MET amplification (METamp) is a noteworthy genomic alteration that can occur in patients with non-small cell lung cancer (NSCLC). It has been demonstrated to occur as a primary oncogenic driver that may exist prior to any treatment and is referred to as de novo METamp. Despite the recognized significance of this genetic alteration, routine large-scale screening for the early detection of de novo METamp is currently lacking in clinical practice and the clinical impact of de novo METamp on NSCLC remains poorly investigated. In this study, we developed a next-generation sequencing-based screening method for detecting and stratifying METamp optimized in silico, validated in a patient cohort (n = 72) and applied to 1932 patients with NSCLC. Clinical outcomes (overall survival [OS] and progression-free survival) were assessed in de novo METamp cases (n = 46). The optimized next-generation sequencing-based method achieved high confidence (F-score > 0.99) during in silico optimization. In vivo validation demonstrated high sensitivity (0.93) and specificity (0.97) compared with fluorescence in situ hybridization. De novo METamp was found in 2.4% of cases stratified into the following distinct amplification groups based on the amplification copy number ratio (CNR): low (1.5 < CNR < 2.2), medium (2.2 < CNR < 4), and high amplification ( CNR > 4). Significant differences in patient outcome (P < .001) were observed between the low- (median OS: 35.9 months), medium- (median OS: 14.3 months) and high-amplification (median OS: 3.3 months) groups. Progression-free survival under chemotherapy was notably reduced in the medium-/high-amplification groups compared with the low-amplification group (P = .001). Screening for METamp detection followed by stratification based on METamp levels may be considered in all patients with NSCLC at diagnosis. This approach could potentially enhance treatment management effectiveness by facilitating inclusion in clinical trials. (c) 2025 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Background: MET amplification (METamp) is a noteworthy genomic alteration that can occur in patients with non-small cell lung cancer (NSCLC). It has been demonstrated to occur as a primary oncogenic driver that may exist prior to any treatment and is referred to as de novo METamp. Despite the recognized significance of this genetic alteration, routine large-scale screening for the early detection of de novo METamp is currently lacking in clinical practice and the clinical impact of de novo METamp in NSCLC remains poorly investigated. Methods: In this study, we developed a NGS-based screening method for detecting and stratifying METamp optimized in silico, validated in a patient cohort (n = 72) and applied to 1,932 NSCLC patients. Clinical outcomes (OS and PFS) were assessed in de novo METamp cases (n = 46). Results: The optimized NGS-based method achieved high confidence (F-score > 0.99) during in silico optimization. In vivo validation demonstrated high sensitivity (0.93) and specificity (0.97) compared to fluorescent in situ hybridization. de novo METamp was found in 2.4% of cases stratified into distinct amplification groups based on the amplification copy number ratio (CNR): Low- (1.5 < CNR ≤ 2.2), Medium- (2.2 < CNR ≤ 4), and High-amplification (CNR > 4). Significant differences in patient outcome (p < 0.001) were observed between the Low- (median OS: 35.9 months), Medium- (median OS: 14.3 months) and High-amplification (median OS: 3.3 months) groups. PFS under chemotherapy was notably reduced in the Medium/High-amplification groups compared to the Low-amplification group (p = 0.001). Conclusions: Screening for METamp detection followed by stratification based on METamp levels may be considered in all NSCLC patients at diagnosis. This approach could potentially enhance treatment management effectiveness by facilitating inclusion in clinical trials.

Purpose: The introduction of PARP inhibitors (PARPis) as a treatment option for patients with high-grade serous ovarian cancer (HGSOC) modified the approach of BRCA testing worldwide. In this study, we aim to evaluate the impact of BRCA1 and BRCA2 variants on treatment response and survival outcomes in patients diagnosed in our institution. Methods: A total of 805 HGSOC samples underwent BRCA1 and BRCA2 variant detection by using next-generation sequencing (NGS). Among them, a pathogenic alteration was detected in 104 specimens. Clinicopathological features and germline status were recovered, and alteration types were further characterized. The clinical significance of variant type in terms of response to chemotherapy and to PARPis as well as overall survival were evaluated using univariate analysis. Results: In our cohort, 13.2% of the HGSOC samples harbored a pathogenic BRCA1 or BRCA2 variant, among which 58.7% were inherited. No difference was observed between germline and somatic variants in terms of the gene altered. Interestingly, patients with somatic variants only (no germline) demonstrated better outcomes under PARPi treatment compared to those with germline ones. Conclusion: The determination of the inheritance or acquisition of BRCA1 and BRCA2 alterations could provide valuable information for improving management strategies and predicting the outcome of patients with HGSOC.

Background: Osimertinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that is highly selective for EGFRT790M subclones in patients with EGFRsensitizing non-small cell lung cancer (NSCLC). Unfortunately, all patients develop resistance through EGFR-dependent or EGFR-independent pathways. Recently, circulating tumoral DNA (ctDNA) analysis has highlighted the usefulness of plasma genotyping for exploring patient survival outcomes after disease progression under osimertinib. Methods: Plasma samples from patients treated with osimertinib as a second-line therapy were collected and the presence of molecular alterations of acquired resistance was evaluated after relapse under osimertinib using ctDNA molecular profiling by next-generation sequencing (NGS) assays. The clinical implications of these genomic alterations for the efficiency of the third-generation TKI were further assessed. Results: Our ctDNA molecular profiling of plasma samples highlighted large number of actionable genomic alterations. According to ctDNA NGS results, patients were classified as having developed an EGFR-dependent or EGFR-independent mechanism of resistance. Thus, patients who developed an EGFR-dependent mechanism of resistance responded longer to osimertinib (13.8 vs. 4.6 months; P<10−4) and have a better post-osimertinib clinical outcome than EGFR-independent resistant patients. Moreover, the development of an EGFR-dependent mechanism of osimertinib resistance was identified as the best fit to determine patients' clinical outcome compared with EGFRT790M status alone (P=0.003). Conclusions: Our study highlights the potential of ctDNA NGS to rapidly select the appropriate drug after osimertinib failure and to determine clinical outcomes of patients. We suggest that ctDNA NGS should be more intensively used in clinical practice to follow patients under third-generation TKIs.

Non-small-cell lung cancer (NSCLC) is the most common form of primary lung cancer. The discovery of several oncogenic driver mutations in patients with NSCLC has allowed the development of personalized treatments based on these specific molecular alterations, in particular in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene. Gefitinib, erlotinib, afatinib, and osimertinib are TK inhibitors (TKIs) that specifically target EGFR and are currently approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as first line treatment for sensitive EGFR-mutant patients. However, these four drugs are associated with severe adverse events (AEs) that can significantly impact patient health-related quality of life and patient monitoring. EGFR-TKIs are commonly used together with other types of medication that can substantially interact. Here, we review approaches used for the management of TKI-AEs in patients with advanced NSCLC to promote the benefits of treatments and minimize the risk of TKI treatment discontinuation. We also consider potential TKI–drug interactions and discuss the usefulness of plasma concentration monitoring TKIs based on chromatographic and mass spectrometry approaches to guide clinical decision-making. Adjusting the most appropriate therapeutic strategies and drug doses may improve the performance therapy and prognosis of patients with advanced EGFR-mutated NSCLC.

Introduction: The proteome is a dynamic system in which protein-protein interactions play a crucial part in shaping the cell phenotype. However, given the current limitations of available technologies to describe the dynamic nature of these interactions, the identification of protein-protein interactions has long been a major challenge in proteomics. In recent years, the development of BioID and APEX, two proximity-tagging technologies, have opened-up new perspectives and have already started to change our conception of protein-protein interactions, and more generally, of the proteome. With a broad range of application encompassing health, these new technologies are currently setting milestones crucial to understand fine cellular mechanisms. Area covered: In this article, we describe both the recent and the more conventional available tools to study protein-protein interactions, compare the advantages and the limitations of these techniques, and discuss the recent advancements led by the proximity tagging techniques to refine our conception of the proteome. Expert opinion: The recent development of proximity labeling techniques emphasizes the growing importance of such technologies to decipher cellular mechanism. Although several challenges still need to be addressed, many fields can benefit from these tools and notably the detection of new therapeutic targets for patient care.

e16189 Background: S-1, an oral fluoropyrimidine, combined with Irinotecan (IRI) or Oxaliplatin (OX) (biweekly schedule) was assessed in Asian populations with acceptable safety profile and efficacy. This phase I trial aimed to evaluate the safety of S-1 in combination with IRI (SIRI) then with IRI and OX (SIRINOX) in patients with aGM. We present here the results of the SIRI step. Methods: Eligible patients had aGM not previously exposed to IRI and/or OX. A 3+3 dose-escalation study with 4 S-1 dose-levels (25, 30, 35 and 40mg/m2, twice/day from day 1 to day 8) combined with IRI 90 min IV 180mg/m2 fixed-dose, every 2 weeks, was performed for SIRI. The primary objectives were to identify the maximum tolerated dose (MTD) and the recommended phase II dose (RP2D) of SIRI. Secondary objectives included response rate (RR), progression-free (PFS) and overall (OS) survival. Results: 14 patients were enrolled. Patients characteristics were: median age, 63 (37-85), 10/14 female; all patients were PS 0 (57%) and 1 (43%). Tumor types were: 10 biliary tract and 2 gastric adenocarcinomas, and 1 esophageal and 1 anal squamous cell carcinomas. Median number of cycles was 5 (1-19). Grade ≥ 3 adverse events (AEs) (Table) and deaths occurred in 7 (50%) and 2 (14%) patients (levels 3 and 4), respectively. Three dose-limiting toxicities were reported in levels 3 (n = 1) and 4 (n = 2). The DMT was reached at level 4 (40mg/m²). The RP2D was set at 35 mg/m². 1 out of 11 patients evaluable for efficacy achieved a tumor response (RR = 7%, 95%CI: 2-34), and 5/11 patients had a stable disease. Median OS and PFS were 5.2 [95%CI: 2.8–not reached] and 2 [95%CI: 1.2-5.2] months, respectively. Treatment is still ongoing (30 cycles) in one patient with stable biliary tract cancer. Conclusions: MTD and RP2D of this SIRI combination were 40mg/m² and 35 mg/m², respectively. The SIRINOX step was planned but the study was prematurely stopped for funding reasons. Clinical trial information: NCT02387138. Acknowledgements This study was supported by Nordic Pharma. Grade ≥ 3 AEs related to treatment 25mg/m2 n = 3 30mg/m2 n = 3 35mg/m2 n = 6 40mg/m2 n = 2 Diarrhea 0 0 1 1 Neutropenia 2 0 1 0 Asthenia 1 0 1 1 Others 0 1 1 1

EAPB0503, belonging to the imidazo[1,2-a]quinoxaline series, is an anticancer drug with antitumoral activity against a variety of tumors. Previous studies have shown that this drug undergoes demethylation and oxygenation reactions. In this paper, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was employed to assess the structures of unknown oxygenated metabolites of EAPB0503. EAPB0503 and its identified demethylated metabolites, EAPB0502 and EAPB0603, were incubated with human, rat, dog and mouse liver microsomes, as well as human, rat and dog hepatocytes. After separation on a C8 analytical column with a gradient elution of acetonitrile-formate buffer, positive ESI-MS/MS experiments were performed. To facilitate metabolite identification, the detailed fragmentation pathways of the parent compounds were first studied using high-resolution MS/MS. Additional hydrogen/deuterium exchange LC-MS/MS experiments were used to support the identification and structural characterization of metabolites. Four hydroxylated metabolites were identified: M'4 and its demethylated derivative M'1 (OH in ortho position on the phenyl substituent in position 1), and M'6 and its demethylated derivative M'3 (OH on the imidazole ring at the C2 position). Three phase II metabolites (Met A, EAPB0602 glucuronide; Met B, M'4 glucuronide; Met C, EAPB0603 glucuronide) were also evidenced. Elucidation of the metabolite structures was performed by comparing the chromatographic behaviors (changes in retention times), by measuring the molecular masses (mass increment), by studying the MS2 spectral patterns of metabolites with those of parent drugs and for M'1 and M'4 by co-analysis with synthetic standards. The results of the present study provided important structural information relating to the metabolism of EAPB0503. (C) 2013 Elsevier B.V. All rights reserved.

For several years, our group has been developing quinoxalinic compounds. Two of them, N-methyl-1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-amine (EAPB0203) and 1-(3-methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-amine (EAPB0503), have emerged as the most promising anticancer drugs. In the present work, we determined metabolism pathways using liver microsomes from four mammalian species including human. We identified the cytochrome P450 isoform(s) involved in the metabolism and then investigated the pharmacokinetics and metabolism of EAPB0203 and EAPB0503 in rat after intravenous and intraperitoneal administration. Biotransformation of the compounds involved demethylation and hydroxylation reactions. Rat and dog metabolized the compounds at a higher rate than mouse and human. In all species, CYP1A1/2 and CYP3A isoforms were the predominant enzymes responsible for the metabolism. From human liver microsomes, unbound intrinsic clearances were approximately 56 ml/(min · g) protein. EAPB0203 and EAPB0503 were extensively bound to human plasma proteins, mainly human serum albumin (HSA) (∼98-99.5%). Thus, HSA could act as carrier of these compounds in human plasma. Scatchard plots showed patterns in which the plots yielded upwardly convex hyperbolic curves. On the basis of the Hill coefficients, there appears to be interaction between the binding sites of HSA, suggesting positive cooperativity. The main in vitro metabolites were identified in vivo. Total clearances of EAPB0203 and EAPB0503 [3.2 and 2.2 l/(h · kg), respectively] were notably lower than the typical cardiac plasma output in rat. The large volumes of distribution of these compounds (4.3 l/kg for EAPB0203 and 2.5 l/kg for EAPB0503) were consistent with extensive tissue binding. After intraperitoneal administration, bioavailability was 22.7% for EAPB0203 and 35% for EAPB0503 and a significant hepatic first-pass effect occurred.

In spite of the development of new anticancer drugs by the pharmaceutical industry, melanoma and T lymphomas are diseases for which medical advances remain limited. Thus, there was an urgent need of new therapeutics with an original mechanism of action. Since several years, our group develops quinoxalinic compounds. In this paper, the first preclinical results concerning one lead compound, EAPB0203, are presented. This compound exhibits in vitro cytotoxic activity on A375 and M4Be human melanoma cell lines superior to that of imiquimod and fotemustine. A liquid chromatography-mass spectrometry method was first validated to simultaneously quantify EAPB0203 and its metabolite, EAPB0202, in rat plasma. Thereafter, the pharmacokinetic profiles of EAPB0203 were studied in rat after intravenous and intraperitoneal administrations. After intraperitoneal administration the absolute bioavailability remains limited (22.7%). In xenografted mouse, after intraperitoneal administration of 5 and 20mg/kg, EAPB0203 is more potent than fotemustine. The survival time was increased up to 4 and 2 weeks compared to control mice and mice treated by fotemustine, respectively. The results of this study demonstrate the relationship between the dose of EAPB0203 and its effects on tumor growth. Thus, promising efficacy, tolerance and pharmacokinetic data of EAPB0203 encourage the development towards patient benefit.

Since several years, our group developed quinoxalinic compounds. Among the synthesized compounds, in the imidazo[1,2-a]quinoxaline series, EAPB0203 has shown interesting activities both on melanoma and lymphoma. The structure of EAPB0203 has been modulated and a new compound, EAPB0503, exhibits an in vitro cytotoxic activity on melanoma cancer cell line 7-9 times higher than EAPB0203. We validated an LC/ESI-MS method to simultaneously quantify EAPB0503 and its metabolite EAPB0603 in human and rat plasma. Chromatography was performed on a C8 Zorbax eclipse XDB column with a mobile phase consisting of acetronitrile and formate buffer gradient elution. LC-MS data were acquired in SIM mode at m/z 305, 291, and 303 for EAPB0503, EAPB0603, and the internal standard, respectively. The drug/internal standard peak area ratios were linked via quadratic relationships to concentrations (low range: 5-300 microg/L, high range: 100-1000 microg/L). The method is precise (precision, < or = 14%) and accurate (recovery, 92-113%). Mean extraction efficiencies, > 72% for each analyte, were obtained. The lower LOQs were 5 microg/L. This highly specific and sensitive method was successfully used to investigate plasma concentrations of EAPB0503 and EAPB0603 in a pharmacokinetic study carried out in rat and would also be useful in clinical trials at a later stage.

Folate, a vitamin of the B group involved in one-carbon group metabolism, plays an important role in DNA synthesis and methylation. Several polymorphisms in the genes involved in folate uptake and biotransformations have been shown to be associated to the risk of cancer and to anticancer drug response. We studied common polymorphisms in MTHFR (N(5,10)-methylene-tetrahydrofolate reductase), MTHFD1 (N(5,10)-methylene-tetrahydrofolate dehydrogenase), MTR (methionine synthetase) and SLC19A1 (reduced folate carrier) in the panel of 60 human tumour cell lines established by the NCI for anticancer drug screening and we tentatively associated these polymorphisms with gene expression and drug cytotoxicity as extracted from the public database of the Developmental Therapeutic Programme. We observed a consistent and highly significant association between the presence of the variant C allele of the A>C1298 polymorphism of MTHFR and the sensitivity to many anticancer drugs belonging to the classes of antifolates, antimetabolites, alkylating agents and, to a lesser extent, topoisomerase inhibitors. In contrast, the T variant allele of the C>T677 variation of MTHFR was rather associated to lower sensitivity of the cell lines towards anticancer drugs (alkylating agents, antifolates and antimetabolites) but with much lower effects than the A>C1298 variation. The polymorphisms of the other genes studied were not associated with differences in anticancer drug sensitivity, but the expression of the SLC19A1 gene was significantly correlated with the sensitivity to several drugs (antifolates, thiopurines, nitrosoureas, and DACH-platinum drugs). We concluded that the NCI-60 panel may constitute a good starting point for implementing clinical studies aimed at discovering and validating predictive genetic markers of drug efficacy and/or toxicity.

BACKGROUND We developed and compared 2 different methods for quantifying uracil (U) and dihydrouracil (UH(2)) in BSA and human plasma. Special attention was paid to the selectivity/specificity and the absence of a matrix effect. The UH(2)/U ratio is intended as a biomarker to identify patients with deficiency in 5-fluorouracil metabolism. METHODS We quantified U and UH(2) with 2 liquid chromatography methods after solid-phase extraction, one with UV detection (LC-UV) and the other with mass spectrometric detection (LC-MS). We selected 2 internal standards to prevent the risk of interferences. Separation was achieved with a Waters Atlantis dC18 column (LC-MS) or a Waters SymmetryShield RP18 column connected with an Atlantis dC18 (LC-UV). Mass spectrometric data were acquired in single-ion monitoring mode. RESULTS Assay imprecision in BSA solution was <15% (LC-UV) and <12% (LC-MS); in plasma, assay imprecision was <9.5% and <9.0%, respectively. Recoveries were 88.2%-110% (LC-UV) and 94.8%-107% (LC-MS). Extraction efficiencies were >or=89.0%. In BSA, the lower limits of quantification for U and UH(2) were 2.5 microg/L and 6.25 microg/L, respectively, for the LC-UV method and 2.5 microg/L and 3.1 microg/L for LC-MS. The corresponding values in plasma were 11.6 microg/L and 21.5 microg/L, and 4.1 microg/L and 12.1 microg/L. CONCLUSIONS To estimate endogenous U and UH(2) concentrations and their ratio, we recommend the use of a drug-free human plasma pool in which baseline U and UH(2) concentrations have previously been measured with the standard-addition method. Our LC-MS method, which has the better test performance and is useful for measuring UH(2)/U ratios in cancer patients, is preferred when this equipment is available.

The objective of this study was to determine the impact of single versus sequential exposure to melphalan on the proliferation of an androgen-independent prostate cell line, PC-3, and to report the results of a pilot phase II study. For exposure to a single bolus dose, the doses were added at the start of the study and cell culture was continued for 96 h. For sequential exposure, 1/9 of the dose was added every 1.5 h over 12 h, followed by cell culture for 84 h. Cell growth inhibition was determined by the MTT assay. The clinical study was carried out on 14 patients with advanced prostate cancer. Melphalan was infused over a 24-h period. The sequential-dose schedule was more effective than the single-dose exposure, IC50 values: 0.074 versus 0.77/microg/ml. Out of the 14 patients (42 courses) enrolled into the study, two patients were removed within the first 2 weeks because of rapid disease progression. The toxicity profile did not differ greatly from that reported after a 1-h infusion. Four PR and two SD were observed. The median survival of the twelve patients was 23 weeks. Melphalan administered over a 24-h period to patients with androgen-independent prostate cancer appeared to provide some clinical benefits with manageable toxicity.