Supplementary Table S7. Tissue informed clinical management in EGFR-mutant NSCLC.

Impact of pCGP and ctDNA dynamics on patient management. A, Swimmer’s plot illustrating the survival of 12 patients with EGFRm NSCLC; these patients were chosen to provide a representative example of situations in which pCGP beyond the first diagnosis was informative for patient management. Examples of how pCGP informed management include the identification of T790M alterations (patients PCGP1, PCGP4, PCGP5, PCGP7, PCGP12); identification of a BRAF alteration (PCGP6); identification of MET amplification (PCGP8 and PCGP9); and identification of a C797S resistance alteration (PCGP2). B and C, The emergence of resistance on pCGP; fish plots illustrate a dynamic, longitudinal representation of molecular alterations identified on pCGP in patients with serial sampling. The Y axis represents the aggregate VAF of all alterations identified at each time point. Each vertical dashed line represents the time point of a pCGP assay, with the number beneath representing the VAF of the most prevalent alteration at that time. B, pCGP serial profiling for a patient with EGFR L858R–driven NSCLC who had five iterations of pCGP. They were on erlotinib up to the second pCGP, which was obtained at the time of radiographic progression. This identified EGFR T790M, and the patient was switched to osimertinib with subsequent suppression of this clone. The third pCGP was at the time of further radiographic progression, no new driver of resistance was identified but EGFR C797S was identified on liver biopsy. They were transitioned from osimertinib to a clinical trial at this point, with corresponding re-expansion of T790M and L858R variants after. Fourth pCGP, after carboplatin/pemetrexed/bevacizumab therapy, identified both C797S and L718Q, an uncommon mutation in EGFR and an additional resistance mechanism. Their final pCGP demonstrated a significant increase in the aggregate VAF of all alterations, and the patient died 2 months later. Separate regimens are separated by semicolons. C, pCGP serial profiling for a patient with ALK-fusion NSCLC that had four iterations of pCGP. Their disease had become resistant to alectinib at the time of the first pCGP, likely attributable to ALK E1210K and I1171T alterations. I1171T is associated with sensitivity to ceritinib, and the patient was switched to ceritinib at this time. After some clinical response with a corresponding contraction of the I1171T variant, expansion of the E1210K variant was seen in addition to an acquired oncogenic PIK3CA resistance variant. The third pCGP is at the time of progression on ceritinib. The patient transitioned to brigatinib initially but progressed after 6 months and transitioned to lorlatinib. Fourth pCGP after 22 months on lorlatinib identified an additional acquired on-target mutation in ALK G1269A. Compound mutations of this kind are common in patients on lorlatinib. Separate regimens are separated by semicolons.

Figure S4. Categorization of EGFR driver variants identified in the EGFRm NSCLC subset.

Figure S3. Actionable alterations identified on first pCGP at first diagnosis (treatment-naive setting).

Supplementary Table S10. Drivers of EGFR TKI resistance.

Supplementary Table S1. Context of first pCGP assays.

Characteristics of study population.

Supplementary Table S5. Summary of tissue informed clinical management in patients with concurrent pCGP and tissue biopsy.

Abstract Plasma comprehensive genomic profiling (pCGP) is implemented in the clinical care of non–small cell lung cancer (NSCLC). Although less well documented, serial pCGP may also guide the management of oncogene-driven NSCLC after first progression. In this study, we assessed the clinical value of serial pCGP, focusing on EGFR-mutant NSCLC. We conducted a retrospective study of 718 patients with NSCLC who underwent pCGP between 2015 and 2022. Clinical genomic data were programmatically extracted from the data workflows of the Johns Hopkins Lung Cancer Precision Medicine Center of Excellence. After variant annotation and actionability characterization, we examined the prevalence and evolving comutation patterns across serial pCGP, focusing on genomic mechanisms of tyrosine kinase inhibitor (TKI)–acquired resistance in EGFR-mutant NSCLC. A total of 718 patients had 818 instances of pCGP, with 79 patients having longitudinal pCGP (range, 2–5). pCGP uniquely informed management in 13% of patients (n = 92), both at initial diagnosis and on serial genotyping. This occurred predominantly through the identification of actionable mutations when tissue testing was unavailable. Among 214 patients with EGFR-mutant NSCLC, pCGP identified PI3K pathway alterations in 11% after first-line therapy. BRAF V600E (3%) and MET exon 14 skipping mutations (3%) emerged after third-generation TKI therapy. After TKI progression, 31 patients (22%) with EGFR-mutant disease had actionable pCGP findings, of whom 18 (58%) were matched to targeted therapy. Serial pCGP can inform treatment decisions in patients with NSCLC. In those with EGFR-mutant disease, pCGP at progression identifies actionable drivers of therapy resistance, enabling therapeutic intervention. Significance: Our study provides critical insights into the routine implementation of serial pCGP within a thoracic oncology program, supported by a precision oncology informatics framework, in a tertiary healthcare institution. We show that pCGP enables genotyping when tissue testing is not feasible and identifies actionable mutations at resistance. The clinical implementation of pCGP can drive improved clinical outcomes by matching patients with effective interventions in a timely and minimally invasive manner.

INTRODUCTION:The phase Ib two-part (dose escalation and expansion) six-cohort TROPION-Lung02 study evaluated datopotamab deruxtecan (Dato-DXd) plus pembrolizumab with or without platinum-based chemotherapy (Pt-CT) in patients with advanced or metastatic NSCLC without actionable genomic alterations. METHODS:Patients received Dato-DXd (4 or 6 mg/kg) plus pembrolizumab 200 mg alone (doublet) or with Pt-CT (triplet; carboplatin AUC 5 or cisplatin 75 mg/m2) once every 3 weeks. The primary objective was safety and tolerability; efficacy was a secondary objective. Exploratory biomarker analyses assessing TROP2 normalized membrane ratio by quantitative continuous scoring were performed in the treatment-naive patient subset. RESULTS:In total, 142 patients received doublet (n = 70) or triplet (n = 72) therapy; 96 were treatment-naive (doublet: n = 42; triplet: n = 54). Grade 3 or higher treatment-related adverse events occurred in 37.1% (doublet) and 59.7% (triplet) of patients. No treatment-related deaths occurred. In treatment-naive patients receiving doublet therapy, the confirmed objective response rate was 54.8%, the median duration of response was 20.1 months, and the median progression-free survival was 11.2 months. With triplet therapy, the confirmed objective response rate was 55.6%, the median duration of response was 13.7 months, and the median progression-free survival was 6.8 months. Tumor responses were observed across programmed death-ligand 1 expression levels for both regimens. Exploratory TROP2 normalized membrane ratio biomarker analyses revealed trends toward improved survival outcomes in patients who were biomarker-positive compared with biomarker-negative. CONCLUSION:Dato-DXd plus pembrolizumab therapy (with and without Pt-CT) exhibited appreciable safety and durable antitumor activity across programmed death-ligand 1 expression levels in patients with advanced or metastatic NSCLC. CLINICAL TRIAL INFORMATION:ClinicalTrials.gov Identifier: NCT04526691.

Figure S6. Plasma-only variants among patients with simultaneous plasma and tissue NGS.

Supplementary Table S8. On-target acquired resistance in EGFR-mutant NSCLC.

Figure S5. Mechanisms of resistance to EGFR TKI according to first line of therapy received.

Supplementary Table S9. Putative uncommon on-target drivers of EGFR TKI resistance.

Figure S2. Actionable alterations identified on all instances of first pCGP.

Circulating tumor DNA (ctDNA) analyses are informative as an early indicator of immunotherapy response in advanced non-small cell lung cancer (NSCLC); however, the clinical value of ctDNA molecular response requires further validation. As part of a prospective clinical protocol ( NCT05995821 ), we conducted targeted error-correction sequencing of ctDNA (n=328) and matched WBC DNA (n=109) from 109 patients with metastatic NSCLC who received anti-PD-(L)1 either as monotherapy or in combination. Following cellular origin resolution of 2,818 variants, landmark molecular response (mR) was defined as undetectable ctDNA within 3-9 weeks of treatment initiation. Pre-treatment ctDNA burden, but not blood tumor mutation burden, predicted survival. Implementing a tumor-naïve WBC DNA-informed approach increased the number of evaluable cases without compromising the overall accuracy of landmark ctDNA molecular responses. A direct comparison of single-timepoint on-therapy ctDNA assessment with ctDNA dynamics from baseline to the 3-9-week interval, along with an analysis of heterogeneity in molecular response within the 3-9-week window, showed that undetectable ctDNA at the landmark timepoint can effectively predict survival outcomes. A significant enrichment in landmark ctDNA mR was noted among patients with progression-free survival (PFS) ≥6 months with immunotherapy (p=2.5e-05) and chemo-immunotherapy (p=0.02). Patients in the landmark mR group had longer progression-free (p=1.6e-06) and overall survival (p=2.5e-05) than those with molecular progression. Landmark ctDNA molecular response provides a real-time, accurate approach for monitoring immunotherapy clinical outcomes. Although not currently validated for regulatory use, these findings demonstrate the potential utility of ctDNA as an early endpoint in clinical trials. Employing circulating tumor DNA (ctDNA) dynamics as an early indicator of immunotherapy response requires a roadmap for the next-generation sequencing approach, definition of molecular response and establishment of its clinical sensitivity. In this study, we introduce the concept of a landmark ctDNA molecular response, determined 3-9 weeks after initiation of immunotherapy, that maximizes the number of evaluable patients without sacrificing the specificity of the approach. Notably, when evaluating heterogeneity in ctDNA detection within the landmark 3-9-week window and assessing the impact of landmark interval dynamics on survival, we found that a single ctDNA assessment performed similarly to multiple ctDNA measurements within the landmark window (most notably, regardless of whether the timepoints were concordant or discordant). Our findings demonstrate that a single assessment of early on-therapy landmark ctDNA molecular response, can identify patients at risk of disease progression and enable future intervention and therapy optimization.

Abstract Background: Circulating tumor DNA (ctDNA) analyses are informative as an early indicator of immunotherapy response in advanced non-small cell lung cancer (NSCLC), however the clinical role of ctDNA molecular response requires further validation. Methods: As part of a prospective clinical protocol (NCT05995821), we performed ctDNA (n=328) and matched white blood cell DNA (WBC; n=109) targeted 521 fixed gene panel next-generation sequencing (Elio Plasma Complete) from 109 patients with advanced/metastatic NSCLC who received anti-PD-(L)1 as monotherapy or in combination with chemotherapy. Following variant cellular origin resolution, landmark molecular response (mR) was defined as undetectable ctDNA within 3-9 weeks of treatment initiation. For a subset of patients (n=34), whole-exome sequencing of baseline tumors was performed and used in benchmarking ctDNA detection and therapy response assessment. Results: Among 2,818 plasma variants, 23% were clonal haematopoiesis-related, which confounded the interpretation of driver gene associations with clinical outcomes and assessment of molecular responses. Implementing a tumor-naïve WBC DNA-informed approach increased the number of evaluable cases while maintaining the overall accuracy of landmark ctDNA molecular responses. Pre-treatment ctDNA burden but not blood tumor mutation burden, predicted survival. Overall, 77 patients were evaluable for landmark molecular response assessment; of these, 29 patients (38%) attained a mR. Landmark evaluation of molecular response enabled evaluation of all cases, was highly specific (92%), and achieved a higher sensitivity (66%) compared to clearance or ctDNA reduction from baseline (sensitivity 59%). In predicting landmark progression-free survival (PFS) at 6 months (durable clinical benefit, DCB), the tumor-agnostic WBC-informed approach strikes a balance between sensitivity (71.4%) and specificity (100%) compared to plasma-only (sensitivity=14.3%, specificity=100%) or tumor-informed (sensitivity=78.6%, specificity=71.4%) approaches. A significant enrichment in landmark ctDNA mR was noted among patients with DCB with immunotherapy (p=2.5e-05) and chemo-immunotherapy (p=0.02). Patients in the landmark mR group attained longer PFS (p=1.6e-06) and overall survival (p=2.5e-05) compared to those with molecular progression. The association between landmark molecular response and survival remained significant (PFS and OS, P < 0.001) after accounting for clinical covariates, line of therapy, and baseline ctDNA levels. Conclusions: Our findings indicate that landmark ctDNA molecular response at 3-9 weeks on treatment provides a real-time and accurate approach for monitoring immunotherapy clinical outcomes. Although not currently validated for regulatory use, these findings demonstrate the potential utility of ctDNA as an early endpoint in clinical trials. Citation Format: Jaime Wehr, Noushin Niknafs, Lavanya Sivapalan, Archana Balan, Gavin Pereira, Samira Hosseini-Nami, Iiasha Beadles, Aliyah Pabani, Kristen Marrone, Qing K. Li, Joseph Christopher Murray, Mark Sausen, Bryan Chesnick, Lorenzo Rinaldi, Christine L. Hann, Susan Combs Scott, Josephine Feliciano, Vincent K. Lam, Benjamin Levy, Patrick M. Forde, Julie R. Brahmer, Valsamo (Elsa) Anagnostou. Clinical utility of landmark ctDNA molecular response as an early indicator of immunotherapy outcomes in 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 110.

Abstract Introduction: Although plasma comprehensive genomic profiling (pCGP) shows significant potential as a minimally invasive alternative to tissue genotyping in non-small cell lung cancer (NSCLC), its precise clinical utility remains insufficiently studied. To bridge this gap, we conducted a single-center retrospective study of 718 patients with NSCLC who had undergone pCGP in our institution from 2015 through 2022. Methods: Integrated clinical and genomic data were extracted from the Precision Medicine Analytics Platform as part of a standard workflow of the Johns Hopkins Lung Cancer Precision Medicine Center of Excellence. Fixed gene panel hybrid capture next-generation sequencing (Guardant 360, n = 572 samples and Guardant 360CDx, n = 246 samples) data were retrieved, followed by variant characterization and annotation by an ensemble approach. Results: Across 718 patients with 818 instances of pCGP, including 79 (11%) patients with serial pCGP, liquid biopsies uniquely informed management in 92 (13%) patients. This primarily occurred when tissue testing was not possible (44%), although pCGP also contributed to clinical decision-making as an adjunct to tissue testing, by improving turnaround time, and by detecting plasma-only mutations. In a subset analysis of first instance of pCGP obtained at the time of diagnosis (n = 427), patients with undetectable ctDNA had significantly prolonged overall survival (OS) (log rank p < 0.0001), with similar results when patients were classified as ctDNA undetectable, 1-2 mutations, or 3 or more detected mutations (log rank p < 0.0001). Importantly, receipt of genotype-matched therapy informed by pCGP was associated with a significant improvement in OS (log rank p < 0.0001). Focusing on 214 patients with EGFR mutant (EGFRm) NSCLC, pCGP uniquely guided patient management in 28% of cases, benefiting both newly diagnosed patients and those on their second or third iteration of genotyping. Among patients progressing on EGFR tyrosine kinase inhibitors (TKI), 31 (22%) had actionable pCGP findings, of whom 18 (58%) were matched to targeted therapy. pCGP captured differential on-target and off-target resistance genomic alterations based on TKI line of therapy and type of TKI (first or second generation vs. third generation). Mutations in PIK3CA most commonly emerged after first-line therapy with third generation TKI, while mutations in the RAS pathway were more frequently detected after third-line therapy, highlighting differential activation of resistance pathways. Conclusions: Serial pCGP captures actionable genomic alterations and the emergence of drivers of therapy resistance, enabling longitudinal care and timely, effective interventions for patients with NSCLC. Citation Format: William Bowers, Michael Conroy, Jaime Wehr, Vivian V. Altiery De Jesus, Archana Balan, Susan Scott, Benjamin Levy, Kristen A. Marrone, Vincent K. Lam, Josephine Feliciano, Christine Hann, Aliyah Pabani, Jarushka Naidoo, Julie R. Brahmer, Patrick M. Forde, Joseph C. Murray, Valsamo (Elsa) Anagnostou. Serial plasma comprehensive genomic profiling detects therapy resistance and guides management of metastatic non-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 1310.

Supplementary Table S6. pCGP-informed clinical management in EGFR-mutant NSCLC.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non-Small Cell Lung Cancer (NSCLC) provide recommendations for the treatment of patients with NSCLC, including diagnosis, primary disease management, surveillance, and subsequent treatment. The panel has updated the list of recommended targeted therapies based on recent FDA approvals and clinical data. This selection from the NCCN Guidelines for NSCLC focuses on treatment recommendations for advanced or metastatic NSCLC with actionable biomarkers.