Objectives: Treatment of esophageal adenocarcinoma has historically involved trimodality therapy. Recently, perioperative chemotherapy has become standard of care due to improved survival. We investigated the implications of this change on surgical and pathologic outcomes. Methods: This multicenter retrospective analysis included adults who underwent esophagogastrectomy for esophageal/gastroesophageal junction adenocarcinoma between 2023 and 2025. The primary outcome was any positive margin on final pathology. Secondary outcomes included rates of nodal upstage and posttreatment tumor characteristics. Fisher exact and Kruskal-Wallis tests were used for comparisons. Results: In total, 110 patients were included; 22 received perioperative chemotherapy (20.0%), 48 chemoradiation (chemoRT, 43.6%), and 40 induction chemotherapy followed by chemoRT (chemo + chemoRT, 36.3%). On final pathology, 36.4% of patients experienced nodal upstaging in the chemotherapy group (n = 8) relative to 16.7% (n = 8) and 7.5% (n = 3) in the chemoRT and chemo + chemoRT groups (P = .030). Similarly, more patients had tumors exhibiting poor treatment response (tumor regression grade 3) in the chemotherapy group (n = 7, 33.3%) compared with the other 2 groups (n = 2 [4.5%] and n = 4 [10.5%]; P = .007). More patients in the chemotherapy group had any positive final margin (n = 8, 36.4%), as compared with the chemoRT (n = 5, 10.4%) or chemo + chemoRT groups (n = 4, 10.0%; P = .017). Early data suggest greater rates of frozen-to-final esophageal margin discrepancies in the chemotherapy cohort (n = 2 [16.6%] vs n = 1 [2.8%] chemoRT vs n = 2 [6.6%] chemo + chemoRT; P = .232). Conclusions: In this small multi-institutional study, chemotherapy-only preoperative regimens seemed to be associated with increased rates of nodal upstage and margin positivity after esophagectomy relative to radiotherapy-inclusive protocols.

In the original publication [...].

Introduction The phase 3 randomized controlled trial of extended pleurectomy decortication and chemotherapy versus chemotherapy alone for pleural mesothelioma (PM) (MARS2) reported “extended pleurectomy decortication was associated with worse survival to 2 years, and more serious adverse events for individuals with resectable PM, compared with chemotherapy alone.” These results have led to considerable discourse regarding the future role of surgery for PM, and there has not been unanimity in the mesothelioma surgical community regarding the trial interpretation. This “perspective” evaluates MARS2 using internationally renowned PM experts who either agreed with the trial interpretation or who found issues with its conduct which may have influenced the results. Methods A facilitator (HP) worked with team leaders (GW, IO) to assemble individuals offering opinions regarding the trial and its conclusions. Arguments agreeing or not agreeing with the trial interpretation were written only after publication of the full trial. Once both arguments were received by the facilitator, the individual team manuscripts were combined and sent to each team allowing editing for changes in perceived factual errors. Findings Insightful arguments include (but were not limited to) the difficulties yet advantages of randomization, quality assurance, selection of histologic subtypes, the timing of randomization, use of preoperative staging, statistical methods, and reasons for surgical mortality. Conclusions The decision to operate for PM in the future will continue to be defined by consensus guidelines and health payer willingness, and the interpretation of MARS2 may play an important role in modulating the role of surgery in the future.

Thymomas are rare anterior mediastinal tumors that can grow large, compressing important thoracic structures. Complete surgical excision remains the “gold standard.” Whereas median sternotomy has traditionally been used, minimally invasive techniques are becoming increasingly favored. Recent studies highlight the safety and efficacy of robotic thymectomy; however, “large” tumors described in the literature have averaged 6 to 8 cm. We present the case of a 68-year-old woman with a 13-cm thymoma resected entirely robotically. Use of various port configurations and enhanced maneuverability of robotic platform instruments enabled adequate visualization and safe dissection. This case highlights that tumor size alone should not preclude robotic thymectomy.

The humoral immune system plays a significant role in the immune response to cancer but is challenging to study at scale. We used programmable phage immunoprecipitation sequencing (PhIP-Seq) to profile the autoantibody repertoire in non-small cell lung cancer (NSCLC) patients for the purpose of training a machine learning-based classifier to distinguish NSCLC patients from healthy controls using 301 primarily early-stage, asymptomatic NSCLC patients and 352 healthy controls. The classifier performed well in cross-validation (average ROC-AUC = 0.94) and in an independently analyzed clinical validation cohort of 134 NSCLC patients and 96 healthy controls (ROC-AUC = 0.84). Classification performance can be maintained with only a few hundred target peptides, provided a sufficiently large cohort is used for optimal training. Our findings suggest the existence of a measurable autoreactive humoral profile in NSCLC and demonstrate the potential for serum-based early detection of cancer independent of nucleic acids. ### Competing Interest Statement AFK, CAD, JRK, DMJ, and JLD are co-inventors on a patent application submitted by the Regents of the University of California and the Chan Zuckerberg Biohub San Francisco related to this work. ### Funding Statement Funding was provided by the Chan Zuckerberg Biohub (San Francisco). LB was supported by grants from the Lung Cancer Research Foundation (LCRF) and the MGH Transformative Scholars Program. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Board of the University of California, San Francisco gave ethical approval for this work (IRB# 11-06107). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All PhIP-Seq data is available on Dryad at < https://doi.org/10.5061/dryad.08kprr5bk >. Code associated with analysis is available on github at < https://github.com/afkung/nsclc-classify >. Normal expression data from the Genotype-Tissue Expression Project (GTEx) used in the analyses described were downloaded from the GTEx Portal and dbGaP accession number phs000424.vN.pN. Lung adenocarcinoma data from The Cancer Genome Atlas Program (TCGA-LUAD) used in the analyses described in this manuscript were downloaded through the UCSC Xena Data Portal (University of California, Santa Cruz).

BACKGROUND:Survival for non-small-cell lung cancer (NSCLC) remains unacceptably low, even in stage IA-IIA. Current guidelines recommend adjuvant treatment for patients considered to be at high risk in stages IB and IIA, but suggest criteria that have not been validated to predict benefit. A previously validated, CLIA-certified 14-gene expression profile has identified patients with high-risk non-squamous NSCLC tumours in stages IA-IIA who benefitted from adjuvant chemotherapy in a non-randomised prospective study. In this prespecified interim analysis, we aimed to assess the efficacy and safety of platinum-based adjuvant chemotherapy in patients with stage IA-IIA molecular high-risk non-squamous NSCLC in a randomised trial. METHODS:AIM-HIGH, a randomised, phase 3 trial, was done at 45 centres in France, Germany, and the USA. Patients aged 18 years or older with stage IA-IIA non-squamous NSCLC, an adequate tumour sample, and an Eastern Cooperative Oncology Group performance status of 0-1 underwent risk stratification with the 14-gene assay. Patients with a molecular high risk, defined as those receiving a high-risk or an intermediate-risk score, were randomly assigned (1:1) to four cycles of platinum-based adjuvant chemotherapy (using local institutional standard of care regimens) or observation. Randomisation was stratified according to age, sex, and tumour size of 4 cm or more. The primary outcomes for the study and for this prespecified interim analysis were 48-month and 24-month disease-free survival, respectively, in the modified intention-to-treat (mITT) population, which was defined as randomly assigned patients who continued to meet eligibility criteria either at chemotherapy initiation or at random assignment to observation; an early interim analysis was prespecified to detect a large difference between groups. This trial is registered at ClinicalTrials.gov, NCT01817192, and is closed to enrolment. FINDINGS:Between Sept 11, 2020, and Feb 7, 2025, 449 patients were enrolled and underwent risk stratification. 236 patients with molecular high risk were randomly assigned to chemotherapy (n=124) or observation (n=112). At the time of the prespecified interim analysis, 87 patients were evaluable in the mITT population (47 [54%] males and 40 [46%] females; median age 63 years [IQR 52-74]) in the chemotherapy group and 107 (58 [54%] males and 49 [46%] females; 66 years [56-76]) in the observation group. 48 (55%) patients in the chemotherapy group and 58 (54%) patients in the observation group had stage IA disease; 34 (39%) and 44 (41%), respectively, had stage IB disease, and five (6%) and five (5%), respectively, had stage IIA disease. Six (3%) of 200 patients in the mITT population had died at the time of the interim analysis. 24-month disease-free survival was 96% (95% CI 92-100) with adjuvant chemotherapy versus 79% (70-90) with observation (hazard ratio 0·22 [0·06-0·76]; p=0·0087). INTERPRETATION:The 14-gene assay identified patients with molecular high risk who benefitted from adjuvant chemotherapy. Use of the assay to determine eligibility for adjuvant therapy in stage IA-IIA non-squamous NSCLC has the potential to substantially improve otherwise persistently poor outcomes. FUNDING:Razor Genomics.

LBA8027 Background: Despite advances in late-stage treatments, survival of early-stage non-small cell lung cancer (NSCLC) remains dismal, with 5-year disease free survival (DFS) of only 65% even in stage Ia. Preliminary, non-randomized clinical data suggest the predictive efficacy of RiskReveal, a 14-gene expression profile, in identifying stage Ia-IIa patients with non-squamous NSCLC who benefit from adjuvant therapy. We undertook an international, multicenter, randomized clinical trial to confirm these findings. Here, we report the results of an early interim analysis that was planned to detect a large discrepancy in outcomes between arms. Methods: 421 patients who underwent resection of stage pIa-IIa NSCLC were categorized as low-, intermediate-, or high-risk by the RiskReveal assay. Intermediate- and high-risk patients were randomized to observation or to 4 cycles of platinum-based adjuvant chemotherapy. The modified intent-to-treat (mITT) population was defined as randomized patients who continued to meet eligibility criteria either at the time of chemotherapy initiation or at randomization to observation. The primary endpoint was DFS in the mITT population, defined as the time from randomization to disease recurrence, exclusive of new primary lung cancer, or death from any cause. DFS was compared between arms using a log-rank test and Kaplan-Meier analysis. An early interim analysis was planned with a type I error rate of 0.02. Results: Of 194 evaluable patients at the time of the interim analysis, 87 had been randomized to adjuvant chemotherapy (55% stage Ia), and 107 to observation (55% stage Ia). There were no significant differences between the groups with respect to age, sex, or tumor size >4 cm; median follow-up was 19.5 months in the chemotherapy arm and 19.0 months in the observation arm. At 24 months, adjuvant chemotherapy significantly improved DFS compared to observation, with a HR of 0.22 (95% CI 0.06, 0.76; p=0.0087). DFS at 24 months was 96% with adjuvant chemotherapy (95% CI 0.92, 1.00) vs. 79% with observation (95% CI 0.70, 0.90). Median DFS was not reached in either group. Conclusion: A 14-gene molecular assay identified a high-risk population of stage Ia-IIa non-squamous NSCLC patients who benefited substantially from adjuvant chemotherapy. Improved survival in lung cancer is best achieved by optimizing outcomes in the earliest stages of disease. Identification of patients who benefit from adjuvant chemotherapy using this predictive test could result in a dramatic improvement in survival for the growing percentage of patients diagnosed in stages I-IIa. Although the study DSMB recommended a halt to enrollment, follow up continues and may provide further confirmation of this benefit. Clinical trial information: NCT01817192 .

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate this, we administered the fibroblast-selective TGFβ1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Unexposed biopsy samples showed higher fibroblast TGFβ1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG significantly downregulated TGFβ1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted Frizzle-like Receptor Protein 2 (sFRP2), an unrecognized TGFβ1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s). In human AEC2-fibroblast coculture organoids, sFRP2 was essential for AEC2 trans-differentiation to basal cells. Precision cut lung slices (PCLS) from normal donors demonstrated that TGFβ1 promoted KRT17 expression and AEC2 morphological change, while sFRP2 was necessary for KRT5 expression in AEC2-derived basaloid cells. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin-related signaling in AEC2s were required for sFRP2-induced KRT5 expression. These findings highlight stage-specific TGFβ1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify the TGFβ1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.

Esophageal cancer is one of the leading causes of cancer deaths globally with an incidence that is concentrated in specific hot spots in Eastern Asia, the Middle East, Eastern Africa, and South America. 10-year overall survival for patients treated with standard of care chemoradiation followed by surgical resection is below 40% highlighting the need for novel therapeutics to treat this disease. We assessed the effect of AMXI-5001, a novel small molecule poly ADP-Ribose polymerase (PARP) inhibitor and microtubule polymerization inhibitor on tumor growth inhibition in both in-vitro and in-vivo murine models. We found that AMXI-5001 was the most potent growth inhibitor of 8 out of 9 different esophageal carcinoma cell lines compared to other clinically available PARP inhibitors, Olaparib, Niraparib, Rucaparib, and Talazoparib. We then confirmed the previously described mechanism of action of AMXI-5001 as a PARP-inhibitor and microtubule polymerization inhibitor using both a PARP trapping assay and immunofluorescence. To further assess AMXI-5001's potential as a therapeutic for esophageal carcinoma we evaluated the effect of AMXI-5001 in combination with standard chemotherapy agents, Cisplatin and 5 Fluorouracil. We showed that AMXI-5001 synergistically inhibits growth in KYSE-70, a squamous esophageal cell line in combination with these drugs. In addition, we found that AMXI-5001 was an effective radiosensitizer, and squamous esophageal carcinoma cell lines treated 24 hours prior to external beam radiation showed significantly more growth inhibition compared to controls. Finally, we assessed the effect of AMXI-5001 monotherapy and in combination with radiotherapy in a xenograft mouse model implanted with subcutaneous KYSE-70 cells. Compared to vehicle control, and those treated with either AMXI-5001 alone or radiation alone, mice treated with both AMXI-5001 and radiation had significant tumor response. In conclusion, AMXI-5001 is an orally bioavailable dual-action PARP and microtubule polymerization inhibitor that holds promise in the treatment of esophageal carcinoma.

Abstract Lung cancer is the leading cause of cancer mortality, and despite improvements in treatment, tumors typically respond incompletely and resume growth after acquisition of drug resistance. Recent completion of a neoadjuvant osimertinib Phase II trial treating patients with surgically resectable stage I-IIIA EGFR-mutated non-small cell lung cancer (EGFRm NSCLC) (NCT03433469) (Blakely et. Al, JCO 2024) has highlighted the importance of further identifying non-genomic mechanisms of persistence and resistance to targeted therapy. Here we analyzed these patient samples to identify transcriptionally regulated signaling patterns enriched at Residual Disease (RD) after neoadjuvant osimertinib treatment compared to treatment naïve samples (TN), as well as signaling patterns enriched at RD in patients who ultimately develop disease recurrence after surgical resection compared to those who remain disease-free. Spatially resolved transcriptomic sequencing was performed, using the 10X Genomics Visium platform, on 36 tissue sections (n=9 TN, n=19 RD, n=4 Progressive Disease (PD), n=4 Tumor Adjacent Normal), from 25 patients. TN and PD samples were from standard-of-care surgical resections. 18 of 19 RD samples were from NCT03433469. The Visium array spots that tile the sequencing capture area are 55 µm in diameter, and thus can contain an estimated 1-10 cells within each array spot. After quality control, 91,582 array spots remained for downstream analysis. We identified high confidence tumor array spots annotated both as “Cancer” by a board-certified pathologist and with tumor characteristic copy number variations. Through dimensionality reduction clustering, we identified 10 major array spot clusters, including the high confidence tumor array spot cluster. These clusters were further subset into 44 high resolution clusters of array spot niches, 32 of which were shared across at least nine patients. Because array spots are not single-cell resolution, these niches represent spots with similar mixed cell type composition and transcriptional signatures. We identified significant increases in the CCL14+ Endothelial Cell Niche, CXCL2+ Endothelial Cell Niche, Lymphocytes + Endothelial Niche, and CXCL12+ Fibroblast Niche in RD samples compared to TN. We identified a cell signaling program defined by spatially co-expressed ligand-receptor pairs associated with focal adhesion, enriched in PD samples. This same pattern was enriched in RD samples from patients who had recurrent disease after surgical resection compared to RD samples from patients who remained disease free. These data describe an increase in immune trafficking in RD samples, as well as identify an EGFR- alternative signaling pathway correlating with osimertinib resistance. Identification of spatially resolved, differential architectural organization and signaling patterns in RD and PD states will expand our understanding of targeted therapy resistance in EGFRm NSCLC, with the potential to identify additional clinically actionable targets. Citation Format: Whitney Tamaki, Daniel L. Kerr, Wei Wu, Grant Eilers, Anatoly Urisman, Yu-Ting Chou, Philippe Gui, Shigeki Nanjo, Johannes R. Kratz, David M. Jablons, Trever G. Bivona, Collin M. Blakely. Identification of cell-cell signaling programs across the tumor-tumor microenvironment ecosystem and associated with clinical status in neoadjuvant osimertinib treated patient samples revealed by spatial profiling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C025.

PURPOSE:To assess the safety and efficacy of the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor osimertinib as neoadjuvant therapy in patients with surgically resectable stage I-IIIA EGFR-mutated non-small cell lung cancer (NSCLC). PATIENTS AND METHODS:This was a multi-institutional phase II trial of neoadjuvant osimertinib for patients with surgically resectable stage I-IIIA (American Joint Committee on Cancer [AJCC] V7) EGFR-mutated (L858R or exon 19 deletion) NSCLC (ClinicalTrials.gov identifier: NCT03433469). Patients received osimertinib 80 mg orally once daily for up to two 28-day cycles before surgical resection. The primary end point was major pathological response (MPR) rate. Secondary safety and efficacy end points were also assessed. Exploratory end points included pretreatment and post-treatment tumor mutation profiling. RESULTS:A total of 27 patients were enrolled and treated with neoadjuvant osimertinib for a median 56 days before surgical resection. Twenty-four (89%) patients underwent subsequent surgery; three (11%) patients were converted to definitive chemoradiotherapy. The MPR rate was 14.8% (95% CI, 4.2 to 33.7). No pathological complete responses were observed. The ORR was 52%, and the median DFS was 40.9 months. One treatment-related serious adverse event (AE) occurred (3.7%). No patients were unable to undergo surgical resection or had surgery delayed because of an AE. The most common co-occurring tumor genomic alterations were in TP53 (42%) and RBM10 (21%). CONCLUSION:Treatment with neoadjuvant osimertinib in surgically resectable (stage IA-IIIA, AJCC V7) EGFR-mutated NSCLC did not meet its primary end point for MPR rate. However, neoadjuvant osimertinib did not lead to unanticipated AEs, surgical delays, nor result in a significant unresectability rate.

8508 Background: Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) effective in treating advanced EGFR-mutated non-small cell lung cancer (NSCLC). Adjuvant osimertinib significantly decreases disease recurrence in stage IB-IIIA EGFR-mutated NSCLC. However, the benefit of neoadjuvant osimertinib prior to surgical resection remains unknown. Methods: This was a multi-institutional phase II trial of neoadjuvant osimertinib for patients with surgically resectable stage I-IIIA (AJCC V7) EGFR-mutated (L858R or exon 19 deletion) NSCLC (NCT03433469). Patients received osimertinib 80 mg orally daily for up to two 28-day cycles prior to surgical resection. The primary endpoint was major pathological response (mPR) rate (≤10% residual viable tumor). 27 evaluable patients provide 87% power to detect a mPR rate of 50% with α = 0.05. Secondary endpoints included pathological response (PR) rate (≤50% residual viable tumor), pathological complete response (pCR) rate, unconfirmed objective response rate (ORR), rate of lymph node downstaging, unanticipated delays to surgery, surgical complication rate, disease-free survival (DFS), overall survival (OS), safety, and tumor mutational profile. Results: A total of 27 patients with early-stage (8 stage IA/B, 10 stage IIA/B, 9 stage IIIA) EGFR-mutated (11 exon 19 del, 16 L858R) NSCLC were treated with neoadjuvant osimertinib for a median 56 days prior to surgical resection. 24 (89%) patients underwent subsequent surgery; 3 (11%) patients were converted to definitive chemoradiotherapy. The mPR rate was 15% (4/27 patients) by intention-to-treat analysis. The PR rate was 48% (13/27). No pCR’s were observed. Partial responses by radiography were observed in 52% (14/27) of patients and stable disease in 44% (12/27) of patients. Lymph node downstaging was achieved in 44% (4/9) of patients with positive lymph nodes. Median DFS after surgical resection was 32 months (95% CI 26-not reached) with a median follow-up of 11 months. OS data are immature. Significant adverse events occurred in 3 patients with grade 2 (G2) dyspnea, grade 3 (G3) pulmonary embolism, and G3 atrial fibrillation. One patient developed G2 treatment-related pneumonitis that resolved without steroids. Perioperative complications occurred in 38% (9/24) of patients; most involved rapidly reversible postoperative G2 atrial fibrillation (6/9) unrelated to study drug. Tumors were evaluable for genetic alterations from 16 patients. 4/6 patients who did not achieve a PR had tumors that harbored loss of function mutations in RBM10 as compared to 0/10 patients who achieved a PR (p < 0.01). Conclusions: Neoadjuvant osimertinib in surgically resectable EGFR-mutated NSCLC achieved a 15% mPR, which did not meet the primary endpoint. Treatment was safe and may induce pathological responses and lymph node-downstaging of disease. Co-mutations in RBM10 may limit response. Clinical trial information: NCT03433469 .

Supplementary Data from Three-Gene Expression Signature Predicts Survival in Early-Stage Squamous Cell Carcinoma of the Lung

The tremendous advances in genetic studies owe a great deal to the completion of human genome project in 2003 and the availability of next-generation sequencing (NGS). Illumina has the biggest NGS market share worldwide. Since Illumina acquired Solexa in 2007, Illumina has led NGS research and applications. Illumina has released many different types of platforms, from a small scale sequencer to a highest throughput sequencer. The first commercialized NGS system shown on the market was 454 pyrosequencing in 2005. For this system, dNTPs are added to the template-bound bead in a PicoTiterPlate. When each dNTP is incorporated into a newly synthesized DNA strand, a pyrophosphate molecule is produced and reacted with adenosine triphosphate (ATP) sulfurylase, leading to a change of adenosine phosphosulfate into ATP. The covered ATP with luciferase changes luciferin into light and oxyluciferin. The chapter discusses several different NGS or third-generation sequencing platforms and sample preparation methods.

BACKGROUND:Genetic changes that drive the transition from lepidic to invasive cancer development within a radiographic ground glass or semi-solid lung lesion (SSL) are not well understood. Biomarkers to predict the transition to solid, invasive cancer within SSL are needed. METHODS:Patients with surgically resected SSL were identified retrospectively from a surgical database. Clinical characteristics and survival were compared between stage I SSL (n = 65) and solid adenocarcinomas (n = 120) resected during the same time period. Areas of normal lung, in situ lepidic, and invasive solid tumor were microdissected from within the same SSL specimens and next generation sequencing (NGS) and Affymetrix microarray of gene expression were performed. RESULTS:There were more never smokers, Asian patients, and sub-lobar resections among SSL but no difference in 5-year survival between SSL and solid adenocarcinoma. Driver mutations found in both lepidic and solid invasive portion were EGFR (43%), KRAS (21%), and DNMT3A (5%). CEACAM5 was the most upregulated gene found in solid, invasive portions of SSL. Lepidic and invasive solid areas had many similarities in gene expression, however there were some significant differences with the gene SPP1 being a unique biomarker for the invasive component of a SSL. CONCLUSIONS:Common lung cancer driver mutations are present in in situ lepidic as well as invasive solid portions of a SSL, suggesting early development of driver mutations. CEACAM5 and SPP1 emerged as promising biomarkers of invasive potential in semi-solid lesions. Other studies have shown both genes to correlate with poor prognosis in lung cancer and their role in evolution of semi-solid lung lesions warrants further study.

Supplementary Data from A Multigene Assay Is Prognostic of Survival in Patients with Early-Stage Lung Adenocarcinoma

OBJECTIVES/GOALS: Smoking is a well-established risk factor for lung cancer, but never smokers account for up to 25% of lung cancer cases. There is mounting evidence that lung cancer in never smokers is biologically distinct. We aim to characterize the genomic and immunologic features of lung adenocarcinoma in never smokers versus smokers. METHODS/STUDY POPULATION: We examined clinical, genomic, and bulk-RNA sequencing data from 499 patients in the TCGA lung adenocarcinoma cohort. Tumor mutation burden was analyzed using maftools (R package). Tumor immune characterization was completed using CIBERSORTx, a digital cytometry tool that uses single cell reference profiles to determine immune cell type frequencies from bulk-RNA sequencing data. Single cell reference profiles for 19 different immune cell types were constructed from sequencing of freshly resected lung tumor tissue from UCSF patients. Partitioning Around Medoids (PAM; R package) was used to identify distinct immune phenotypes based on immune cell composition. Fisher’s exact test was used to evaluate for associations between immune phenotypes and smoking status. RESULTS/ANTICIPATED RESULTS: Of the 499 TCGA lung adenocarcinoma patients, 75 were never smokers, 269 were female, and 246 were over the age of 65. Never smokers had lower tumor mutation burden and lower predicted neoantigen burden compared to smokers (p < 0.001). There was no difference in total tumor immune cell infiltration between never smokers and smokers. PAM yielded 2 distinct clusters/immune phenotypes. The first was enriched in M1 Macrophages, cytotoxic T Cells, helper T Cells, regulatory T Cells, and Plasma Cells. The second was enriched in plasmacytoid Dendritic Cells, M2 Macrophages, and exhausted cytotoxic T Cells. Never smoking status was associated with an increased odds of having the first immune phenotype (OR 1.95, 95% CI: 1.15 - 3.35) and this association was statistically significant (p = 0.0086). DISCUSSION/SIGNIFICANCE: Our findings suggest that never smokers have an immune phenotype that is distinct from that observed in smokers. The distinct immune characteristics we observed could explain clinical trial data suggesting immune checkpoint inhibitors are less effective in never smokers and hold implications for tailoring therapy.