Supplementary Fig. 6. Longitudinal analysis of on-treatment fold changes (log2) in peripheral regulatory T-cell subsets in tobemstomig-treated patients, stratified by cancer type.

Table S5. snRNA-Seq sample overview.

Early-generation TRK tyrosine kinase inhibitors (TKIs) approved for treating NTRK fusion-positive (NTRK+) solid tumors provide clinical benefit; however, resistance emerges. Repotrectinib is a next-generation ROS1/TRK TKI with a compact macrocyclic structure designed to improve durability of response. TRIDENT-1 is a registrational phase 1/2 trial assessing repotrectinib, a next-generation ROS1/TRK TKI, in adults with advanced solid tumors, including NTRK+ disease. The primary endpoint was confirmed objective response; secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival and safety. Median follow-up ranged between 21.3 months and 25.7 months. In the TKI-naive cohort (n = 51; 95% confidence interval (CI)), the response rate was 59% (44-72); the median DOR was not estimable (NE); and the median PFS was 30.3 months (9.0-NE). In the TKI-pretreated cohort (n = 69; 95% CI), the response rate was 48% (36-60); the median DOR was 9.8 months (7.4-13.0); and the median PFS was 7.4 months (3.9-9.7). Of 30 TKI-pretreated patients with NTRK solvent front mutations, 16 had a response (53%; 95% CI: 34-72). Intracranial responses were observed in two of three TKI-naive patients and in four of six TKI-pretreated patients with measurable intracranial disease at baseline. Among all treated patients (n = 565), the most common any-grade treatment-related adverse event (TRAE) was dizziness (57%); most TRAEs were low grade; and 4% discontinued repotrectinib due to a TRAE. Here repotrectinib demonstrated durable systemic and intracranial responses with generally low-grade adverse events in patients with NTRK+ solid tumors, including those with previous TRK TKI treatment and solvent front mutations. These results support the use of repotrectinib to treat patients with NTRK+ solid tumors. ClinicalTrials.gov identifier: NCT03093116 .

Table S8. Response assessment per RECIST version 1.1.

Abstract Purpose: The immunoglobulin G1–based bispecific antibody tobemstomig (RO7247669) simultaneously targets and blocks programmed cell death protein 1 and lymphocyte activation gene-3 expressed on activated T cells. Patients and Methods: This first-in-human, open-label, phase I clinical trial of tobemstomig included a dose-escalation part in patients with advanced and/or metastatic solid tumors and an expansion part with three tumor-specific cohorts, enrolling checkpoint inhibitor (CPI)–experienced patients with melanoma and non–small cell lung cancer (NSCLC) and CPI-naïve patients with esophageal squamous cell carcinoma (ESCC). Primary and secondary objectives included safety/tolerability, maximum tolerated dose (MTD) and/or recommended dose for expansion (RDE), pharmacokinetics (PK), drug receptor occupancy, and preliminary antitumor activity. Results: Thirty-five (dose-escalation) and sixty-nine patients (expansion) were enrolled. Tobemstomig was well tolerated up to the highest tested dose of 2,100 mg once every 2 weeks. The MTD was not reached, and 2,100 mg once every 2 weeks was established as the RDE. Tobemstomig exhibited linear PK across the studied dose range. Partial responses were achieved by 2 of 4 (600 mg) and 4 of 13 (2,100 mg) patients during dose escalation, 6 of 41 patients with CPI-experienced melanoma [objective response rate (ORR): 15%; 95% confidence interval (CI), 6.6–26.9], and 1 of 8 patients with CPI-naïve ESCC (ORR: 12.5%; 90% CI, 0.6–47.1). Proof of mechanism was demonstrated in patients with CPI-experienced melanoma based on increases in the amounts of CD8+ T cells, expansion of stem-like CD8+ T cells, and the acquisition of cytotoxic effector functions, with limited changes in the regulatory T-cell compartment. Conclusions: Tobemstomig had a tolerable and manageable safety profile across various advanced solid tumor indications. The encouraging antitumor activity associated with pharmacodynamic activity and proof of mechanism in patients with CPI-experienced melanoma indicates the therapeutic potential of tobemstomig and supports further investigation in earlier disease treatment settings.

Background: The predictive value of PD-L1 to select patients for immunotherapy in resectable NSCLC remains imprecise, confounded by different assays used across trials and intra-tumoural heterogeneity (ITH). We sought to compare the concordance between 3 PD-L1 antibodies stratified by EGFR mutation status, evaluate ITH and implications on survival outcomes. Methods: Tissue microarrays were constructed from stage IA-IIIA NSCLC with 3 tumour cores per patient. Tumour proportion score (TPS) was evaluated by 3 pathologists for SP263, SP142, 22C3 and analysed in tertiles of < 1 %, 1-49 % and >= 50 %. ITH was defined as discordant TPS in >= 2/3 tumour cores. Cohen's kappa test was used to assess agreement. Survival outcomes were estimated using Kaplan-Meier. Results: A total of 561 patients were included, 59.5% (334/561) were EGFR-mutant. Stage IA comprised 45.5%(255/561), IB 24.1%(135/561), IIA 12.7%(71/561), IIB 4.5%(25/561) and IIIA 13.4%(75/561). Across 1683 tumour cores, SP263 and 22C3 had the highest concordance (Kappa = 0.689), followed by 22C3 and SP142 (Kappa = 0.354), then SP263 and SP142 (Kappa = 0.284), similar between EGFR-mutant and EGFR-wildtype. Agreement between pathologists was almost perfect. ITH by SP263 was observed in 14.1 % of EGFR-mutant versus 24.2 % in EGFR-wildtype(p = 0.002). Discordance was highest among TPS 1-49 % at 92.6 % (88/95) followed by >= 50 % at 37.8 % (14/37) and least among < 1 % at 0 % (0/429) (p < 0.001). For tumour cores scored 1-49 %, 63 %/70 % of adjacent cores were scored < 1 % for EGFR-wildtype/mutant respectively. Histological grade was the only independent predictor of PD-L1 ITH on multivariable analysis. PD-L1 ITH was not associated with survival on multivariable analysis. Conclusion: PD-L1 scoring by SP263 and 22C3 are interchangeable but not SP142 regardless of EGFR status. PD-L1 ITH was more common in EGFR-wildtype versus EGFR-mutant tumours. Extra care should be taken to select the most representative tumour core for tumours with high histological grade or TPS 1-49% as this may influence peri-operative treatment decisions.

AIM:Plasma Epstein-Barr virus (EBV) DNA is an archetypal biomarker for endemic nasopharyngeal carcinoma (NPC) employed for disease surveillance and early detection of recurrences. However, its accuracy is unknown. We utilised a single institution dataset with homogeneous surveillance procedures and routine EBV DNA testing using a harmonised assay to investigate its accuracy for recurrence detection. METHODS:We utilised a cohort of patients with histologically-confirmed, non-metastatic NPC treated from February, 2017 to July, 2023. All patients had ≥ 1 EBV DNA test using the harmonised BamHI-W polymerase chain reaction-based assay within 3 years post-radiotherapy (RT). We analysed the negative (NPV) and positive predictive values (PPV) for recurrence over a 1-year window (at 3-monthly intervals - windows 1-4) for each test performed. Recurrence was ascertained by radiological imaging and/or histopathological confirmation. RESULTS:Of 1746 EBV DNA readings from 393 patients diagnosed between January, 2017 to May, 2023, 1385 (79.3 %), 294 (16.8 %), and 67 (3.9 %) were recorded as negative, low (< 500 copies/mL), and high EBV DNA (≥ 500 copies/mL), respectively. NPVs of the assay were high (range: 97.6-99.3 %). PPVs were modest, highest at window 1 (range: 26.3-34.3 %) compared with the other 3 windows (range: 2.6-10.5 %). Sensitivity analyses revealed that PPVs were threshold-dependent; 71.4-100 % at window 1 for ≥ 500 copies/mL versus 16.9-23.1 % for < 500 copies/mL, which corresponded to median time of recurrence onset (0.8 vs 4.8 months, respectively). CONCLUSIONS:Negative EBV DNA test results have high NPVs, suggesting that patients may be routinely surveyed, while a positive result of ≥ 500 copies/mL indicates high recurrence risk.

Purpose To investigate the efficacy of metastasis-directed therapy (MDT) when added to camrelizumab (Cam) in patients with recurrent or metastatic nasopharyngeal carcinoma (R/M-NPC). Methods and Materials We conducted a randomized, controlled, multicenter, phase 2 trial in 3 centers from China (NCT04830267). Patients with R/M-NPC, without prior exposure to immunotherapy, who presented with ≥2 lesions, and at least 1 measurable lesion were randomized 1:1 to either Cam alone or Cam plus MDT (Cam+MDT). Patients randomized to the MDT group must have 1 lesion that is amendable to stereotactic body radiation therapy (SBRT) prescribed to 27 Gy in 3 fractions every other day. The primary endpoint was objective response rate (ORR) of unirradiated lesions using Response Evaluation Criteria in Solid Tumors v1.1. Results Between April 2021 and August 2023, 39 patients were randomly assigned to receive either Cam (n = 20) or Cam+MDT (n = 19). In total, 17 out of 39 (43.6%) patients had oligometastatic disease (≤3 lesions), 18 out of 39 (46.2%) had liver involvement, and 3 out of 39 (7.7%) had locoregional recurrent disease. ORR of unirradiated lesions did not differ between the treatment groups (26.3% [Cam+MDT] vs 30.0% [Cam], P = 1.0). The disease control rate of unirradiated lesions was 73.7% in the Cam+MDT group compared with 60.0% in the Cam group (P = .571). After a median follow-up of 25.8 months, median progression-free survival was 9.3 (95% CI, 6.2-not reached [NR]) months in the Cam+MDT group and 8.8 (95% CI, 3.3-NR) months in the Cam group (P = .750). Exploratory analyses suggested a longer overall survival (OS) with Cam+MDT for patients with >3 lesions (HR, 0.23; 95% CI, 0.07-0.77; P = .009). G3 and above adverse events were comparable between the treatment groups (15.8% [Cam+MDT] vs 20.0% [Cam]). The overall rate of capillary proliferation was 17.9% (7/39) for the trial. Conclusions Our study did not meet its primary endpoint of superior ORR of unirradiated lesions with the addition of MDT to Cam in patients with R/M-NPC.

The year 2024 is the 20 th anniversary of the discovery of activating epidermal growth factor receptor ( EGFR ) mutations in non -small cell lung cancer (NSCLC). Since then, tremendous advances have been made in the treatment of NSCLC based on this discovery. Some of these studies have led to seismic changes in the concept of oncology research and spurred treatment advances beyond NSCLC, leading to a current true era of precision oncology for all solid tumors. We now routinely molecularly profile all tumor types and even plasma samples of patients with NSCLC for multiple actionable driver mutations, independent of patient clinical characteristics nor is profiling limited to the advanced incurable stage. We are increasingly monitoring treatment responses and detecting resistance to targeted therapy by using plasma genotyping. Furthermore, we are now profiling early-stage NSCLC for appropriate adjuvant targeted treatment leading to an eventual potential "cure" in early-stage EGFR+ NSCLC which have societal implication on implementing lung cancer screening in never-smokers as most EGFR+ NSCLC patients are never-smokers. All these advances were unfathomable in 2004 when the five papers that described "discoveries" of activating EGFR mutations (del19, L858R, exon 20 insertions, and "uncommon" mutations) were published. To commemorate this 20 th anniversary, we assembled a global panel of thoracic medical oncology experts to select the top 20 papers (publications or congress presentation) from the 20 years since this seminal discovery with December 31, 2023 as the cutoff date for inclusion of papers to be voted on. Papers ranked 21 to 30 were considered "honorable mention" and also annotated. Our objective is that these 30 papers with their annotations about their impact and even all the ranked papers will serve as "syllabus" for the education of future thoracic oncology trainees. Finally, we mentioned potential practice-changing clinical trials to be reported. One of them, LAURA was published online on June 2, 2024 was not included in the list of papers to be voted on but will surely be highly ranked if this consensus survery is performed again on the 25 th anniversay of the discovery EGFR mutations (i.e. top 25 papers on the 25 years since the discovery of activating EGFR mutations).

INTRODUCTION:Nasopharyngeal carcinoma (NPC) remains an endemic disease in certain parts of the world, with many patients presenting with advanced disease on diagnosis. Chemotherapy had remained the standard of care with minimal progress made until recent years. This review aims to provide an overview of recent significant breakthroughs and up-and-coming novel strategies in treating this deadly disease. AREAS COVERED:This review focuses on the latest clinical development of promising investigational agents in the treatment of advanced NPC. These include anti-vascular agents, signaling pathways inhibitors and immunotherapy. EXPERT OPINION:The addition of immune-checkpoint inhibitors (CPI) to platinum-based chemotherapy has undoubtedly changed the therapeutic landscape of R/M NPC in the first-line setting. This leaves much room for further research on the optimal treatment strategy in subsequent-line settings, likely including the addition of CPI to anti-vascular agents or novel CPI combinations, with or without chemotherapy as a backbone. Other potential approaches include optimal CPI maintenance therapy after first-line CPI-chemotherapy combination. Potential novel agents on the horizons are antibody-drug conjugates, bi-specific antibodies and signaling inhibitors, with several phase II/III studies currently underway.

Supplemental Figure S3. Best percentage change from baseline in sum of diameters of target lesions for patients treated with spartalizumab (A) and chemotherapy (B)

<p>Supplemental Figure S1. CONSORT diagram</p>

<p>Supplemental Figure S2. Adverse events in crossover group</p>

Supplemental Table S3. Interval between last prior chemotherapy and first dose of study treatment

Nasopharyngeal carcinoma (NPC) is a high mortality, Epstein-Barr virus (EBV)-driven cancer with limited treatment options. Although EBV is highly antigenic and detectable in nearly all NPC tumor cells, it remains unclear why NPC tumors evade immune targeting despite high levels of immune infiltration. Here, we investigate the phenotypes of peripheral EBV-specific T cells with the purpose of identifying clinically relevant, cancer-associated EBV-specific T cell phenotypes. We developed a 34-marker mass cytometry and multiplexed MHC-I tetramer panel to detect and phenotype CD8 T cells of up to 55 antigen-specificities, including EBV and other known viral and cancer-antigen epitopes. We used this approach to profile peripheral blood mononuclear cells (PBMCs) from treatment-naïve NPC patients (n=51). Compared to other antigen-specific T cells detected in NPC patients and EBV-specific T cells in healthy individuals, EBV-specific T cells in NPC patients expressed high levels of activation markers (CD38, HLA-DR), co-inhibitory markers (CD39, TIGIT, PD-1), and migratory markers (ITB7, CD103). Using unsupervised clustering, we found that the frequency of EBV-specific T cells in a phenotypic cluster characterized by high CD103, CD39, HLA-DR, and CD38 expression positively correlated with plasma EBV-DNA titer and gross tumor volume. Remarkably, we observed that regardless of antigen specificity, NPC patients had a higher frequency of total CD8 T cells with this activated/exhausted cluster phenotype. CD8 frequencies of this cluster were also positively correlated with plasma EBV-DNA titer and stage (TNM). To investigate the gene expression profiles and TCR clonal diversity of this cancer-associated phenotype, we performed single-cell multi-omics analysis on peripheral CD8 T cells from NPC patients (n=17). We identified that cell subsets within this phenotype had distinct gene expression patterns indicative of potential associations with the tumor and we also assessed the clonality of these subpopulations. Overall, this study identified a unique cancer-associated peripheral EBV-specific T cell phenotype in the context of nasopharyngeal carcinoma that is positively correlated with advanced disease. Frequencies of peripheral T cells with this phenotype could also be useful as a blood-based prognostic marker for NPC. Citation Format: Nandita Kumar, Long Nguyen, Saumya Jani, Darren WT Lim, Joe PS Yeong, Melvin LK Chua, Amit Jain, Evan W Newell. Comprehensive EBV-specific T cell profiling and multi-omics analysis of nasopharyngeal carcinoma CD8 T cells reveals novel cancer-associated phenotype [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A052.

<p>Supplemental Table S4. Post-treatment antineoplastic medication</p>

Supplemental Figure S1. CONSORT diagram

Introduction: To compare the performance of droplet digital polymerase chain reaction (ddPCR) and plasma next -generation sequencing (NGS) in detecting clearance of plasma EGFR (pEGFR) mutations. Methods: Patients with treatment-naive advanced EGFR-mutated lung cancer treated with first-line tyrosine kinase inhibitors (TKIs) were included. pEGFR were measured at baseline and first response assessment using ddPCR and NGS. Clearance of pEGFR was defined as undetectable levels after a positive baseline result. Results were correlated with time-to-treatment failure (TTF). In exploratory analysis, corresponding change in carcinoembryonic antigen (CEA) levels was evaluated. Results: Between January 1, 2020, and December 31, 2021, 27 patients were recruited. Ex19del comprised 74% (20 of 27) and L858R 26% (seven of 27). Osimertinib was used in 59% (16 of 27), dacomitinib 4% (one of 27), and gefitinib/ erlotinib 37% (10 of 27). Sensitivity of ddPCR and NGS in detecting pEGFR mutation at baseline was 70% (19 of 27) and 78% (21 of 27), respectively (p = 0.16). All patients with detectable pEGFR by ddPCR were detected by NGS. At a median of 8 (range 3-24) weeks post-TKI initiation, clearance of pEGFR was achieved in 68% (13 of 19) and 71% (15 of 21) using ddPCR and NGS, respectively. Concordance between ddPCR and NGS was 79% (kappa = 0.513, p = 0.013). Clearance of pEGFR was associated with longer median TTF (not reached versus 6 months, p = 0.03) and median decrease in CEA levels by 70% from baseline. In another cohort of 124 patients, decrease in CEA levels by greater than 70% within 90 days of TKI initiation was associated with doubling of both TTF and overall survival. Conclusions: Plasma NGS trended toward higher sensitivity than ddPCR in detecting pEGFR, although both had similar concordance in detecting pEGFR clearance. Our results support using NGS at diagnosis and interchangeability of NGS and ddPCR for monitoring, whereas CEA could be explored as a surrogate for pEGFR clearance. (c) 2023 The Authors. Published by Elsevier Inc. on behalf of the International Association for the Study of Lung Cancer. This is an open access article under the CC BY-NC-ND li-cense (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

OBJECTIVES:To characterize longitudinal changes in Epstein-Barr virus (EBV) DNA post-radiotherapy in nasopharyngeal carcinoma (NPC) patients, and investigate whether an early (0-2 weeks) or delayed (8-12 weeks) EBV DNA result better predicts for disease-free survival (DFS). MATERIALS AND METHODS:Histologically-confirmed NPC patients with ≥1 EBV DNA test quantified using the harmonized BamHI-W polymerase chain reaction-based assay at 0-2 and 8-12 weeks post-radiotherapy were included. RESULTS:We identified 302 patients with EBV DNA measured at 0-2 weeks post-radiotherapy; of which, 110 (36.4 %) underwent a repeat test at 8-12 weeks post-treatment. Patients harboring a detectable EBV DNA at 0-2 weeks experienced an inferior DFS (adjusted HR1-264 copies 1.72 [95 %CI: 1.05-2.83], P = 0.031; AHR≥265 copies 4.39 [95 %CI: 1.68-11.44], P = 0.002 relative to 0 copies/mL). At 8-12 weeks, we observed substantial shifts in EBV DNA readings from 0 to 2 weeks; 76/110 (69.1 %) and 34/110 (30.9 %) patients at 0-2 weeks versus 90/110 (81.8 %) and 20/110 (18.2 %) at 8-12 weeks recorded undetectable and detectable EBV DNA, respectively. Positive EBV DNA at 8-12 weeks was strongly associated with relapse (73.3 % [11/15] for 1-264; 80.0 % [4/5] for ≥265 subgroups had relapses versus 15.6 % [14/90] for 0 copies/mL). Area under receiver operating curve values for 2-year relapse rates were 0.817 (95 %CI: 0.725-0.909) for stage + EBV DNA8-12w versus 0.654 (95 %CI: 0.542-0.765) for stage + EBV DNA0-2w. CONCLUSION: EBV DNA is dynamic post-radiotherapy, and delayed EBV DNA testing better enriched for higher-risk NPC patients. This implicates trials investigating adjuvant chemotherapy intensification based on early EBV DNA testing.