Glioblastoma (GBM) is the most common adult primary brain tumor for which new therapeutic strategies are desperately needed. Monopolar spindle 1 (MPS1) is a mitotic kinase that plays a pivotal role in the spindle assembly checkpoint (SAC). GBM appears to be dependent on SAC fidelity, as MPS1 is overexpressed in many GBM patients. Thus, inhibiting MPS1 seems a viable therapeutic strategy to enhance mitotic cell death by attenuating SAC fidelity. NTRC 0066-0 is an MPS1 inhibitor that combines low nanomolar potency with a relatively long on-target residence time. We here investigate the potential of NTRC 0066-0 as monotherapy and in combination with chemo-radiation for treatment of GBM using various in vitro and orthotopic in vivo models. We show that NTRC 0066-0 efficiently induces GBM cell death in vitro, following continuous exposure with IC50s in the low nanomolar range. In vivo, we demonstrate that NTRC 0066-0 has a high brain penetration, although it is a substrate of the efflux transporter P-glycoprotein at the blood-brain barrier. However, despite using recipient Abcb1a/b; Abcg2−/− mice with superior brain penetration and administering NTRC 0066-0 using a dose-dense regimen, we did not observe antitumor efficacy against an orthotopic GBM mouse model, neither as monotherapy nor in combination with standard-of-care temozolomide chemotherapy and radiotherapy. These data indicate that developing MPS1 inhibitors for treatment of GBM will be challenging and would require further understanding of in vivo determinants of translating SAC inhibition to antitumor efficacy.

Background: Therapeutic Drug Monitoring optimizes oral anticancer drug treatment by measuring plasma levels. Volumetric absorptive microsampling (VAMS) allows home sampling with a minimal blood sample. However, methods for converting whole blood into plasma are required to interpret these results. This study aimed to establish conversion methods for abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and their metabolites, while assessing the differences between venous and capillary blood. The feasibility of home sampling was also evaluated. Methods: Plasma and VAMS samples, both from venipuncture-collected whole blood tubes and from a finger prick, were collected from each patient. The VAMS samples were deemed comparable if their concentrations were within ±20% of each other for ≥2/3rd of the patients. The Passing–Bablok regression and conversion factor methods were tested for the plasma and VAMS finger prick samples. The estimated plasma concentrations using both methods were required to be within ±20% of the measured plasma concentrations for ≥2/3rd of the pairs. Results: Overall, 153 patients were enrolled in this study. Conversion methods were applied to the VAMS samples, and the acceptance criteria were met for alectinib-M4, cabozantinib, imatinib, N-desmethyl imatinib, olaparib, sunitinib, and N-desethyl sunitinib but not for abiraterone, D4A, or alectinib. The capillary and venous VAMS concentrations were similar, except for that of D4A. Patients were positive toward home sampling. Conclusions: The established VAMS conversion methods for 7 out of 10 oral targeted anticancer drugs or metabolites met the acceptance criteria. Future studies need to validate the conversion methods with an independent cohort and integrate home sampling via VAMS to provide patients with an alternative to venipuncture at the outpatient clinic.

Understanding the target site pharmacokinetics (PK) of the nitroimidazole analog DNDI-0690, a potential drug for the neglected parasitic disease leishmaniasis, is important due to the diversity of infected tissue sites and potential drug penetration variability. An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for quantifying DNDI-0690 in murine biomatrices (plasma, liver, spleen, skin, and skin microdialysate). The method used three protein precipitation sample preparation procedures, tailored for different biomatrices, utilizing a surrogate biomatrix approach. Murine tissues were enzymatically homogenized with a Collagenase A mixture. Chromatographic detection was performed on a C18 column using gradient elution, coupled to a QTRAP6500 quadrupole MS, operating in positive ionization mode. The method demonstrated accurate and precise quantification of all murine biomatrices on the surrogate biomatrix calibration standards, with a high and reproducible total recovery ranging from 75.9% to 94.2% (CV% ≤ 2.5%). Matrix interferences were mitigated with a deuterated internal standard. Stability experiments demonstrated that DNDI-0690 remained stable in all biomatrices under various conditions. This validated UHPLC-MS/MS method was successfully used to quantify DNDI-0690 in a target site murine infection model, demonstrating its suitability for future target site PK studies involving DNDI-0690.

BACKGROUND:Abiraterone, an active metabolite of abiraterone acetate, is used for the treatment of prostate cancer. Therapeutic drug monitoring (TDM) of abiraterone could improve treatment outcomes. However, its stability in plasma for only 4 hours at room temperature, is making the TDM implementation difficult in clinical practice. Stabilization experiments were performed in our laboratory using esterase inhibitors for the stabilization of abiraterone acetate in preclinical samples. The esterase inhibitor bis(4-nitrophenyl) phosphate (BNPP) stabilizes abiraterone acetate and abiraterone as well. Therefore, we investigated whether the esterase inhibitor BNPP could stabilize abiraterone in fresh human plasma. METHODS:BNPP at 1 and 10 mM were evaluated for its stabilizing effects on abiraterone in fresh human K 2 EDTA plasma. The samples were analyzed using a validated liquid chromatography-mass spectrometry (LC-MS/MS) method. A partial validation assessed BNPP's impact on accuracy, precision, selectivity, and specificity within the fully validated LC-MS/MS method. RESULTS:BNPP at 10 mM, but not 1 mM, effectively prevented abiraterone degradation in fresh human K 2 EDTA plasma, maintaining stability for at least 5 days at room temperature. Partial validation confirmed that all results met the acceptance criteria of the European Medicines Agency guidelines and the US Food and Drug Administration guidance. CONCLUSIONS:We demonstrated that the esterase inhibitor BNPP effectively stabilizes abiraterone in fresh human K 2 EDTA plasma. BNPP had no significant effect on the accuracy, precision, selectivity, or specificity of LC-MS/MS for abiraterone detection. The addition of BNPP to clinical abiraterone samples may be helpful in implementing abiraterone TDM in daily clinical practice.

Leishmaniasis is a neglected parasitic infection that continues to pose a significant global health challenge, with currently limited effective treatment options. DNDI-6174 is a novel orally-active, investigational drug with antileishmanial properties. Herein, a novel ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify DNDI-6174 in relevant murine biomatrices, i.e., K2EDTA plasma and enzymatically-homogenized skin, spleen and liver to support the translational pharmacokinetic-pharmacodynamic model-informed drug development. The chromatographic system consisted of a gradient elution on a standard C18 column connected to a triple quadrupole MS, operating in positive ionization mode. Pre-processing of murine tissues with collagenase A led to a superior homogenization and analyte extraction compared to mechanical disruption. Human K2EDTA plasma served as a surrogate matrix, enabling accurate (bias between -12.0 % and 9.8 %) and precise (relative standard deviation (RSD) ≤ 12.5 %) quantification of DNDI-6174 in the various murine biomatrices. Sample processing with tert-methylbutyl ether resulted in a reproducible recovery between 70.0 % and 93.8 % (RSD ≤ 4.0 %) with an absolute matrix factor between 0.89 and 1.00 for all biomatrices. DNDI-6174 was stable under various conditions, including under tissue homogenization conditions, in all biomatrices investigated. This method was successfully applied in a translational study using a murine cutaneous leishmaniasis skin infection model to assess the target site pharmacokinetics of DNDI-6174, supporting its development as clinical candidate.

A rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantifying ABT-751, an anticancer agent targeting microtubules. Sample preparation involved protein precipitation using acetonitrile and formic acid (100:1, v/v), providing efficient ABT-751 extraction with minimal ion suppression. Buparlisib (BKM-120) served as the internal standard. Chromatographic separation was achieved on a Zorbax Extend C18 column, with gradient elution from 20 to 95% methanol in 0.1% (v/v) formic acid in water, and MS/MS detection was performed in positive ionization mode. This assay was validated for human plasma, mouse plasma, and various mouse tissues, including brain, liver, lung, and kidney homogenates. Calibrants were prepared in each respective blank biological matrix, except for mouse tumor tissue, and curves were fitted by quadratic regression from 5 to 10,000 nM. For mouse tumor tissue we used human plasma as surrogate matrix for calibrants. Precision and accuracy for intra-day and inter-day measurements were within acceptable limits across low, medium, and high concentrations for all matrices. Stability concerns with ABT-751 in mouse plasma and tissue homogenate samples that were stored for more than 8 months were identified and addressed. A pilot pharmacokinetic study in mice demonstrated the applicability of this validated LC-MS/MS method.

Over the past two decades, marked progress has been made in treating non-small cell lung cancer (NSCLC) patients with EGFR-, ALK-, ROS1- and KRASG12C-targeted inhibitors. NSCLC patients very often develop brain metastases. Despite the continuous development of newer and better inhibitors, the survival outcomes of NSCLC patients with brain metastases remain significantly worse than those of patients without. The main challenges in these pharmacotherapies are the development of resistance mutations, and, potentially, the presence of the blood-brain barrier (BBB). The outcomes of clinical studies show the improved efficacy of later-generation targeted inhibitors. The increase in progression free survival (PFS) in patients treated with these later-generation inhibitors is largely attributed to their efficacy against multiple resistance mutations, and possibly due to enhanced brain penetration. This review explores the different aspects hindering the targeted treatment of NSCLC and especially of brain metastases, focusing on recent clinical trials and emerging resistance mutations and the influence of the BBB on the efficacy of EGFR, ALK, ROS1 and KRASG12C inhibitors. The role of the ABCB1 and ABCG2 drug transporters in differential efflux of the targeted drugs at the BBB is also discussed, since preclinical studies indicate that they may reduce the efficacy of transported inhibitors.

Home-sampling for therapeutic drug monitoring (TDM) for oral targeted anticancer drugs offers a promising alternative to traditional hospital-based sampling methods, though it presents challenges. This review aims to summarize the state-of-the-art of home-sampling methods for TDM and evaluates the analytical and clinical validation challenges. A comprehensive search was conducted across Embase, Medline, and Scopus. Eligible articles described analytical and/or clinical validation of home-sampling methods for oral targeted anticancer drugs. ASReview was used to process unique references and to identify relevant studies. Of the 39 included articles, 32 detailed on analytical validation experiments, while 27 covered clinical validation experiments. Dried blood spot and volumetric absorptive microsampling were the primary sampling methods. Key challenges were ensuring robust sample collection, sample pretreatment, hematocrit effects, and sample stability, which were generally thoroughly investigated. Clinical validation yielded promising results for most analytes, although external validation remains crucial for confirming reliability. Home-sampling methods for TDM of oral targeted anticancer drugs show promising results for clinical implementation. Methods for well-studied drugs may be clinically implemented immediately, while others require further external validation. Future research should address device-specific challenges and assess patient feasibility to facilitate the routine use of home-sampling in clinical practice.

Tumor-infiltrating lymphocyte (TIL) medicinal products (MPs) show promise for treating solid tumors, especially metastatic melanoma, in the clinical trial setting. Through these studies, TIL developers have gained an immunological perspective into the mechanism of action (MoA) and infusion product characteristics that influence clinical response. However, to reach marketing authorization for any of the TIL MPs, it will be beneficial to gain a pharmaceutical (process) development perspective as well, from which control of the TIL MPs manufacturing process can be demonstrated and a suitable control strategy can be developed. To do this, a well-defined TIL MP must be established. Defining and optimizing MPs from a pharmaceutical perspective is done by identifying and improving product characteristics or quality attributes (QAs) thought to impact safety and efficacy. Through awareness of the QAs relevant to TIL MPs and considering them throughout pharmaceutical development, improvements and changes can be validated. This approach to pharmaceutical development is part of the quality-by-design workflow, of which this review tackles the first steps. Here, the QAs are structured within a quality target product profile (QTPP), and the corresponding regulatory expectations are considered, spanning quantity, identity, purity, microbiological assays, and biological activity. Based on the regulatory expectations and available literature, the (critical) QAs and points of consideration are proposed when developing TIL MPs. The active pharmaceutical ingredient of the TIL MP is defined as the CD45+CD3+ cells. By analyzing identity attributes correlated to clinical efficacy, four broadly applicable in vivo functionalities associated with TIL MPs MoA and clinical effectiveness are described: tumor recognition, cytotoxic capacity, tumor homing, and persistence. How these in vivo functionalities are quantified in potency assays and the limitations of their methods/readouts are also discussed. The QTPP is a foundation for developing a robust, substantiated control strategy for regulatory approval and increasing patient access. Harmonizing TIL MP development under a unified QTPP applicable in different settings could also facilitate comparisons and, therefore, the development of safer and more efficacious TIL MP variations.

Abstract BACKGROUND Non-small cell lung cancer (NSCLC) represents 85 percent of all lung cancers. Among NSCLC patients, 1-2 percent harbor mutations in the ROS1 proto-oncogene (ROS1). The most prevalent fusion mutations observed include SLC34A2-, CD74- and EZR-ROS1. Approximately 30 percent of the NSCLC patients with these ROS1 fusions develop brain metastases over time, posing significant challenges in treatment efficacy, due to the blood-brain barrier (BBB). ATP-binding cassette (ABC) transporters in the BBB restrict the penetration of compounds into the brain, reducing treatment effectiveness. To address this critical gap in treatment efficacy, a model that accurately mimics NSCLC brain metastases might be of great value. Currently, no SLC34A2-ROS1 driven intracranial tumor model exists, which might limit the clinical development of targeted agents in ROS1-fusion brain metastases. Therefore, the development and characterization of a mouse intracranial brain tumor model utilizing HCC78 cells, which have an intrinsic ROS1-SLC34A2 fusion, could provide valuable insights into targeted therapy efficacy and enhance understanding of NSCLC disease progression. METHOD The publicly available HCC78 cells will be transduced with a mCherry-Luciferase tag, to enable tumor growth visualization over time. Nude FVB mice will be injected with 250.000 HCC78 cells intracranially. Tumor growth will be assessed using IVIS imaging and MRI scanning. When the tumor size reaches its endpoint, the mice will be sacrificed and perfused with Texas Red. The brain will be stained for different markers, to assess tumor proliferation, immune infiltration, ROS1 status and the presence of ABC-transporters. RESULTS Preliminary results show that HCC78 cells are able to grow intracranially. No significant side effects have been observed in the mice. Further characterization of this model is ongoing.

Supplementary Fig. S5. The set of eight genes from the MPAS signature was highly enriched in a GSEA analysis using as input the list of genes sorted on the results from the differential analysis “on treatment” vs. “baseline.”

Supplementary Fig. S2. Vorinostat plasma concentrations over time on days 1 and 14 of the first treatment cycle.

Supplementary Table S3. Results of RNA sequencing.

Developing an oral formulation for the chemotherapeutic cabazitaxel might improve its patient-friendliness, costs, and potentially exposure profile. Cabazitaxel oral availability is restricted by CYP3A-mediated first-pass metabolism, but can be substantially boosted with the CYP3A inhibitor ritonavir. We here tested whether adding the ABCB1/P-glycoprotein inhibitor elacridar to ritonavir-boosted oral cabazitaxel could further improve its tissue exposure using wild-type, CYP3A4-humanized and Abcb1a/b-/- mice. The plasma AUC0-2h of cabazitaxel was increased 2.3- and 1.9-fold in the ritonavir- and ritonavir-plus-elacridar groups of wild-type, and 10.5- and 8.8-fold in CYP3A4-humanized mice. Elacridar coadministration did not influence cabazitaxel plasma exposure. The brain-to-plasma ratio of cabazitaxel was not increased in the ritonavir group, 7.3-fold in the elacridar group and 13.4-fold in the combined booster group in wild-type mice. This was 0.4-, 4.6- and 3.6-fold in CYP3A4-humanized mice, illustrating that Abcb1 limited cabazitaxel brain exposure also during ritonavir boosting. Ritonavir itself was also a potent substrate for the Abcb1 efflux transporter, limiting its oral availability (3.3-fold) and brain penetration (10.6-fold). Both processes were fully reversed by elacridar. The tissue disposition of ritonavir-boosted oral cabazitaxel could thus be markedly enhanced by elacridar coadministration without affecting the plasma exposure. This approach should be verified in selected patient populations.

Supplementary Table S2. Tissue and ctDNA results.

Background: Volumetric Absorptive Microsampling (VAMS) is a useful tool for therapeutic drug monitoring (TDM) of oral targeted anticancer agents. VAMS aims to improve safety and efficacy by enabling at-home blood sample collection by patients. This study aimed to develop and validate an ultra-high performance liquid chromatography–tandem mass spectrometry method for the quantitative determination of abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and the metabolites, Δ(4)-abiraterone (D4A), alectinib-M4, imatinib-M1, and N -desethyl sunitinib, in dried whole blood samples using VAMS to support TDM. Methods: After the collection of 10 μL of whole blood sample using the VAMS device, the analytes were extracted from the tip using methanol with shaking, evaporated, and reconstituted in acetonitrile:0.1 mol/L ammonium hydroxide in water (1:1, vol/vol). The extracts were then analyzed using ultra-high performance liquid chromatography–tandem mass spectrometry. Validation experiments based on the ICH M10 guideline were carried out, and stability was evaluated under shipping and storage conditions. VAMS specimens were collected in the outpatient clinic to demonstrate the applicability of the assay. Results: The validated range of the method was considered accurate and precise for all analytes. Accordingly, the validation experiments met the relevant requirements, except for cross-analyte interference. Based on the stability data, shipment can be performed at room temperature within 14 days after sample collection and the VAMS specimen can be stored up to 9 months at −20 and −70°C. Samples from 59 patients were collected at the hospital. Conclusions: The developed method could be used to successfully quantify the concentrations of abiraterone, D4A, alectinib, alectinib-M4, cabozantinib, imatinib, imatinib-M1, olaparib, sunitinib, and N -desethyl sunitinib within the validated range using VAMS. Therefore, the method can be used to estimate the dried whole blood-to-plasma ratios for TDM in the clinic.

Introduction Post-kala-azar dermal leishmaniasis (PKDL) arises as a dermal complication following a visceral leishmaniasis (VL) infection. Current treatment options for PKDL are unsatisfactory, and there is a knowledge gap regarding the distribution of antileishmanial compounds within human skin. The present study investigated the skin distribution of miltefosine in PKDL patients, with the aim to improve the understanding of the pharmacokinetics at the skin target site in PKDL.Methods Fifty-two PKDL patients underwent treatment with liposomal amphotericin B (20 mg/kg) plus miltefosine (allometric dosing) for 21 days. Plasma concentrations of miltefosine were measured on study days 8, 15, 22 and 30, while a punch skin biopsy was taken on day 22. A physiologically based pharmacokinetic (PBPK) model was developed to evaluate the distribution of miltefosine into the skin.Results Following the allometric weight-based dosing regimen, median miltefosine concentrations on day 22 were 43.73 mu g/g (IQR: 21.94-60.65 mu g/g) in skin and 33.29 mu g/mL (IQR: 25.9-42.58 mu g/mL) in plasma. The median individual concentration ratio of skin to plasma was 1.19 (IQR: 0.79-1.9). In 87% (45/52) of patients, skin exposure was above the suggested EC90 PK target of 10.6 mg/L associated with in vitro susceptibility. Simulations indicated that the residence time of miltefosine in the skin would be more than 2-fold longer than in plasma, estimated by a mean residence time of 604 versus 266 hours, respectively.Conclusion This study provides the first accurate measurements of miltefosine penetration into the skin, demonstrating substantial exposure and prolonged retention of miltefosine within the skin. These findings support the use of miltefosine in cutaneous manifestations of leishmaniasis. In combination with parasitological and clinical data, these results are critical for the future optimization of combination therapies with miltefosine in the treatment of PKDL.

Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500-500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at -70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

Carboxylesterase 2 (CES2) is expressed mainly in liver and intestine, but most abundantly in intestine. It hydrolyzes carboxylester, thioester, and amide bonds in many exogenous and endogenous compounds, including lipids. CES2 therefore not only plays an important role in the metabolism of many (pro-)drugs, toxins and pesticides, directly influencing pharmacology and toxicology in humans, but it is also involved in energy homeostasis, affecting lipid and glucose metabolism. In this study we investigated the pharmacological and physiological functions of CES2. We constructed Ces2 cluster knockout mice lacking all eight Ces2 genes (Ces2-/- strain) as well as humanized hepatic or intestinal CES2 transgenic strains in this Ces2-/- background. We showed that oral availability and tissue disposition of capecitabine were drastically increased in Ces2-/- mice, and tissue-specifically decreased by intestinal and hepatic human CES2 (hCES2) activity. The metabolism of the chemotherapeutic agent vinorelbine was strongly reduced in Ces2-/- mice, but only marginally rescued by hCES2 expression. On the other hand, Ces2-/- mice exhibited fatty liver, adipositis, hypercholesterolemia and diminished glucose tolerance and insulin sensitivity, but without body mass changes. Paradoxically, hepatic hCES2 expression rescued these metabolic phenotypes but increased liver size, adipose tissue mass and overall body weight, suggesting a "healthy" obesity phenotype. In contrast, intestinal hCES2 expression efficiently rescued all phenotypes, and even improved some parameters, including body weight, relative to the wild-type baseline values. Our results suggest that the induction of intestinal hCES2 may combat most, if not all, of the adverse effects of metabolic syndrome. These CES2 mouse models will provide powerful preclinical tools to enhance drug development, increase physiological insights, and explore potential solutions for metabolic syndrome-associated disorders.

BACKGROUND:The role of antibiotics in malignancies treated with immune checkpoint inhibitors (ICI) remains unclear. Several studies suggested a detrimental impact of antibiotic use on the response to ICI, but were susceptible to confounding by indication. Our objective was therefore to assess whether the relationship between antibiotic use and ICI response is causative or merely associative.METHODS:A large, single-center observational cohort study was performed with individuals treated for either non-small cell lung carcinoma (NSCLC) or metastatic melanoma. An effect modification approach was used, aiming to estimate the association between antibiotic use and overall survival (OS) and compare these estimates between individuals receiving first-line ICI treatment versus those receiving first-line tyrosine kinase inhibitors (TKIs). Exposure of interest was antibiotic use within 30 days before the start of anticancer treatment. HRs for OS were estimated for antibiotics versus no antibiotics in each cohort using multivariable propensity adjusted analysis. The "true antibiotic effect" within the ICI versus TKI cohort was modeled using an interaction term.RESULTS:A total of 4534 patients were included, of which 1908 in the ICI cohort and 817 in the TKI cohort. Approximately 10% of patients in each cohort used antibiotics within 30 days before the start of anticancer treatment. Our results demonstrate a lack of synergistic interaction between current antibiotic use and ICI therapy in relation to OS: although antibiotic use was significantly associated with OS decline in the ICI cohort (HR=1.26 (95% CI 1.04 to 1.51)), a similar magnitude in OS decline was found within the TKI cohort (HR=1.24 (95% CI 0.95 to 1.62)). This was reflected by the synergy index (HR=0.96 (95% CI 0.70 to 1.31)), which implied no synergistic interaction between current antibiotic use and ICI.CONCLUSION:This study strongly suggests that there is no causal detrimental association between antibiotic use and ICI therapy outcome when looking at OS in individuals with malignant melanoma or NSCLC. The frequently observed inverse association between antibiotics and ICI response in previous studies is most likely driven by confounding by indication, which was confirmed by the findings in our reference TKI cohort.