Baseline Plasma Samples Research Articles

Differences in the Circulating Proteome in Individuals with versus without Sickle Cell Trait.

Sickle cell trait affects approximately 8% of Black individuals in the United States, along with many other individuals with ancestry from malaria-endemic regions worldwide. While traditionally considered a benign condition, recent evidence suggests that sickle cell trait is associated with lower eGFR and higher risk of kidney diseases, including kidney failure. The mechanisms underlying these associations remain poorly understood. We used proteomic profiling to gain insight into the pathobiology of sickle cell trait. We measured proteomics ( N =1285 proteins assayed by Olink Explore) using baseline plasma samples from 592 Black participants with sickle cell trait and 1:1 age-matched Black participants without sickle cell trait from the prospective Women's Health Initiative cohort. Age-adjusted linear regression was used to assess the association between protein levels and sickle cell trait. In age-adjusted models, 35 proteins were significantly associated with sickle cell trait after correction for multiple testing. Several of the sickle cell trait-protein associations were replicated in Black participants from two independent cohorts (Atherosclerosis Risk in Communities study and Jackson Heart Study) assayed using an orthogonal aptamer-based proteomic platform (SomaScan). Many of the validated sickle cell trait-associated proteins are known biomarkers of kidney function or injury ( e.g. , hepatitis A virus cellular receptor 1 [HAVCR1]/kidney injury molecule-1 [KIM-1], uromodulin [UMOD], ephrins), related to red cell physiology or hemolysis (erythropoietin [EPO], heme oxygenase 1 [HMOX1], and α -hemoglobin stabilizing protein) and/or inflammation (fractalkine, C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 [MCP-1], and urokinase plasminogen activator surface receptor [PLAUR]). A protein risk score constructed from the top sickle cell trait-associated biomarkers was associated with incident kidney failure among those with sickle cell trait during Women's Health Initiative follow-up (odds ratio, 1.32; 95% confidence interval, 1.10 to 1.58). We identified and replicated the association of sickle cell trait with a number of plasma proteins related to hemolysis, kidney injury, and inflammation.

Impact of ramucirumab plus erlotinib on circulating cell-free DNA from patients with untreated metastatic non-small cell lung cancer with EGFR-activating mutations (RELAY phase 3 randomized study).

An exploratory, proof-of-concept, liquid biopsy addendum to examine biomarkers within cell-free DNA (cfDNA) in the RELAY phase 3, randomized, double-blind, placebo-controlled study was conducted. RELAY showed improved progression-free survival (PFS) with ramucirumab (RAM), a human immunoglobulin G1 vascular endothelial growth factor receptor 2 antagonist, plus erlotinib (ERL), a tyrosine kinase inhibitor, compared with placebo (PL) plus ERL. Treatment-naïve patients with endothelial growth factor receptor (EGFR)-mutated metastatic non-small cell lung cancer were randomized (1:1) to RAM + ERL or PL + ERL. Plasma samples were collected at baseline, on treatment, and at 30-day post-study treatment discontinuation follow-up. Baseline and treatment-emergent gene alterations and EGFR-activating mutation allele counts were investigated by next-generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR), respectively. cfDNA concentration and fragment size were evaluated by real-time polymerase chain reaction and the BioAnalyzer. Patients with a valid baseline plasma sample were included (70 RAM + ERL, 61 PL + ERL). TP53 mutation was the most frequently co-occurring baseline gene alteration (43%). Post-study treatment discontinuation EGFR T790M mutation rates were 54.5% (6/11) and 41.2% (7/17) by ddPCR, and 22.2% (2/9) and 29.4% (5/17) by NGS, in the RAM + ERL and PL + ERL arms, respectively. EGFR-activating mutation allele count decreased at Cycle 4 in both treatment arms and was sustained at follow-up with RAM + ERL. PFS improved for patients with no detectable EGFR-activating mutation at Cycle 4 vs. those with detectable EGFR-activating mutation. Total cfDNA concentration increased from baseline at Cycle 4 and through to follow-up with RAM + ERL. cfDNA fragment size was similar between treatment arms at baseline [mean (standard deviation) base pairs: RAM + ERL, 173.4 (2.6); PL + ERL, 172.9 (3.2)] and was shorter at Cycle 4 with RAM + ERL vs. PL + ERL [169.5 (2.8) vs. 174.1 (3.3), respectively; P<0.0001]. Baseline vs. Cycle 4 paired analysis showed a decrease in cfDNA fragment size for 84% (48/57) and 23% (11/47) of patient samples in the RAM + ERL and PL + ERL arms, respectively. EGFR-activating mutation allele count was suppressed, total cfDNA concentration increased, and short fragment-sized cfDNA increased with RAM + ERL, suggesting the additional anti-tumor effect of RAM may contribute to the PFS benefit observed in RELAY with RAM + ERL vs. PL + ERL. ClinicalTrials.gov; identifier: NCT02411448.

183-OR: Metabolic Signatures of Type 2 Diabetes Subtypes in UK Biobank

Type 2 diabetes (T2D) is a complex disease with heterogeneous pathophysiology. Five distinct subtypes of T2D were previously identified in the UK Biobank and validated in the All of Us cohort through a novel clustering algorithm: Cluster 1-5, characterized by early-onset of T2D, late-onset of T2D, severe obesity, poor glycemic control, and poor renal function, respectively. We aim to establish the subtype-specific signatures of metabolites. Nuclear magnetic resonance spectroscopy-derived metabolomic profiling was undertaken on baseline plasma samples in 12,271 UK Biobank participants with T2D. Among a subset of 2828 participants with metabolomic data available, elastic net regularized regressions with 10-fold cross-validation were applied to identify subtype-specific metabolic signatures. Of the 168 named metabolites used in the analyses, we selected a combination of 39 metabolites most significantly associated with subtypes of T2D (P&amp;lt;0.0001, FDR&amp;lt;0.05). We observed subtype-specific highest levels of creatinine in the impaired renal function characterized Cluster 5 and the lowest level in Cluster 1, featured by early-onset of T2D (mean difference= 2.46 [SD=0.09], P&amp;lt;0.0001). Cluster 4 characterized by poor glycemic control had increased levels of glucose and decreased levels of glutamine, indicating a greater level of hyperglycemia. Individuals in Cluster 3 characterized by severe obesity exhibited elevated levels of inflammation metabolites biomarker (glycoprotein acetyls) and obesity-related amino acids (tyrosine and valine). The plasma unsaturation degree of fatty acid was also lower in Cluster 3 compared to other clusters. Cluster 2 patients had lower levels of glucose and glycoprotein acetyls and higher levels of glutamine, omega-3 fatty acids, and docosahexaenoic acids. Overall, Cluster 1 and 2 had the least metabolic dysregulation among the T2D subtypes. We have identified metabolic signatures for T2D subtypes, revealing subtype-specific metabolic mechanisms for precision interventions. Disclosure Q.Xue: None. X.Li: None. X.Wang: None. H.Ma: None. Y.Heianza: None. L.Qi: None.

317-OR: Metabolomic Analyses Identify Novel Predictors of Diabetic Kidney Disease in Youth-Onset Type 2 Diabetes

Background: Diabetic kidney disease (DKD) develops by young adulthood in up to 50% of people with youth-onset type 2 diabetes (Y-T2D), increasing risk of dialysis and premature death. Understanding mechanisms responsible for early DKD is key to management and prevention; accordingly, we sought to identify metabolite signatures of DKD in Y-T2D. Methods: We measured 57 metabolites in 374 baseline plasma and urine samples from the Treatment Options for type 2 Diabetes in Adolescents and Youth (TODAY) study, using mass spectrometry with a targeted ZipChip based assay. Urine albumin-to-creatinine ratio (UACR) was assessed annually for up to 15 years. Incident moderate and severe albuminuria were defined as UACR ≥30 and ≥300 mg/g, respectively, on ≥2 of 3 measures. We evaluated prediction of moderate and severe albuminuria in separate Cox proportional hazards models adjusted for HbA1c, triglycerides, systolic blood pressure, and estimated insulin sensitivity. Urine metabolites were normalized by urine creatinine. Results: Participants were 14±2 years of age, 37% male; 43% developed either moderate or severe albuminuria. Four urine metabolites predicted time to moderate albuminuria, while 8 urine metabolites predicted time to severe albuminuria, with 3 metabolites in common: 2-hydroxybutyric acid (moderate: HR: 0.82 per 1 SD [95% CI 0.70, 0.96]; severe: 0.70 [0.55, 0.88]), glycine (moderate: 0.81 [0.69, 0.94]; severe: 0.62 [0.45, 0.85]), citric acid (moderate: 0.78 [0.67, 0.91]; severe: 0.76 [0.58, 0.99]). Four plasma metabolites predicted time to moderate albuminuria and severe albuminuria, such as glutamic acid for moderate albuminuria (1.23 [1.03, 1.47]) and citric acid for severe albuminuria (1.39 [1.09, 1.77]). Conclusion: Higher urine metabolites involved in mitigating oxidative stress (glycine), glomerular epithelial cell injury (2-hydroxybutyric acid) and preserving mitochondrial function (citrate) predicted lower risk of albuminuria in Y-T2D. Disclosure L.Pyle: None. L.M.Laffel: Advisory Panel; Medtronic, Lilly Diabetes, Novo Nordisk, Vertex Pharmaceuticals Incorporated, Roche Diagnostics, Provention Bio, Inc., Consultant; Dexcom, Inc., Janssen Pharmaceuticals, Inc., Medscape. A.S.Shah: None. K.Drews: None. J.R.Ryder: None. R.Gubitosi-klug: None. K.Sharma: Advisory Panel; Reata Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc. P.Bjornstad: Advisory Panel; AstraZeneca, Novo Nordisk, Lilly, Horizon Therapeutics plc, Boehringer Ingelheim (Canada) Ltd., LG Chem, Consultant; Bayer Inc., Bristol-Myers Squibb Company. T.B.Vigers: None. L.El ghormli: None. I.De boer: Advisory Panel; AstraZeneca, Boehringer Ingelheim and Eli Lilly Alliance, Boehringer Ingelheim International GmbH, Otsuka America Pharmaceutical, Inc., Bayer Inc., Consultant; George Clinical, Gilead Sciences, Inc., Medscape, Research Support; Dexcom, Inc. R.G.Nelson: None. S.Waikar: None. H.L.Heerspink: Consultant; AstraZeneca, Boehringer Ingelheim International GmbH, Bayer Inc., Eli Lilly and Company, Chinook Therapeutics Inc., CSL Behring, Gilead Sciences, Inc., George Clinical, Merck &amp; Co., Inc., Janssen Research &amp; Development, LLC, Traveere Pharmaceuticals, Novo Nordisk. N.White: None. K.L.Tommerdahl: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (U01DK61242)

1095-P: Proteomic Analyses Identify Novel Predictors of Loss of Glycemic Control in Youth-Onset Type 2 Diabetes

The basis for the more aggressive loss of β-cell function in youth-onset type 2 diabetes (Y-T2D) compared to adult-onset T2D and its unresponsiveness to interventions remain incompletely understood. Given the increasing incidence of Y-T2D, there is an urgent need to better understand its pathophysiology and develop appropriate interventions. Accordingly, we sought to identify multiprotein signatures that predict loss of glycemic control in Y-T2D. We measured 7604 Aptamers in 374 baseline plasma samples from the Treatment Options for type 2 Diabetes in Adolescents and Youth (TODAY) study, using the SomaScan 7K Proteomic (SomaLogic) platform. Loss of glycemic control was defined as HbA1c ≥8% for 6 months or inability to wean from temporary insulin after acute metabolic decompensation. We evaluated associations with time to loss of glycemic control in Cox proportional hazards models adjusted for body mass index (BMI) and estimated insulin sensitivity. Gene set enrichment analysis identified pathways of interest. The false discovery rate was controlled at 5% and we report q-values. Participants were 14±2 years of age, 37% male; 72% experienced loss of glycemic control. Seventy-four proteins were associated with time to loss of glycemic control, with sixty-seven proteins remaining associated with time to loss of glycemic control after multivariable adjustments, such as: plexin-B2 (HR: 1.54 per 1 SD [95% CI 1.35, 1.76], q&amp;lt;0.0001) and Ephrin-A3 (HR: 1.25 [1.13, 1.39], q=0.012). Twenty gene sets, including HIF-1 signaling, metabolic and cell adhesion pathways, were positively related and 13 gene sets were negatively related to time to loss of glycemic control. Novel proteins, including those involved in regulation of insulin secretion and ligand-receptor pairing in β-cells, predict loss of glycemic control in Y-T2D, independently of BMI and estimated insulin sensitivity. Future work should include validation of these findings and investigating potential therapeutic targets. Disclosure T.B.Vigers: None. L.M.Laffel: Advisory Panel; Medtronic, Lilly Diabetes, Novo Nordisk, Vertex Pharmaceuticals Incorporated, Roche Diagnostics, Provention Bio, Inc., Consultant; Dexcom, Inc., Janssen Pharmaceuticals, Inc., Medscape. A.S.Shah: None. K.Drews: None. J.R.Ryder: None. R.Gubitosi-klug: None. P.Bjornstad: Advisory Panel; AstraZeneca, Novo Nordisk, Lilly, Horizon Therapeutics plc, Boehringer Ingelheim (Canada) Ltd., LG Chem, Consultant; Bayer Inc., Bristol-Myers Squibb Company. L.Pyle: None. L.El ghormli: None. I.De boer: Advisory Panel; AstraZeneca, Boehringer Ingelheim and Eli Lilly Alliance, Boehringer Ingelheim International GmbH, Otsuka America Pharmaceutical, Inc., Bayer Inc., Consultant; George Clinical, Gilead Sciences, Inc., Medscape, Research Support; Dexcom, Inc. R.G.Nelson: None. K.Sharma: Advisory Panel; Reata Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc. S.Waikar: None. H.L.Heerspink: Consultant; AstraZeneca, Boehringer Ingelheim International GmbH, Bayer Inc., Eli Lilly and Company, Chinook Therapeutics Inc., CSL Behring, Gilead Sciences, Inc., George Clinical, Merck &amp; Co., Inc., Janssen Research &amp; Development, LLC, Traveere Pharmaceuticals, Novo Nordisk. N.White: None. K.L.Tommerdahl: None.

Apo CIII Proteoforms, Plasma Lipids, and Cardiovascular Risk in MESA.

Apo CIII (apolipoprotein CIII) is an important regulator of triglyceride metabolism and was associated with cardiovascular risk in several cohorts. It is present in 4 major proteoforms, a native peptide (CIII0a), and glycosylated proteoforms with zero (CIII0b), 1 (CIII1, most abundant), or 2 (CIII2) sialic acids, which may differentially modify lipoprotein metabolism. We studied the relationships of these proteoforms with plasma lipids and cardiovascular risk. Apo CIII proteoforms were measured by mass spectrometry immunoassay in baseline plasma samples of 5791 participants of Multi-Ethnic Study of Atherosclerosis, an observational community-based cohort. Standard plasma lipids were collected for up to 16 years and cardiovascular events (myocardial infarction, resuscitated cardiac arrest, or stroke) were adjudicated for up to 17 years. Apo CIII proteoform composition differed by age, sex, race and ethnicity, body mass index, and fasting glucose. Notably, CIII1 was lower in older participants, men and Black and Chinese (versus White) participants, and higher in obesity and diabetes. In contrast, CIII2 was higher in older participants, men, Black, and Chinese persons, and lower in Hispanic individuals and obesity. Higher CIII2 to CIII1 ratio (CIII2/III1) was associated with lower triglycerides and higher HDL (high-density lipoprotein) in cross-sectional and longitudinal models, independently of clinical and demographic risk factors and total apo CIII. The associations of CIII0a/III1 and CIII0b/III1 with plasma lipids were weaker and varied through cross-sectional and longitudinal analyses. Total apo CIII and CIII2/III1 were positively associated with cardiovascular disease risk (n=669 events, hazard ratios, 1.14 [95% CI, 1.04-1.25] and 1.21 [1.11-1.31], respectively); however, the associations were attenuated after adjustment for clinical and demographic characteristics (1.07 [0.98-1.16]; 1.07 [0.97-1.17]). In contrast, CIII0b/III1 was inversely associated with cardiovascular disease risk even after full adjustment including plasma lipids (0.86 [0.79-0.93]). Our data indicate differences in clinical and demographic relationships of apo CIII proteoforms, and highlight the importance of apo CIII proteoform composition in predicting future lipid patterns and cardiovascular disease risk.

#3115 THE ASSOCIATION OF FIBROBLAST GROWTH FACTOR-23 AND CARDIORENAL SYNDROME IN PATIENTS WITH DIABETES MELLITUS

Abstract Background and Aims Previous studies suggest that interactions between the heart and the kidney can contribute to the progressive dysfunction of both organs. Recently, there has been an increase in the prevalence of cardiovascular disease (CVD) and chronic kidney disease (CKD) due to increasing diabetes mellitus rate. It is known that fibroblast growth factor-23 (FGF-23) has been found to be related to kidney metabolism especially as a biomarker and a key factor to be used in the kidney field. The aim of our study was to assess potential therapeutic approaches of the use of FGF-23 in clinical settings as a potential circulating biomarker for CVD and CKD. Method A total of 155 patients with DT2, aged 34 to 84 years (60 [60;72]), were examined. Control group included 94 healthy people the same age. All patients underwent standard clinical and laboratory examination, with an assessment of the levels of FGF-23 in baseline plasma samples. Renal function was assessed based on the levels of serum creatinine, cystatin C, eGFR, which was calculated according to the CKD-EPI formula, and albuminuria, which was assessed as albumin/creatinine ratio (A/C). An echocardiographic examination was conducted according to the standard protocol with the calculation of dimensional, volume and speed characteristics. Results The levels of FGF-23 were significantly higher in DT2 patients with CKD 5 in comparison with stages 1–4. There were positive strong significant association between FGF-23 and creatinine (r = 0.71, p&amp;lt;0.001). In both unadjusted and adjusted analyses, probability of decreased eGFR was associated strongly with FGF-23 (COR; 95% CI 1.890; 1.362-2.622, p&amp;lt;0,001). When evaluating the dependence of the probability of decreased eGFR on the FGF-23 using the ROC analysis, the cut-off value of FGF-23 was 0,9 pmol/l. The sensitivity and specificity of the method were 75.3% and 74.5%, respectively (AUC 0.832±0.035 with 95% CI: 0.764-0.901, p&amp;lt;0.001). DT2 patients with left ventricular hypertrophy (LVH) were characterized by higher levels of FGF-23 (2,45 [0,65;7,43] vs 0,57 [0,19;2,19] pmol/l in control group). DT2 patients with LVH and natriuretic peptides levels ≥ 35 pg/ml for BNP and/or ≥125 ng/ml for NT-proBNP had significantly high concentrations of FGF-23 than patients with normal levels of natriuretic peptides (3,22 [1,09;9,55] vs. 0,52 [0,27;1,08] pmol/l, p&amp;lt;0,0001). In order to assess the diagnostic significance of the FGF-23 for predicting left ventricular mass index (LVMI) thickening and increasing the proBNP concentration, a ROC analysis was performed. Thus, at the level of FGF-23 (AUC-0.702) = 0.9 pmol/l, the sensitivity and specificity for LVMI thickening were 66.9% and 67.3%. Conclusion Our study identifies FGF-23 as a promising target of novel therapeutic interventions in cardiorenal syndrome, which should be investigated in future clinical studies.

Molecular correlation of response to pyrotinib in advanced NSCLC with HER2 mutation: biomarker analysis from two phase II trials

BackgroundNon-small cell lung cancer (NSCLC) with HER2 mutation has entered into the era of targeted therapy. However, both anti-HER2 antibody–drug conjugates (ADCs) and tyrosine kinase inhibitors (TKIs) showed moderate objective response rate (ORR) and median progression-free survival (PFS). The aim of this study was to investigate the molecular features of responders to pyrotinib in advanced NSCLC with HER2 mutation.MethodsPatients from our two previous phase II trials were pooled analyzed. Their circulating tumor DNA (ctDNA) were detected by next-generation sequencing (NGS) panels, and the correlation with the efficacy of pyrotinib was investigated.ResultsThis pooled analysis included 75 patients, and 50 of them with baseline plasma samples were finally enrolled with a median age of 57 years old. The overall ORR and median PFS were 28% and 7.0 months respectively. Biomarker analysis showed that 5 patients were ctDNA nonshedding. Patients with TP53 wild type were significantly associated with higher disease control rate (97.1%vs. 68.8%, p = 0.010), PFS (median 8.4 vs. 2.8 months, p = 0.001) and overall survival (OS, median 26.7 vs. 10.4 months, p < 0.001) than those with mutations. ctDNA of nonshedding and clearance exhibited significantly longer PFS (median: 10.2 vs. 9.8 vs. 5.6 months, p = 0.036) and a trend of longer OS (median: 35.3 vs. 18.1 vs. 14.6 months, p = 0.357) than those not.ConclusionPatients with TP53 wild type, ctDNA nonshedding, or clearance showed superior efficacy of pyrotinib in patients with HER2-mutated advanced NSCLC, which might be helpful to guide the utility of pyrotinib in clinical setting.Trial registration: The patients were from two registered clinical trials (ClinicalTrials.gov: NCT02535507, NCT02834936).

A Nested Case-Control Study to Explore the Association between Immunoglobulin G N-glycans and Ischemic Stroke

ObjectiveThis study prospectively investigates the association between immunoglobulin G (IgG) N-glycan traits and ischemic stroke (IS) risk. MethodsA nested case-control study was conducted in the China suboptimal health cohort study, which recruited 4,313 individuals in 2013–2014. Cases were identified as patients diagnosed with IS, and controls were 1:1 matched by age and sex with cases. IgG N-glycans in baseline plasma samples were analyzed. ResultsA total of 99 IS cases and 99 controls were included, and 24 directly measured glycan peaks (GPs) were separated from IgG N-glycans. In directly measured GPs, GP4, GP9, GP21, GP22, GP23, and GP24 were associated with the risk of IS in men after adjusting for age, waist and hip circumference, obesity, diabetes, hypertension, and dyslipidemia. Derived glycan traits representing decreased galactosylation and sialylation were associated with IS in men (FBG2S2/(FBG2 + FBG2S1 + FBG2S2): odds ratio (OR) = 0.92, 95% confidence interval (CI): 0.87–0.97; G1n: OR = 0.74, 95% CI: 0.63–0.87; G0n: OR = 1.12, 95% CI: 1.03–1.22). However, these associations were not found among women. ConclusionThis study validated that altered IgG N-glycan traits were associated with incident IS in men, suggesting that sex discrepancies might exist in these associations.

Mobocertinib efficacy in patients with NSCLC and EGFR exon 20 insertion mutations (ex20ins) identified by next-generation sequencing (NGS) of circulating tumor DNA (ctDNA).

9082 Background: EGFR ex20ins are distinct targetable driver mutations present in ~2% of all NSCLC. While most EGFR TKIs have limited effectiveness, mobocertinib is the first oral therapy approved for this unmet need. EGFR ex20ins are heterogeneous ( &gt; 60 variants), and PCR testing detects a limited number of variants, leading to undetected disease in ~50% of cases, whereas NGS can identify all such mutations. Testing plasma ctDNA is a noninvasive approach for detection of genomic variants, such as EGFR ex20ins. Methods: In the mobocertinib phase 1/2 study (NCT02716116), patients were enrolled based on EGFR ex20ins detected by various local tissue or liquid clinical trial assays (CTAs). Baseline plasma samples were collected and processed to extract cell-free DNA and were evaluated for EGFR ex20ins status using FoundationOne Liquid CDX (F1LCDX), an NGS-based plasma ctDNA test. Concordance between F1LCDX and the CTAs was demonstrated by testing patient samples from the phase 1/2 trial and paired tissue and plasma samples from commercially acquired and stage-matched patients with NSCLC (N = 305). Efficacy analyses by assay method were performed in EGFR ex20ins–positive platinum-pretreated patients (PPP; n = 114 [FDA approved population]; data cutoff: 1 Nov 2021) in the phase 1/2 trial. Results: Concordance between F1LCDX and CTAs was demonstrated with samples from the CTA-positive (n = 159) and CTA-negative populations (n = 87) tested by F1LCDX where tissue and plasma were assessable; the point estimate of positive percentage agreement (95% CI) was 68.6% (61.0–75.3), and the point estimate of negative percentage agreement was 100% (95.8–100). F1LCDX detected 34 individual EGFR ex20ins variant types. In the response evaluable population, patients who tested EGFR ex20ins positive by F1LCDX (n = 55) demonstrated confirmed ORR (34.5%) and disease control rate (74.5%), which was comparable to those observed among all PPP in the phase 1/2 trial (confirmed ORR: 28%; confirmed disease control rate: 78%). Conclusions: Mobocertinib has shown efficacy in PPP with EGFR ex20ins NSCLC. F1LCDX effectively identified patients with EGFR ex20ins who may benefit from mobocertinib, providing an additional noninvasive diagnostic option to guide treatment. Clinical trial information: NCT02716116 .

Longitudinal mutational analysis of TP53 in plasma circulating tumor DNA (ctDNA) in patients (pts) with solid tumors in a phase I study of BI 907828, an MDM2–p53 antagonist.

3036 Background: Inactivation of p53 can occur due to TP53 mutations or downregulation of wild-type p53 by its primary negative regulator, MDM2. Targeting the MDM2–p53 interaction may therefore restore p53 function. BI 907828, a highly potent MDM2–p53 antagonist, is being evaluated in a phase Ia/b study in pts with advanced solid tumors (NCT03449381). During dose escalation (phase Ia), BI 907828 demonstrated a manageable safety profile and early signs of efficacy. Here we present data from a longitudinal mutational analysis of TP53 using ctDNA. The objective of the analysis was to identify if mutations in TP53 can be associated with possible acquired resistance to BI 907828. Methods: Collection of blood samples for ctDNA analyses was optional and sampled longitudinally from pts at baseline and every cycle until end of treatment (EoT). ctDNA was purified from plasma and analyzed using tumor-specific next generation sequencing (NGS; custom-made 8-gene panel using KAPA HyperCap technology, including TP53) to identify tumor-derived somatic mutations that may be relevant to understanding resistance mechanisms to BI 907828. The limit of detection was determined to be 0.5% mutant allele frequency; common polymorphisms were filtered out. Results: Plasma samples for ctDNA analysis were available from 44/54 (81%) pts enrolled to phase Ia. Baseline plasma samples from 26/54 (48%) pts and EoT samples from 41/54 (76%) pts were analyzed. Mutations were found in 6/26 (23%) samples at baseline. In 2 pt samples, mutations were found in both tumor and ctDNA, but concordant results were limited to 1 pt. At EoT, no (0) mutations were found in 16/41 (39%) samples, 1 mutation in 11/41 (27%), 2 mutations in 6/41 (15%), 3 mutations in 2/41 (5%) and 4 mutations in 6/41 (15%). No sample had more than 4 mutations. The most frequent mutation at EoT (in 9/41 [22%] samples) was R175H, a known p53 loss of function (LOF) mutation, followed by R248Q/W LOF (in 6/41 [15%) samples). Mutations in non- TP53 genes were detectable independent of TP53 mutation status, suggesting ctDNA was present in all samples. No clear association of induction of mutations could be identified for any BI 907828 dose or dosing schedule, treatment duration, or tumor type. 9/20 (45%) pts with no detectable mutations at baseline maintained wild-type TP53 status at EoT. Signs of efficacy were observed in pts with acquired TP53 LOF mutations, suggesting that these mutations have no effect on efficacy. Analysis of TP53 mutational status at EoT in conjunction with correlative clinical efficacy will be presented. Conclusions: This mutation analysis represents one of the most comprehensive assessments of longitudinal ctDNA by NGS for TP53 from a clinical trial of an MDM2–p53 antagonist. Preliminary data suggest that BI 907828 does not systematically lead to broad acquisition of resistance by inducing alterations in TP53. Clinical trial information: NCT03449381 .

Clinical application of ctDNA detection in Chinese pediatric patients with soft tissue sarcoma (STS).

10041 Background: Circulating tumor DNA (ctDNA) is an effective and noninvasive supplementary approach for assessing minimum tumor burden in a range of solid tumors; however, studies on pediatric soft tissue sarcoma (STS) are limited. The purpose of the study was to assess the utility of ctDNA detection in predicting patient response and recurrence, and to inform treatment strategies through dynamic monitoring of ctDNA during the treatment of various types of pediatric STS. Methods: From January 2022 to January 2023, a total of 27 tissues and 112 plasma samples were obtained at baseline and during treatment. Utilizing the next-generation sequencing approach, ctDNA profiling was performed on tissue and plasma samples from 48 pediatric STS ( &lt; 18 years old) with a panel of 481 sarcoma-specific genes (Nanjing Geneseeq Technology Inc.). Results: The study included thirty patients with rhabdomyosarcoma, nine Ewing sarcoma, and nine other subtypes of sarcoma, with 52.1% (n = 25) female. Seven patients were stages I-II, and 24 were stages III-IV. At baseline, ctDNA was identified in 56.25 % (18/32) of plasma before chemotherapy and 87.50 % (14/16) of plasma before surgery. Comparable mutational burdens were seen in baseline tissue and plasma samples, with an average of 3.8 and 3 mutations per sample, respectively. The concordance of detected mutations between tissue and plasma was 64.3%. The average plasma abundance (p = 0.007), maximum abundance (p = 0.0045), ctDNA concentration (hGE/ml) (p = 0.0005), and relative ctDNA abundance (p = 0.0083) of stage IV patients (n = 5) were higher than those of stage I-III patients (n = 6). Following surgical resection, four patients (25 %, 4/16) had detectable ctDNA. In the longitudinal monitoring, we found a high correlation (75.86 %, 22/29) between ctDNA trend and clinical response. Notably, ctDNA positive in Ewing sarcoma had the strongest correlation with clinical response. The inconsistency between ctDNA detection and clinical response could be attributed to the presence of bone lesions (57.1%, 4/7). One patient was observed to have positive ctDNA before any radiological findings. Conclusions: Our findings showed that it is feasible to use ctDNA to dynamically monitor tumor burden in pediatric soft tissue sarcoma, especially Ewing sarcoma. ctDNA may serve as a safe, non-invasive option complementary to imaging assessment of sarcoma, and further clinical benefits are being investigated.

Safety and efficacy of trastuzumab biosimilar plus irinotecan or gemcitabine in patients with previously treated HER2 (ERBB2)-positive non-breast/non-gastric solid tumors: a phase II basket trial with circulating tumor DNA analysis

Human epidermal growth factor receptor 2 (HER2) (ERBB2)-directed agents are standard treatments for patients with HER2-positive breast and gastric cancer. Herein, we report the results of an open-label, single-center, phase II basket trial to investigate the efficacy and safety of trastuzumab biosimilar (Samfenet®) plus treatment of physician's choice for patients with previously treated HER2-positive advanced solid tumors, along with biomarker analysis employing circulating tumor DNA (ctDNA) sequencing. Patients with HER2-positive unresectable or metastatic non-breast, non-gastric solid tumors who failed at least one prior treatment were included in this study conducted at Asan Medical Center, Seoul, Korea. Patients received trastuzumab combined with irinotecan or gemcitabine at the treating physicians' discretion. The primary endpoint was the objective response rate as per RECIST version 1.1. Plasma samples were collected at baseline and at the time of disease progression for ctDNA analysis. Twenty-three patients were screened from 31 December 2019 to 17 September 2021, and 20 were enrolled in this study. Their median age was 64 years (30-84 years), and 13 patients (65.0%) were male. The most common primary tumor was hepatobiliary cancer (seven patients, 35.0%), followed by colorectal cancer (six patients, 30.0%). Among 18 patients with an available response evaluation, the objective response rate was 11.1% (95% confidence interval 3.1% to 32.8%). ERBB2 amplification was detected from ctDNA analysis of baseline plasma samples in 85% of patients (n= 17), and the ERBB2 copy number from ctDNA analysis showed a significant correlation with the results from tissue sequencing. Among 16 patients with post-progression ctDNA analysis, 7 (43.8%) developed new alterations. None of the patients discontinued the study due to adverse events. Trastuzumab plus irinotecan or gemcitabine was safe and feasible for patients with previously treated HER2-positive advanced solid tumors with modest efficacy outcomes, and ctDNA analysis was useful for detecting HER2 amplification.

Phase II trial of lazertinib in patients with epidermal growth factor receptor (EGFR) mutation-positive (M+), metastatic non-small cell lung cancer (NSCLC) with asymptomatic or mild symptomatic brain metastases after failure of EGFR tyrosine kinase inhibitor (TKI) (KCSG LU20-15).

9054 Background: Central nervous system (CNS) metastases occur not infrequently, ultimately devastating consequences in advanced EGFR M+ NSCLC. Lazertinib, a 3rd-generation (G) EGFR-TKI, has shown potent antitumor activity in EGFR-mutant NSCLC. We conducted a phase II single-arm study of lazertinib in EGFR M+ NSCLC patients (pts) having CNS metastases to evaluate its CNS activity. Methods: The patients exhibiting CNS progression upon 1st or 2nd-G EGFR-TKIs with least one measurable CNS lesion were enrolled. The primary endpoint was intracranial objective response rate (iORR) in evaluable for response set (ERS). Secondary endpoints included iORR without T790M mutation, overall ORR (oORR), disease control rate (DCR), intracranial progression-free survival (iPFS) in ERS. Overall survival (OS) and safety analyses were performed in pts who received at least one dose of lazertinib. Cerebrospinal fluid (CSF) penetration was measured as exploratory biomarker study. Results: Of 40 pts enrolled, 38 were evaluable for tumor response. Nineteen pts harbored exon 19 deletion and 21 pts for exon 21 L858R and 30% of pts had radiologic evidence of leptomeningeal carcinomatosis (LM) with 2 cytological confirmed cases. Forty-five percent of pts received afatinib as first line treatment, followed by 42.5% of gefitinib and 12.5% of erlotinib. Baseline plasma samples of all the 40 patients obtained for liquid-based next-generation sequencing (Guardant360) and T790M was found in 5 pts prior to lazertinib treatment. iORR was 57.9% (22/38). iORR with T790M-negative, oORR, and DCR were 54.5%, 39.5%, and 97.4%, respectively. With median follow-up of 13.6 months, median iPFS and OS were not reached. Treatment-related adverse events (TRAEs) emerged in 85.0% of pts with most common TRAEs being skin rash (50.0%) and paresthesia (42.5%). Grade 3 or 4 TRAEs were reported in 10.0%. The CSF penetration rates of lazertinib and its metabolite (YH26334) were 46.2% and 33.1%, respectively in paired CSF and plasma samples, suggesting high CNS penetration efficacy. Conclusions: Lazertinib has substantial CNS activity regardless of T790M status against the progression of intracranial metastases ± LM during the treatment of 1st or 2nd-G EGFR-TKIs in metastatic EGFR M+ NSCLC pts. These results suggest that using lazertinib instead of brain local treatment would be a potential strategy in EGFR M+ NSCLC, who progressed on CNS metastases after prior EGFR-TKI. Clinical trial information: NCT05326425 . [Table: see text]

Landscape of FGFR2/3 alterations in genitourinary cancer.

e15047 Background: FGFR2/3 is frequently activated by mutation or overexpression, and it is a validated therapeutic target in several cancers. However, the molecular profile of FGFR2/3 in genitourinary cancer is still lacking. In this study, we assessed FGFR2/3 alterations for better understanding therapeutic implications in genitourinary cancer. Methods: Patients diagnosed with genitourinary cancer were enrolled in the study. Tumors and plasma samples of participants were sequencing for next-generation sequencing from Chinese cohort and TCGA database. Results: A total of 3858 patients were enrolled with 1640 renal cell cancer (RCC), 1300 prostate cancer (PC), and 918 urothelial cancer (UC), containing 2482 tumor tissues and 1376 baseline plasma samples were collected. A total of 14.6% patients (563/3858) harbored at least one FGFR2/3 alterations . 24.1% (221/918) UC carried the most FGFR2/3 alterations , including somatic mutation (18.2%), CNV (5.7%), and fusion (2.1%). Furthermore, the altered FGFRs targets are mainly S249C (6.5%), Y373C (2.5%), FGFR3-TACC3 fusion (1.6%). R248C (1.2%), and G370C (0.5%) in UC. FGFR2/3 alterations were found in 14.0% (182/1300) PC, including somatic mutation (8.2%), CNV (5.8%), and fusion (0.4%). However, only 2 patients were found to have altered FGFRs targets including R248C mutation and FGFR3-TACC3 fusion (n = 1 each). Regarding of RCC, only 9.8% (160/1640) patients had FGFR3 alterations , including somatic mutation (5.8%), CNV (5.0%), and fusion (0.5%). Furthermore, the altered FGFRs targets are mainly R248C, S249C, Y373C (n = 4 each). Moreover, patients with FGFR2/3 alterations harbored a significantly higher TMB than those in patients without FGFR2/3 alterations (Mean: 13.3 vs 9.4; p &lt; 0.001). There was a significant difference in TMB among different cancers with FGFR2/3 alterations , with UC being the highest and PC the lowest (Mean: 17.0 vs 13.0 vs 10.2; p &lt; 0.001). Conclusions: FGFR2/3 alterations are frequently altered in genitourinary cancer, especially UC. Comprehensive genomic profiling of FGFR2/3 alterations highlights the clinical and therapeutic implication.

RAS testing of circulating cell-free DNA (cfDNA) from tissue RAS/BRAF wild-type metastatic colorectal carcinoma (mCRC) patients: Preliminary data from the Liquid Biopsy Monoclonal Antibodies in mCRC (LIBImAb) study.

3046 Background: In mCRC patients RAS/BRAF testing is usually performed on tissue biopsy in order to select patients for treatment with anti-EGFR monoclonal antibodies. Several studies described discordant RAS status between tissue and cfDNA testing from the same patients. In addition, up to 30% of patients with secondary resistance to anti-EGFR therapies become RAS mutant at progression. The LIBImAb study is a phase III, randomized, open-label, comparative, multi-centre trial to assess the superiority in terms of efficacy of bevacizumab versus cetuximab in combination with FOLFIRI chemotherapy in patients with mCRC, RAS/BRAF wild-type on tumor tissue and RAS mutant (RASmut) at liquid biopsy. This study is supported by the Italian Drug Agency (AIFA). We report preliminary data of liquid biopsy testing. Methods: Plasma samples from enrolled patients are analyzed for variants in exons 2, 3 and 4 of KRAS and NRAS genes and for codon 600 BRAF mutations using the fully automated Idylla system (Biocartis). Patients RASmut on liquid biopsy at baseline are randomized to receive FOLFIRI plus Cetuximab or FOLFIRI plus Bevacizumab. Patients RAS wild-type on cfDNA at baseline, are treated with FOLFIRI plus Cetuximab up to 8 cycles and, if not progressed, they are retested for RAS mutations on cfDNA. RASmut patients at re-screening are randomized to continue Cetuximab or to switch to Bevacizumab. Results: Plasma samples from 169 tissue RAS/BRAF wild-type mCRC patients at baseline and 75 patients at 8 cycles of treatment have been tested as of January 31, 2023. Analysis of baseline plasma samples detected 16 RAS mutations in 16/169 patients (9.5%). In particular, 12 KRAS variants (7.1%, 8 in exon 2 and 4 in exon 3) and 4 NRAS variants (2.3%, all in exon 2) were found. The Idylla test also detected 3 BRAFV600 mutations (1.8%) in 3 additional patients. The overall concordance between tissue and cfDNA testing for RAS/BRAF variants was 88.8%. RAS mutations were also detected in 6/75 (8%) plasma samples obtained at 8 cycles of FOLFIRI/cetuximab treatment. The cfDNA test revealed the presence of 5 KRAS (6.7%, 3 in exon 2 and 1 in exon 3) and 1 NRAS mutation (1.3%, exon 2). A BRAFV600 variant was detected in 1 additional patient (1.3%). The rate of RAS/BRAF mutant cases at 8 cycles of treatment was 9.3%. Conclusions: These preliminary data suggest that liquid biopsy might better recapitulate the heterogeneity of mCRC and might be useful in clinical practice to complement tissue testing. The clinical relevance of RAS variants detected in cfDNA will be determined in the LIBImAb trial.

Vaccination prior to SARS-CoV-2 infection reduces autoreactive antibody induction

Abstract SARS-CoV-2 infection leads to diverse outcomes ranging from asymptomatic infection to death. Several studies reported elevated autoreactive antibodies in SARS-CoV-2-infected individuals compared to healthy controls, suggesting SARS-CoV-2 infection promotes autoantibodies, which could increase the risk of autoimmunity. However, it is not known whether autoreactive antibodies observed after infection were present in individuals prior to infection. Further, the prevalence, duration, and vaccine impact on autoreactive antibodies are unresolved. Since elevated autoreactive antibodies precede the onset of autoimmune disease, it is important to investigate the impact of SARS-CoV-2 infection on self-reactive antibodies. We established a longitudinal study to collect baseline PBMC and plasma samples from participants at enrollment, beforeSARS-CoV-2 infection or vaccination. Participants were screened weekly by nasal swab PCR to detect asymptomatic infection. Additional samples were collected after confirmed SARS-CoV-2 infection and assessed for autoreactive antibodies predictive of various autoimmune disorders. Preliminary analysis showed that a subset of individuals exhibited an increase in autoreactive antibodies following infection, while other individuals unexpectedly displayed decreased levels. Notably, individuals infected after vaccination displayed fewer changes in autoreactive antibodies compared to infection prior to vaccination. Our data suggest that SARS-CoV-2 infection increases specific autoreactive antibody levels but does not induce a widespread increase in all autoreactive antibodies. Moreover, SARS-CoV-2 vaccination prior to infection reduced autoantibody induction. Supported by grants from NIH CEIRS HHSN272201400006C and Household Respiratory Virus SARS-CoV-2 Transmission and Immunity Sub-Study (HRTS) 3U01AI144616-02S1

Abstract CT216: Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial

Abstract Background: 1L pembro + platinum-based chemo has shown clinical activity in metastatic NSCLC regardless of tissue tumor mutational burden (tTMB) status. bTMB assessed using circulating tumor DNA in plasma may be a surrogate for tTMB. The single-arm, phase 2 KEYNOTE-782 study (NCT03664024) evaluated the correlation of bTMB with efficacy of 1L pembro + chemo in nonsquamous NSCLC. Methods: Eligible patients (pts) had histologically or cytologically confirmed stage IV nonsquamous NSCLC with measurable disease per RECIST v1.1 and were not eligible for EGFR-, BRAF-, ROS1-, or ALK-directed therapy. Pts were not previously treated for advanced or metastatic disease, had an ECOG PS of 0 or 1, and had an evaluable biopsy sample for biomarker analysis. All pts received intravenous pembro 200 mg Q3W + platinum chemo doublet Q3W (pemetrexed 500 mg/m2 + 4 cycles of carboplatin AUC 5 mg/mL/min or cisplatin 75 mg/m2). The primary objective was to evaluate the association of baseline bTMB with ORR per RECIST v1.1 by investigator assessment. Secondary objectives were to determine safety and the association of baseline bTMB with PFS per RECIST v1.1 by investigator assessment and OS. A study-specific next-generation sequencing-based assay using a 1.9 Mb/654-gene cancer panel, which includes specific lung cancer-associated gene targets, was used to measure bTMB (continuous scale) in cell-free DNA extracted from baseline plasma samples. Paired white blood cell DNA sequencing was also performed to eliminate potential clonal hematopoiesis-derived somatic mutations. Database cutoff: November 5, 2021. Results: 117 pts were enrolled; median age was 64.0 years (range, 37-85), and the majority were male (60.7%), had an ECOG PS of 1 (69.2%), and no brain metastases at baseline (92.3%). Median time from first dose to data cutoff was 19.3 months (range, 1.0-35.5). ORR was 40.2% (95% CI, 31.2-49.6; 6 CRs, 41 PRs), median PFS was 7.2 months (95% CI, 5.6-9.8), and median OS was 18.1 months (95% CI, 13.5-25.6). Treatment-related adverse events (TRAEs) occurred in 113 pts (96.6%) and grade 3-5 TRAEs occurred in 56 (47.9%). Eight pts (6.8%) died due to a TRAE (febrile neutropenia and pneumonitis [n = 2 each], and septic shock, pulmonary sepsis, general physical health deterioration, and neutropenia [n = 1 each]). bTMB data were available for 101 pts. The area under the receiver operating curve for bTMB as a continuous variable to discriminate response was 0.47 (95% CI, 0.36-0.59). The posterior probabilities for a positive association of bTMB with PFS and OS were 16.8% and 7.8%, respectively. Conclusions: Baseline bTMB showed no evidence of an association with ORR, PFS, or OS in pts with nonsquamous NSCLC treated with 1L pembro + chemo. These findings indicate no clinical utility of bTMB in this patient population and treatment setting. No new safety signals were observed. Citation Format: Jair Bar, Emilio Esteban, Delvys Rodríguez-Abreu, Santiago Ponce Aix, Zsuzsanna Szalai, Enriqueta Felip, Maya Gottfried, Mariano Provencio Pulla, Andrew Robinson, Andrea Fülöp, Suman B. Rao, D. Ross Camidge, Giovanna Speranza, Steven M. Townson, Julie Kobie, Mark Ayers, Elisha J. Dettman, Robert McDaniel, Byoungsok Jung, David Burkhardt, Ruth Mauntz, Tibor Csőszi. Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT216.

Abstract 6452: A plasma metabolomic signature of healthy lifestyles and risk of colorectal cancer in the UK Biobank

Abstract Background: Lifestyle factors contribute to the etiology of colorectal cancer (CRC). We aimed to derive a metabolomic signature of adherence to healthy lifestyles and examine its association with CRC risk. Methods: In the analysis, we included 98,898 UK Biobank participants with 168 metabolomic biomarkers measured by nuclear magnetic resonance spectroscopy from baseline plasma samples. A healthy lifestyle score (HLS) was derived from 8 lifestyle factors according to the American Cancer Society (ACS) guidelines. We applied elastic net regularized regressions to identify a metabolomic signature from 130 biomarkers. Cox proportional hazard models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association of metabolomic signature with CRC risk after adjusting for known confounders. Results: During a median follow-up time of 8.2 years, 818 incident primary CRC cases were identified after participants were enrolled for more than two years. We observed a strong partial correlation between the metabolomic signature and HLS (r = 0.34), as well as waist-to-hip ratio, sedentary time, red and processed meat intake, and tobacco smoking aspects of the HLS (r = 0.14-0.33). A higher HLS was characterized by a signature of increased levels of linoleic acid, total cholines, and polyunsaturated fatty acids and decreased levels of omega-6 fatty acids, phospholipids in intermediate-density lipoprotein, and phosphatidylcholines. The metabolomic signature showed a significantly inverse association with CRC risk (HR = 0.84; 95% CI = 0.80-0.89 per standard deviation increment). The significantly inverse associations were observed irrespective of sex, educational attainment, and CRC subsite. Stronger associations were seen among participants aged between 50 and 60 years at study enrollment, and those diagnosed with CRC at 60 years old or later. Conclusion: We identified a metabolomic signature characterizing adherence to the ACS guidelines and showed that the signature was inversely associated with CRC risk. Citation Format: Fangcheng Yuan, Danxia Yu, Wanqing Wen, Wei Zheng. A plasma metabolomic signature of healthy lifestyles and risk of colorectal cancer in the UK Biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6452.

Abstract 4329: Combined extracellular vesicle PD-L1 and radiomics as predictors of response in patients with advanced lung cancer undergoing immunotherapy

Abstract Background: Immune-checkpoint inhibitors (ICIs) have improved the clinical benefit of only a subset of patients with advanced non-small cell lung cancer (NSCLC), since tissue PD-L1 has showed low predictive accuracy. We have previously showed that extracellular vesicle (EV) PD-L1 identified durable responders to ICIs and that the radiomics analysis of baseline CT scans improved the EV PD-L1 predictive model. Here, we aim to validate this combination model to identify non-responders to ICIs. Methods: We enrolled 3 cohorts of patients, cohort (1) included 27 patients receiving ICIs, cohort (2) 17 on ICIs + chemotherapy (ChT), and cohort (3) with 13 undergoing ChT. Baseline and 3rd cycle plasma samples were collected, EV were isolated by ultracentrifugation, and EV PD-L1 dynamics was analyzed by immunoblot. 800 radiomics features were evaluated in baseline CT scans and a set of 6 features identified in cohort 1, were analyzed in cohort 2 and 3. Early and durable responses were assessed in CT scans after the 3rd and the 6-8 treatment cycle, respectively, using RECIST v1.1. Results: Increased expression of EV PD-L1 during treatment showed good predictive value at identify non-responders to ICIs and was not associated with ChT response. The addition of radiomics features improved the predictive model for early and durable response to ICIs, while similar AUC was observed in particular for durable response in cohort 2. Moreover, these biomarkers outperformed tissue PD-L1 and were unspecific for response to Docetaxel (Table 1). No predictive models could be evaluated for early response to ChT. Conclusions: We present for the first time, validated evidence of the combination of EV PD-L1 and radiomics as a highly accurate minimally invasive biomarker for predicting response to ICIs. This combinatorial approach outperformed the current standard-of-care tissue PD-L1 and is a promising tool for the stratification of patients to receive ICIs. Table 1. Predictive models for early and durable response: A: Predictive models for early RECIST response (3 treatment cycles) Cohort: 1: ICIs (n=27) 2: ICIs + Docetaxel (n=17) 3: Docetaxel (n=13) Biomarker: AUC Sensitivity Specificity AUC Sensitivity Specificity AUC Sensitivity Specificity ΔEV PD-L1 + Radiomics 73.3% 83.3% 73.3% 75.0% 100.0% 75.0% NA ΔEV PD-L1 61.7% 50.0% 73.3% 71.9% 100.0% 56.3% NA Radiomics 67.8% 83.3% 53.3% 90.6% 100.0% 87.5% NA Tissue PD-L1 ≥1% 57.5% 75.0% 40.0% 62.5% 100.0% 25.0% NA B: Predictive models for durable RECIST response (6-8 treatment cycles) Cohort: 1: ICIs (n=27) 2: ICIs + Docetaxel (n=17) 3: Docetaxel (n=13) Biomarker: AUC Sensitivity Specificity AUC Sensitivity Specificity AUC Sensitivity Specificity ΔEV PD-L1 + Radiomics 81.3% 75.0% 90.9% 76.7% 100.0% 66.7% 53.6% 42.9% 83.3% ΔEV PD-L1 72.7% 56.3% 81.8% 83.3% 50.0% 100.0% 54.8% 28.6% 83.3% Radiomics 71.0% 50.0% 90.9% 61.7% 50.0% 80.0% 57.1% 42.9% 83.3% Tissue PD-L1 ≥1% 60.2% 75.0% 54.5% 63.3% 100.0% 26.7% 52.4% 28.6% 66.6% Citation Format: Murat Ak, Diego De Miguel Perez, Priyadarshini Mamindla, Alessandro Russo, Vishal Peddagangireddy, Mehmet E. Er, Şerafettin Zenkin, Muthukumar Gunasekaran, Luis Lara-Mejia, Francesco Buemi, Feliciano Barron, Marisol Arroyo-Hernández, Sunjay Kaushal, Andres F. Cardona, Aung Naing, Vincenzo Adamo, Oscar Arrieta, Rivka R. Colen, Christian Rolfo. Combined extracellular vesicle PD-L1 and radiomics as predictors of response in patients with advanced lung cancer undergoing immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4329.