatic sites (P=.01). The presence of circulating mutated DNA was also strongly correlated with serum LDH activity (P

e21026Background: Circulating tumor DNA is an alternative source of tumor DNA that could be used for noninvasive identification of BRAF mutations at various time-points during the course of the dis...

PIK3CA-related overgrowth spectrum (PROS) includes rare genetic conditions due to gain-of-function mutations in the PIK3CA gene. There is no approved medical therapy for patients with PROS, and alpelisib, an approved PIK3CA inhibitor in oncology, showed promising results in preclinical models and in patients. Here, we report for the first time the outcome of two infants with PROS having life-threatening conditions treated with alpelisib (25 mg) and monitored with pharmacokinetics. Patient 1 was an 8-mo-old girl with voluminous vascular malformation. Patient 2 was a 9-mo-old boy presenting with asymmetrical body overgrowth and right hemimegalencephaly with West syndrome. After 12 mo of follow-up, alpelisib treatment was associated with improvement in signs and symptoms, morphological lesions and vascular anomalies in the two patients. No adverse events were reported during the study. In this case series, pharmacological inhibition of PIK3CA with low-dose alpelisib was feasible and associated with clinical improvements, including a smaller size of associated complex tissue malformations and good tolerability.