Supplemental Figure 7: A) Emm55 specific peptides responsible for IgG responses pre and post treatment on individual patients.

Emm55 specific peptides responsible for IgG responses pre and post treatment on individual patients.

Abstract Background: Delta-like Ligand 3 (DLL3), a Notch receptor ligand, is highly expressed in small cell lung cancer (SCLC) and has emerged as a promising therapeutic target. Circulating tumor cells (CTCs), abundant in SCLC, interact with diverse immune cell populations in peripheral blood. This study investigates the potential of DLL3 as a biomarker to enhance CTC detection and monitor immunotherapy response in SCLC. Methods: We enrolled 17 patients with extensive-stage (ES) SCLC and 5 with stage IV non-small cell lung cancer (NSCLC; as controls), all scheduled for an immune checkpoint inhibitor or a bispecific T-cell engager. Whole blood (6 mL) was collected before and during treatment. CTCs were enriched using CTCeptorTM, a fully automated, negative depletion-based continuous centrifugal microfluidic system, and classified into three phenotypes: pan-cytokeratin (CK)+/DLL3-/CD45-, CK-/DLL3+/CD45-, or CK+/DLL3+/CD45-. Baseline tumor samples were analyzed by immunohistochemistry for SCLC molecular subtypes and DLL3. Results: DLL3+/CD45- CTCs were detected in all ES-SCLC patients (100%) prior to treatment with atezolizumab plus chemotherapy or tarlatamab, with a median count of 23 (range 7-121) and a median size of 9.71 μm (7.33-12.18). In contrast, no DLL3+/CD45- CTCs were detected in NSCLC patients, where only CK+/DLL3-/CD45- CTCs were observed (1-4 cells). Among ES-SCLC patients, CK-/DLL3+/CD45- cells were the predominant subtype (63.3%, 522/825), while CK+/DLL3+/CD45- CTCs accounted for 27.7% (228/825) of all detected CTCs, representing additional CTCs not captured by CK-based detection alone. ASCL1 was the most prevalent molecular subtype (12/17), with DLL3 exhibiting the highest H-score (median, 185; range, 20-300). Notably, no correlation was observed between the number or proportion of DLL3+CTC and the DLL3 H-score (r = -0.163, p = 0.578), while DLL3+CTCs remained prevalent in NERUDO1 and POU2F3 subtypes. Furthermore, both total and DLL3+CTC counts markedly decreased within 2 cycles of atezolizumab chemotherapy or 1 week after tarlatamab treatment. Conclusion: DLL3+CTCs can be efficiently and specifically detected in ES-SCLC patients using CTCeptorTM, serving as a complementary marker to CK for CTC identification. DLL3 appears more frequently in CTCs than in tumor tissue. These findings support DLL3 as a potential biomarker for CTC-based monitoring in ES-SCLC and warrant further validation in larger cohorts, including analyses of immunologic features. Citation Format: Hyung-Joo Oh, Hyun-Ju Cho, Seung-Hee Song, Yoo-Duk Choi, Woo-Jae Son, Jin-Han Bae, In-Jae Oh, Young-Chul Kim, Hae Ung Lee, Cheol-Kyu Park. Delta-like ligand 3 (DLL3)-based enrichment enables sensitive and specific detection of circulating tumor cells during immunotherapy in small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1071.

Box plot of genes depicted in Figure 1.

Supplemental Figure 2: Cytokine/chemokines increased or decreased after therapy in individual patients with the IO panel. The

ABSTRACT MET exon 14 skipping is an actionable oncogenic driver in non–small cell lung carcinoma (NSCLC); however, noncanonical splice‐region variants are frequently classified as variants of unknown significance (VUS), which may result in missed therapeutic opportunities and highlight limitations in current DNA next‐generation sequencing (NGS) reporting criteria. We report a patient with pulmonary sarcomatoid carcinoma harboring a noncanonical MET splice donor–proximal indel (c.3022_3028 + 13delinsA), initially interpreted as a VUS, who achieved a rapid and durable response to capmatinib. Subsequent RNA sequencing and droplet digital PCR (ddPCR) confirmed MET exon 14 skipping, supporting the value of orthogonal transcript‐level validation for exon‐adjacent variants.

Supplemental Figure 6: Emm55 specific peptides responsible for IgM responses pre and posttreatment on individual patients.

Supplemental Figure 4: Emm55 specific peptides responsible for IgM responses pre and post treatment on individual patients.

Background:Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors improve outcomes in EGFR T790M-positive non-small cell lung cancer (NSCLC), but the prognostic value of plasma-detected T790M remains uncertain. We evaluated the clinical significance of baseline plasma T790M in patients treated with lazertinib, accounting for metastatic distribution and coexisting genomic alterations. Methods:In this prospective multicenter cohort, we analyzed 117 patients with EGFR-mutant NSCLC who received lazertinib after T790M confirmation in tissue or plasma. Plasma EGFR mutations were profiled using next-generation sequencing before treatment. Progression-free survival (PFS) and overall survival (OS) were compared by plasma T790M status, metastatic sites, and co-alterations. Results:Of 117 patients, 92 were plasma T790M-positive and 25 were plasma T790M-negative. Plasma T790M positivity was associated with shorter PFS (10.0 vs. 23.0 months, P=0.03) and OS (20.0 months vs. not reached, P=0.006). All patients with liver or adrenal metastases were plasma T790M-positive, and involvement of either site predicted poorer outcomes than in patients without these metastases. Bone metastasis also portended a worse prognosis, irrespective of plasma T790M status. Among co-alterations, EGFR C797S or MYC alterations correlated with shorter PFS. Conclusions:Baseline plasma T790M, interpreted alongside metastatic distribution, provided prognostic information in EGFR-mutant NSCLC treated with lazertinib. Liver and adrenal metastases occurred exclusively in plasma T790M-positive patients and were associated with markedly worse outcomes, consistent with a ctDNA-shedding phenotype. Bone metastasis was an adverse prognostic factor independent of plasma T790M, underscoring the combined prognostic impact of molecular and metastatic features.

Objectives: This study reports the effects of comprehensive Korean medicine treatment as palliative care in a patient with terminal hepatocellular carcinoma (HCC) and discusses the practical challenges of implementing do-not-resuscitate (DNR) orders in a Korean medicine hospital.Methods: A 61-year-old man with stage IV HCC who discontinued immunotherapy received comprehensive Korean medicine treatment, comprising Saenggangeonbi-tang-gami, Gunchil-dan, and acupuncture, for palliative symptom management from July 14 to August 22, 2025.Results: Korean medicine treatment was associated with symptom control during the terminal phase. Acute hepatotoxicity was not observed, suggesting an acceptable safety profile. However, the independent contribution of Korean medicine treatment could not be determined because of the concurrent use of conventional medications and the difficulty in distinguishing treatment effects from the natural course of terminal malignancy.Conclusion: Comprehensive Korean medicine treatment may be considered a component of palliative care in patients with terminal HCC. However, further institutional development and systematic end-of-life care guidelines are required to ensure appropriate DNR implementation in Korean medicine hospitals.

Seizure-related homolog 6 (SEZ6) is expressed in small cell lung cancer (SCLC) and neuroendocrine neoplasms. In an open label, phase 1 trial, ABBV-706, an antibody-drug conjugate with a SEZ6-directed antibody linked to topoisomerase-1 inhibitor (Top1i), was administered intravenously every 3 weeks (Q3W) to 288 patients with advanced solid tumors; 240 received monotherapy, including 124 with relapsed/refractory (R/R) SCLC. Primary objectives of dose escalation (part 1, advanced solid tumors), dose optimization and expansion (part 2, R/R SCLC only) and dose expansion (part 4, central nervous system tumors and high-grade neuroendocrine neoplasms only) were to evaluate the safety, tolerability, pharmacokinetics (PK), immunogenicity and antitumor activity of ABBV-706 monotherapy and, from parts 1 and 2, to determine the recommended phase 2 dose (RP2D) of ABBV-706 in R/R SCLC. In the monotherapy cohort (N = 240), the most common treatment-related adverse events (TRAEs) at any grade were anemia (61%) and fatigue (38%). Grade 3 or higher TRAEs occurred in 61% of patients and were dose dependent (39% at 1.8 mg kg-1 and 70% at 2.5 mg kg-1). In the R/R SCLC monotherapy cohort (n = 124), any-grade and grade 3 or higher TRAEs occurred in 93% and 61% of patients, respectively. ABBV-706 demonstrated promising preliminary efficacy in patients with R/R SCLC, with an objective response rate (ORR) of 52% (65/124). In patients with R/R SCLC receiving monotherapy in dose optimization and expansion part 2, ORR was similar between 1.8 mg kg-1 and 2.5 mg kg-1 doses (56% (23/41) and 59% (23/39), respectively), with a duration of response that was highest at the 1.8 mg kg-1 dose and with most patients achieving rapid and durable tumor reduction. Although exploratory, long-term efficacy measures were an important consideration in the RP2D determination, and in R/R SCLC monotherapy, overall survival (OS) was highest at the 1.8 mg kg-1 dose, with a median OS of 12.4 months. Based on the totality of available data, including, but not limited to, safety, preliminary efficacy measures and PK, 1.8 mg kg-1 Q3W was confirmed as the optimal RP2D for patients with R/R SCLC. ClinicalTrials.gov: NCT05599984 .

Supplemental Figure 8: Nucleic Acid Sequence of pAc/emm55 Construct with Amino Acid Translation

Supplemental Table 1: Demographics; related/unrelated adverse events.

Number of lesions injected, Prior lines of therapy, time on post-protocol anti-PD1 therapy.

12 Background: Previous studies utilizing multiplex Immunofluorescence (mIF) have described cell density patterns of immune exhaustion implicating both innate and adaptive immune system components across varying stages of penile squamous cell carcinoma (PSCC). Here we studied geospatial clustering patterns of various immune effector cells within the tumor immune microenvironment (TIME). Methods: Tissue microarray (TMA) was constructed for 57 cases of invasive PSCC and immunostained for CD20, CD3, CD4, CD8, CD45RO, CD68, CD206, CD163, NKp46, FOXP3 using OPAL TM 7 kit (AKOYA Biosciences). Areas of tumor and stroma were identified using an image analysis system (InForm 2.2.4). Spatial analysis and co-clustering of cell phenotypes was performed using Ripley’s K. Survival curves were determined using Kaplan Meier method and tested using logrank and cox regression. Univariate and bivariate analysis describes co-clustering and proximity to tumor cells, respectively. Results: 57 PSCC patients (median age 60, [IQR (interquartile range) 31-92]); 60% HPV negative, and 31/57 with pN+ had mIF analysis. A high clustering pattern of CD68+, a general marker for macrophages, was associated with a significant OS benefit on univariate analysis (162 vs 36 mos, p=0.006) and an OS, RFS, and CSS benefit on bivariate analysis (84 vs 27 mos; NA vs 14 mos; NA vs 27 mos p=0.007, 0.014, 0.001). High clustering of CD3+CD4+, a marker for T-helper-lymphocytes, within the tumor and the tumor-stroma interface was associated with an OS benefit on bivariate analysis (84 v 20 mos, p=0.009). Low intra-tumoral clustering of CD68+CD163+, marker for M2 protumor macrophages, was associated with improved RFS on bivariate analysis (NA v 15 mos, p=<0.01). Low clustering of CD68+CD163+ in HPV+ patients was associated with a CSS benefit compared to high clustering in HPV- patients (p=0.024). High versus low clustering of CD68+CD163+ was inconsequential in HPV- patients (p=0.66).Overall, low intratumoral clustering pattern of FOXP3+, a marker for regulatory immune-suppression, was associated with an improved CSS on bivariate analysis (p=0.017). Low clustering of FOXP3+ in HPV+ patients was associated with an improved CSS versus low clustering of FOXP3+ in HPV- patients (p=0.041). Conclusions: Spatial analysis shows that proximity of CD68+ and CD3+CD4+, a general marker for macrophages and T-helper cells, to tumor cells has a positive impact survival in PSCC. Interestingly, proximity of protumor M2 macrophages to cancer cells confers a survival benefit specifically in HPV+ tumors and has no impact on survival in HPV- tumors. Decreased intratumoral FOXP3+ activity, a marker for regulatory immune-suppressive T cells, shows a preferential survival benefit in in HPV+ tumors. These findings point to diverging tumorigenic pathways related to HPV status that may be attributable to their poorer prognosis.

BACKGROUND:Second-generation anaplastic lymphoma kinase (ALK) inhibitors, including alectinib and brigatinib, are widely used in patients with ALK-positive non-small cell lung cancer (NSCLC) who develop resistance or progress on crizotinib. However, real-world data comparing their efficacy and safety remain limited. This multicenter, prospective cohort study compared the clinical outcomes of alectinib and brigatinib in this setting. METHODS:Patients with stage IV ALK-positive NSCLC who progressed on crizotinib were enrolled and treated with either brigatinib or alectinib. The primary endpoint was the progression-free survival (PFS) rate. RESULTS:Sixty patients were included (brigatinib, n = 34; alectinib, n = 26). Median follow-up durations were 26.5 and 30.0 months. Disease progression or death occurred in 50.0% (brigatinib) and 46.2% (alectinib), respectively. The 3-year PFS was 51.5% (brigatinib) vs. 62.1% (alectinib), with no significant difference at 5 years (40.0% vs. 42.5%; p = 0.260). Overall response rates were similar (58.8% vs. 46.2%; p = 0.475). However, intracranial outcomes appeared more favorable with alectinib: the 3-year intracranial PFS was 70.5% vs. 31.7% (p = 0.023), and intracranial ORR was 94.4% vs. 64.3% (p = 0.028). More patients in the brigatinib group had prior whole-brain radiotherapy (21.4% vs. 5.6%), while radiosurgery was more frequent in the alectinib group (55.6% vs. 35.7%). Treatment discontinuation rates due to adverse events were comparable between the two groups. CONCLUSIONS:In crizotinib-refractory ALK-positive NSCLC, systemic efficacy was not significantly different between brigatinib and alectinib; however, alectinib was associated with more favorable intracranial PFS and ORR, which may be partly explained by differences in prior brain-directed local treatments.

Lignans have garnered significant interest in various fields of pharmaceuticals, nutrition, and pesticides due to their diverse biological activities. This chapter primarily focuses on the extraction and purification methods of lignans from Schisandra, followed by an extensive examination of qualitative and quantitative analytical techniques, including thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Additionally, methods for structural analysis using nuclear magnetic resonance (NMR) are briefly summarized. The chapter also details approaches for determining the relative and absolute composition of lignan compounds, with illustrative examples provided.

Lung cancer remains the leading cause of cancer-related mortality worldwide. The primary reason for this poor prognosis is late-stage diagnosis, as approximately 50% of lung cancer cases are detected at stage 4 when curative treatment is no longer an option, and the 5-year survival of this stage is approximately 15%. While low-dose computed tomography (LDCT) has significantly improved early detection, its high false-positive rate (60-70%) leads to unnecessary invasive procedures and patient anxiety. Therefore, a complementary, highly specific, and non-invasive biomarker is urgently needed. Liquid biopsy, particularly circulating tumor DNA (ctDNA) analysis, has emerged as a promising diagnostic tool for lung cancer screening. Unlike conventional tissue biopsies, ctDNA-based tests provide a minimally invasive means to detect cancer-specific genomic alterations, including somatic mutations, DNA methylation patterns, somatic copy number alterations (CNAs), and fragment size variations. While ctDNA analysis has demonstrated clinical utility in advanced lung cancer, its application in early-stage disease remains challenging due to the low abundance of ctDNA in circulation. Strategies such as whole-genome methylation profiling, CNA analysis, and fragmentomics have been explored to enhance sensitivity and specificity for early detection. This review summarizes recent advances and challenges in ctDNA-based lung cancer screening, focusing on the strengths and limitations of different approaches. We highlight the need for integrating ctDNA assays with current LDCT screening protocols to improve diagnostic accuracy. Future large-scale prospective studies are essential to validate the clinical utility of ctDNA-based liquid biopsy in early lung cancer detection. A multi-modal biomarker strategy that combines genetic, epigenetic, and structural features holds great promise for transforming lung cancer screening and improving clinical outcomes.

577 Background: Approximately 30% of patients with ccRCC present with metastatic disease (stage IV) and close to half of patients with stage III disease will recur during follow-up surveillance. Two of the most common and morbid sites of metastatic development are brain and bone metastasis. Existing treatment guidelines do not recommend routine brain or bone directed imaging during either initial staging or surveillance in the absence of clinical signs or symptoms. A group of recurrently altered aberrant splice variants in primary ccRCC tumors associated with metastatic progression have previously been identified. Our study aims to investigate metastatic organotropism of the FGD1 -splice variant ( FGD1 -SV) to refine risk stratification and therapeutic decision-making as cabozantinib is drug with activity at these disease sites. Methods: This study leveraged the ORIEN AVATAR network, a data-sharing alliance of 18 NCI-designated cancer centers, to evaluate the presence of FGD1 -SV in a cohort of 1,001 clear cell renal cell carcinoma (ccRCC) patients using bulk RNA-sequencing (RNA-seq). Samples with three or more FGD1 -SV reads were classified as positive. Clinical and survival data were collected, and Kaplan-Meier curves were generated. Odds ratios (ORs) evaluated the presence of FGD1 -SV on brain and bone metastases, while hazard ratios (HRs) assessed association with cabozantinib response. Of 1,037 RNA-seq samples, 84 were metastatic tumor specimens. The relationship between FGD1 -SV positivity and metastasis to common sites was assessed with Fisher's exact test. Results: Brain and bone metastases demonstrated the highest FGD1 -SV positivity (50% and 44%, respectively). A grouped comparison of brain and bone metastases versus all other common metastatic sites (adrenal, lymph node, pancreas, lung, and liver) revealed significant enrichment of FGD1 -SV in these metastases (OR 5.33, 95% CI [1.64 - 18.42], p=0.002). FGD1 -SV positivity in primary tumors was associated with greater risk of recurrence after surgical resection (p=0.0029). Furthermore, FGD1 -SV positive tumors were associated with poor survival when not treated with cabozantinib (HR 2.04, 95% CI [1.20 - 3.45], p=0.01). Conclusions: FGD1 -SV positivity is significantly associated with brain and bone metastatic organotropism in ccRCC. Our findings warrant prospective studies to validate these results and investigate the integration of FGD1 -SV into clinical decision-making, potentially guiding surveillance and therapeutic approaches in high-risk and metastatic patients.