Tumor Genome and Microenvironment Alteration by Trastuzumab Deruxtecan as Neoadjuvant Therapy for HER2 Mutant NSCLC

Tumor Genome and Microenvironment Alteration by Trastuzumab Deruxtecan as Neoadjuvant Therapy for HER2 Mutant NSCLC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tumor Genome and Microenvironment Alteration by Trastuzumab Deruxtecan as Neoadjuvant Therapy for HER2 Mutant NSCLC Jiangyang Li, Xianfeng Lu, Shuai Yue, Ruyi Hang, Xiaoyan Dai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8854444/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Non-small cell lung cancer (NSCLC) patients carrying HER2 20ins exon 20 insertion (HER2 20ins) are poorly respond to conventional therapies and pan HER inhibitors. Trastuzumab Deruxtecan (T-DXd) has been approved in second line treatment in advanced NSCLC, but its efficacy in operable patients remains unclear. Here, we report a case of a patient with operable, HER2 20ins NSCLC who received neoadjuvant T-DXd. We utilized whole-genome sequencing (WGS) and immune microenvironment analysis to investigate the treatment's impact. WGS analysis revealed significant reduction in tumor clones with genome instability and extrachromosomal DNA (ecDNA) following T-DXd treatment. Meanwhile, immune profiling demonstrated substantially increased CD8 + T-cell infiltration in tumor cores with elevated PD1 expression. In contrast, conventional chemotherapy did not significantly alter CD8 + T cell infiltration or PD1 expression in another matched HER2 20ins patient. These findings suggest that neoadjuvant T-DXd may offer a well-tolerated and efficient therapeutic strategy for locally advanced NSCLC with HER2 20ins mutations. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology HER2 (ERBB2) NSCLC Deruxtecan (T-DXd DS-8201) Genomic instability Immune microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction HER2, encoded by ERBB2 gene, exon 20 insertion (HER2 20ins), predominantly Y772_A775dupYVMA, is the most common HER2 gene mutation in non-small cell lung cancer (NSCLC), with the incidence of 2–4% in non-squamous NSCLC. 1 , 2 Although the standard of care for patients with advanced disease carrying HER2 mutations in first line is chemotherapy, they usually have superior responses to the treatment than patients with or without other actionable driver mutations. 3 Meanwhile, traditional HER2-targeted or pan HER-targeted therapies, including monoclonal antibodies and small molecules, failed to show robust efficacies and acceptable toxicities in this cohort of NSCLC patients. 4 Trastuzumab deruxtecan (T-DXd, 8201) is the first FDA-approved HER2 mutation-targeted antibody drug conjugate (ADC) for NSCLC in second-line setting, based on the results of DESTINY-Lung02 study which demonstrated an ORR of 54.9% and a median progression-free survival (mPFS) of 8.2 months. 5 The DESTINY-Lung05 study further confirmed comparable benefits and toxicities in the Chinese population (ORR 58.3%, mPFS 10.8 months). 6 However, the efficacy and safety of T-DXd in locally advanced patients, particularly those who are unable to tolerate chemotherapy as a neoadjuvant treatment, are yet to be established. This study presents the first reported case of an elderly patient with stage IIIA HER2 20ins NSCLC who achieved significant tumor regression and underwent successful surgical resection after two cycles of neoadjuvant T-DXd therapy. More importantly, comprehensive profiling of the tumor genome and microenvironment was performed using WGS, RNA-seq, and multiplex immunofluorescence. This approach demonstrated the promising efficacy of neoadjuvant T-DXd and provided insights into alterations in the tumor genome and microenvironment following treatment. These findings may facilitate the future development of combination therapies with PD-1 inhibitors. 2 Materials and Methods 2.1 Clinical samples collection All human sample collection procedures were conducted in accordance with the principles of the Declaration of Helsinki and were granted exemption from ethical review by the Ethics Committee of the Army Medical Center of the PLA. All specimens were sourced from the Thoracic Surgery Department, Daping Hospital & Army Medical Center of PLA, Army Medical University. For the study subject, biopsy samples were collected prior to T-DXd treatment, while surgical specimens including tumor tissue, proximal peritumor tissue (within 3 cm of the tumor margin), and distal peritumor tissue (beyond 15 cm from the tumor margin) were obtained following T-DXd therapy from the same patient. Additionally, paired pre- and post-treatment FFPE samples were collected from control patients who underwent conventional chemotherapy. 2.2 DNA Extraction and Whole-Genome Sequencing Total DNA was extracted from both formalin-fixed paraffin-embedded (FFPE) biopsy samples and fresh frozen (FF) surgical specimens, including tumor tissues and matched adjacent non-tumor tissues(3 cm from tumor margin). Adjacent samples were used to filter out germline variants. High-quality genomic DNA was extracted using a commercial DNA extraction kit (Qiagen) following the manufacturer’s protocol. Libraries were constructed with the TruSeq Nano DNA Library Prep Kit (Illumina) and subjected to paired-end sequencing (2 × 150 bp) on the Illumina HiSeq X platform. High-quality reads were aligned to the UCSC human reference genome (hg38) using Burrows−Wheeler Aligner (BWA). 16 The resulting alignments were sorted, converted to BAM format, and indexed using Samtools. 17 All downstream preprocessing (duplicate marking and base quality recalibration) and somatic variant calling were executed using GATK4. 18 Functional annotation of somatic variants was performed with ANNOVAR. 19 Copy number variations (CNVs) were analyzed using CNVkit to identify amplified and deleted genomic regions. 20 Structural variants (SVs) were detected with Delly. 21 Complex amplicon structures such as extrachromosomal DNA (ecDNA) and breakage–fusion–bridge (BFB) cycles were identified with AmpliconArchitect. 10 Clonal evolution analysis was performed using PhyloWGS. 22 The genomic landscape of somatic mutations was visualized using Circos. 23 Kataegis hypermutation foci were identified and analyzed with Maftools R package. 24 Chromothripsis events were identified using ShatterSeek with SV and CN data. 25 Mutational signatures were extracted and deconvoluted with MutationalPatterns R package. 26 All statistical analyses were conducted in R v4.3.1. 2.3 RNA Isolation and RNA-Sequencing Total RNA was extracted from post-treatment surgical specimens, including tumor tissue, proximal peritumor tissue (within 3 cm of the tumor margin), and distal peritumor tissue (beyond 15 cm from the tumor margin). High-quality total RNA was isolated using a commercial RNA extraction kit (Qiagen) according to the manufacturer’s instructions. RNA sequencing was performed on the Illumina HiSeq X Ten platform to generate high-throughput paired-end sequencing data. Gene expression quantification with FeatureCounts 27 was followed by differential expression analysis via DESeq2. 28 Functional enrichment of differentially expressed genes was performed for Gene Ontology (GO) 29 trems and Kyoto Encyclopedia of Genes and Genomes (KEGG) 30 pathways. Immune cell infiltration in the samples was further evaluated using CIBERSORTx to characterize alterations in the immune microenvironment. 31 2.4 Immunohistochemical Analysis Immunohistochemical (IHC) staining was performed on formalin-fixed, paraffin-embedded (FFPE) tissue 4 µm-thick sections. The sections were incubated with the following primary antibodies: anti-PD-1 antibody (Zhongshan Goldenbridge Biotechnology; dilution 1:200) and anti-c-Kit antibody (Selleck Chemicals; dilution 1:400). Antigen retrieval was conducted using citrate buffer (pH 6.0) under high-temperature and high-pressure conditions. After blocking endogenous peroxidase activity with 3% hydrogen peroxide, the sections were treated with non-immune serum to reduce nonspecific binding. The antibody-antigen complexes were visualized using a horseradish peroxidase (HRP)-labeled polymer detection system and diaminobenzidine (DAB) as the chromogen. Finally, the sections were counterstained with hematoxylin, dehydrated, cleared, and mounted. All stained slides were independently evaluated by two pathologists to ensure consistency and accuracy. Analysis was performed on randomly chosen immunohistochemical images of equal dimensions utilizing the ImageJ software package. 2.5 Multiplex Immunofluorescence Analysis After deparaffinization and antigen retrieval, sections were blocked with 3% BSA for 30min. The following primary antibodies were sequentially applied and incubated overnight at 4℃: anti-CD8(Abcam, ab178089), anti-pan Cytokeratin (panCK) (Abcam, ab215838), anti-CD4(Abcam, ab133616), anti-CD20(Abcam, ab64088), anti-FoxP3(Abcam, ab20034), and anti-CD68(Abcam, ab192847). After each primary antibody incubation, slides were incubated with HRP-conjugated secondary antibodies at room temperature for 30 min, followed by a 5 min incubation with fluorophore-conjugated tyramide signal amplification reagent. Each staining cycle was followed by PBS washing and 100 ℃heat-induced antibody stripping. Finally, slides were counterstained with DAPI, and images were acquired using the AKOYA Vectra Polaris system. Quantification of positively stained cells in multiplex immunofluorescence was performed utilizing the digital pathology platforms Q-path and Phenochart within the analysis workflow. 2.6 H&E staining Conventional hematoxylin and eosin (H&E) staining was performed on paraffin-embedded tissue sections. Briefly, sections were first baked at 65°C for 2 hours, followed by deparaffinization and hydration. Subsequently, the nuclei were stained with hematoxylin and the cytoplasm was counterstained with eosin. After dehydration, the sections were mounted with neutral balsam and imaged under an inverted microscope. 2.7 Statistical analysis Statistical analysis was performed with GraphPad Prism 8.0. All values are presented as means±standard deviation (SD). Student’s t-test was used to determine statistical differences between two groups. And P < 0.05 was considered statistically significant (*:P < 0.05, **: P < 0.01, ***: P < 0.001; NS: no significance). 3 Results 3.1 Clinical Courses Case 1 A 74-year-old male, heavy smoker, was hospitalized due to accidentally finding of an nodule in the right lower lung during preoperative examination for "vocal cord cyst". Enhanced computed tomography scan revealed a 4.0×2.1 cm lesion in the dorsal segment of the right lower lobe, with enlarged mediastinal lymph nodes in station 4L and 7. Needle biopsy at lung lesion was then validated by pathological evaluation of lung adenocarcinoma with clinical stage as cT2aN2M0, stage IIIA. The driver genes status was then revealed HER2 exon 20 insertion mutation (YVMA) by ARMS methodology using quantitative PCR. In regard of a complex medical history of sick sinus syndrome, coronary atherosclerotic heart disease, hypertension (grade 3, very high risk), syphilis, and chronic hepatitis B, multidisciplinary team (MDT) consultant was suggested the lesions are potentially resectable with effective preoperative systemic treatment. After discussion with patients and family members, patient refused to receive standard of care immuno-chemotherapy with concerns about the age and multiple comorbidities. After acquiring informed consent from the patient, we offered trastuzumab deruxtecan (T-DXd) amonotherapy at dose of 5.4 mg/kg, administered once every 3 weeks, for a total of 2 cycles as neoadjuvant therapy. Only grade 1 constipation was observed during the neoadjuvant treatment.The first tumor evaluation revealed a partial response was achieved through neoadjuvant T-DXd with a tumor shrinkage of 47%, but the tumor was still in very active status with SUVmax of 13.99 by positron emission tomography scan. The second round of MDT were conducted and definitive surgery was recommended. Right lower lobectomy and hilar/mediastinal lymph node dissection was successfully performed 1 week after the second dose of T-DXd. Moderate tissue adhesion is observed during operation (Fig. 1 ). Pathological evaluation revealed after induction of T-DXd, the TNM stage was mildly downstaged as ypT1N2M0 with residual tumor cell was 60% which categorized to pathological regression grade 3 (CAP/NCCN) (Fig. 2 A). After 9 cycles of adjuvant T-DXd therapy, patient discontinued receiving further doses due to the economic reason and followed up every 3 months. As of the latest follow-up at October 2025 (16 months disease-free), no signs of recurrence or metastasis have been observed. Case 2 46-year-old female presented with headache and was diagnosed with left upper lung adenocarcinoma (clinical stage cT3N2M1b, IVB) carrying HER2 exon 20 insertion mutation, complicated by left hilar, mediastinal lymph node, and right occipital lobe (brain) metastases. Initial conversion therapy (bevacizumab + pemetrexed/cisplatin, 1 cycle) achieved stable disease (SD). She then underwent navigational microscope-assisted right parieto-occipital craniotomy for right parietal lobe brain metastasis resection under general anesthesia. One month later, video-assisted thoracoscopic surgery (VATS) was performed, involving left upper lobectomy, systematic lymph node dissection, and pleural adhesion lysis. Postoperatively, 7 cycles of adjuvant therapy were completed, with stable disease control at follow-up. 3.2 Cancer genome profiling before and after T-DXd treatment WGS analysis of paired samples from a T-DXd-treated patient revealed genomic instability in the pre-treatment sample (Figure S1A) . In particular, ERBB2 gene copy number gain (CN = 13.34) and multiple known driver gene alterations were detected, such as EP300, KMT2B, KDM5C, ABL1, and MED12. After T-DXd treatment, the tumor underwent significant genomic remodeling, including a decrease in ploidy from 2.94 to 2.22, normalization of the ERBB2 copy number (CN = 2.1), and clearance of the vast majority of driver gene alterations. This indicates that the tumor underwent rapid evolution under the pressure of T-DXd treatment, further implying its robust capability to eradicate tumor clones with unstable genomes ( Fig. 3 C ) . WGS data revealed the full landscape of genomic rearrangements, including structural variations (SVs), somatic copy-number alterations (SCNAs), extrachromosomal DNA (ecDNA), and genome-wide catastrophic events (kataegis and chromothripsis). A substantial burden of SVs (Deletion: 8444, Duplication: 1800, Inversion: 92232, Insertion: 1366, Breakend: 1971) was identified in pre-treatment tumor tissues. In contrast, post-treatment samples exhibited a pronounced reduction in SVs (Deletion: 9099, Duplication: 1761, Inversion: 1051, Insertion: 1582, Breakend: 2252). Our analysis of single base substitution (SBS) mutational signatures revealed that SBS1, SBS5, and SBS46 present prior to treatment were significantly reduced post-treatment, with only a small number of SBS5 mutations remaining (Figure S1C) . This suggests that tumor subclones harboring SBS1 and SBS46 were selectively eradicated through drug-mediated clearance during treatment. Furthermore, analysis of CNAs revealed a markedly higher degree of intratumoral copy number heterogeneity in pre-treatment samples compared to post-treatment samples, indicative of an effective cytotoxic response to T-DXd ( Fig. 3 A ) . eccDNA (extrachromosomal circular DNA) is a form of circular DNA that originates from chromosomal DNA but exists independently of it, whereas ecDNA (extrachromosomal DNA) specifically refers to larger circular DNA found in tumors, which has been further analyzed due to its reported roles in amplifying oncogenes and enabling accelerated tumor evolution during tumorigenesis and therapeutic resistance. 7 – 9 Due to the absence of centromeres, ecDNA is randomly distributed during cell division, resulting in easier copy number gain or loss of ecDNA under survival stress. 9 , 10 Analysis of ecDNA amplification dynamics revealed that the tumor exhibited both ecDNA-mediated amplification and complex non-circular amplification events (including CDK12/ERBB2/MIEN1) prior to treatment. Following treatment, only a non-circular amplification structure retaining AGAP2, CDK4, and MDM2 genes was detected ( Fig. 3 B ) . Notably, this structure essentially originated from the evolution of pre-existing chr12 ecDNA, and prominent chromothripsis was observed in the same genomic region both before and after treatment. Assessment of clustered alterations detected kataegis and chromothripsis (Figure S1B,D) , predominantly on chromosome 12 (chr12), in both pre-treatment and post-treatment samples. A concerted reduction in the burden of kataegis and the genomic extent of chromothripsis after T-DXd treatment indicates that the underlying subclones bearing these catastrophic events were effectively eliminated, likely due to their high drug sensitivity. Transcriptomic analysis showed sustained abnormal activation of the PI3K-AKT pathway after treatment. 3.3 Tumor Microenvironment Analysis Tissue samples were collected both pre- and post-treatment to analyze the effects of T-DXd. Multiplex immunofluorescence revealed a significant increase in CD8 + T cell infiltration post-treatment. Similarly, immunohistochemistry showed a substantial upregulation of PD-1 expression in the tumor core (Fig. 2 A,C,D). Transcriptomic immune infiltration analysis of post-treatment samples also revealed this phenomenon (Fig. 4 B). In contrast, a sample from a comparable HER2 exon 20 insertion mutant patient who received conventional chemotherapy exhibited minimal CD8 + T cell infiltration and no significant change in PD-1 expression after treatment (Fig. 2 B,C,D). This stark difference suggests that T-DXd, unlike chemotherapy, can remodel the tumor immune microenvironment and potentially sensitize tumors to PD-1 checkpoint inhibitors. Furthermore, transcriptome analysis revealed that many effector cells (NK cells, CD4 + T cells CD8 + T cells and NKT cells) are infiltrated into tumor core after T-DXd treatment. Aligned with mIF results (Fig. 2 A, 4 B), these results collectively indicate immune-cold tumor could turn into immune-hot tumor by T-DXd treatment. In addition, neutrophil extracellular traps (NETs) have been documented to promote tumorigenesis and metastasis and dampen immunotherapy efficacy. 11 As expected, we further found neutrophils appeared to be significantly more enriched in peritumoral and distant regions instead of tumor core, reflecting the more activated immune environment and may synergize with immunotherapy. Taken together, these results suggest that T-DXd treatment can induce a highly activated TME, which may hold significant potential for improving immunotherapy outcomes. 3.4 Mechanism of adverse effects by T-DXd treatment Patients receiving T-DXd treatment are at risk of pulmonary adverse events, particularly interstitial lung disease (ILD), with a predominant incidence of interstitial pneumonitis. 12 This patient also displayed a interstitial changes in right lower lobe according to CT scan after 2 cycles of T-DXd, without any symptoms (Fig. 4 A). In this regards, distal and peritumor normal lung tissues were also under further investigation using RNA-seq and pathological analysis. A study has shown that alveolar macrophages residing in the perivascular microenvironment play a key role in the development of T-DXd-related ILD, with SPP1 (osteopontin) identified as a critical mediator in this process. 13 Through transcriptome analysis, we found that SPP1 expression in cancer tissues was significantly higher than in the adjacent normal lung tissues, both adjacent (> 3 cm) and distal (> 15 cm) to the tumor. Pathological analysis revealed substantial infiltration of inflammatory cells in all three distinct regions(Fig. 4 C). Immune microenviroment analysis by RNA-seq further suggested that mast cell infiltration was more pronounced in the distal cancer-adjacent tissues compared to the proximal tissues, a phenomenon potentially associated with the development of ILD. Immunohistochemical results corroborated the increased mast cell population in the distal cancer-adjacent tissues(Fig. 4 C,D). These findings suggest that mast cells may contribute to the development of T-DXd-related ILD by promoting fibrosis in the distal cancer-adjacent tissues. 4 Discussion While T-DXd has demonstrated efficacy in advanced HER2-mutant NSCLC, its role in neoadjuvant therapy for locally advanced disease remains underexplored. To our knowledge, this is the first report to describe a case of locally advanced (cT2aN2M0 IIIA) HER2 20ins mutation (A775_G776insYVMA) carrying NSCLC patient who successfully underwent surgical resection after receiving neoadjuvant T-DXd treatment and achieving partial response. This case demonstrates that T-DXd has significant efficacy with acceptable safety. WGS results show that dominant tumor clones with highly unstable genome and enriched ecDNA were cleared. More importantly, T-DXd treatment induced significant CD8 + T cell infiltration in the tumor core area and upregulated PD1 expression (Fig. 2 A), suggesting that sequential or concurrent addition of PD-1 inhibitors may further enhance therapeutic efficacy. Recent reports have shown that T-DXd combined with PD-1 inhibitors successfully induced pathological complete response (pCR) and achieved R0 resection in stage IIIB HER2-mutant NSCLC patients (PD-L1 50%). Although the patient's tumor exhibited a typical "cold" immune phenotype prior to treatment, neoadjuvant T-DXd therapy significantly remodeled the tumor microenvironment, leading to enhanced infiltration of CD8 + T cells. Furthermore, multiplex immunofluorescence analysis revealed that PD-1 expression followed a pattern similar to that of CD8 + T cell infiltration, with both showing significant upregulation after treatment. These findings suggest that T-DXd may exert its antitumor effects by remodeling the immune microenvironment, thereby providing a rationale for its combination with PD-1 inhibitors. Despite its significant efficacy, T-DXd therapy increases the risk of interstitial lung disease (ILD), which is notably higher in lung cancer patients compared to those with breast or gastric cancer. 4 The development of T-DXd-related ILD/pneumonitis may be attributed to HER2 protein expression in the bronchial and bronchiolar epithelium of the lung or a direct cytotoxic effect of T-DXd on lung tissue. 14 Prior studies using mouse models have revealed that alveolar macrophages (AMs) serve as the primary target cells of T-DXd in the pulmonary microenvironment and contribute to the development of T-DXd-related interstitial lung disease. 13 RNA-seq analysis and immunohistochemical profiling of CD117 revealed a higher level of mast cell infiltration in distal lung tissue (> 15 cm) compared to adjacent lung tissue (> 3 cm)(Fig. 4 B,C). This spatial distribution suggests a potential role for mast cells in ILD pathogenesis. Recent studies have indicated that mast cells, as a potential source of fibrotic factors such as TGF-β, play an important role in the fibrosis process. The tryptase they secrete can stimulate the proliferation of pulmonary fibroblasts and enhance extracellular matrix (ECM) synthesis, thereby promoting the pathological progression of fibrosis. 15 Pathological examination of resected specimens revealed a pro-fibrotic response, characterized by significant chronic inflammatory cell infiltration, multinucleated giant cell reactions, and calcified fibrous proliferative changes in adjacent lung tissue. These findings suggest that T-DXd can induce fibrotic responses, underscoring the need for heightened vigilance regarding ILD during treatment and surgery. The primary limitation of this study is its single-case design. While providing valuable mechanistic insights, the findings require validation in larger prospective cohorts to determine the generalizability of the immune responses observed. Our findings specifically support trials investigating combination therapy with PD-1 inhibitors to enhance efficacy. Additionally, the identified fibrotic response and mast cell infiltration highlight the need for translational research focused on predicting and preventing T-DXd-related ILD. In conclusion, this report highlights the potent efficacy and manageable toxicity of T-DXd in the neoadjuvant treatment of HER2-mutant NSCLC, while also demonstrating its immune-modulating effects. Additionally, mast cell-mediated fibrotic responses were identified as a potential mechanism for T-DXd-related ILD, providing a foundation for future studies aimed at optimizing the therapeutic potential of T-DXd and mitigating its associated risks. Abbreviations NSCLC Non-small cell lung cancer T-DXd Trastuzumab Deruxtecan WGS whole-genome sequencing ecDNA extrachromosomal DNA eccDNA extrachromosomal circluar DNA HER2 Human Epidermal Growth Factor Receptor 2 ERBB2 Epidermal Growth Factor Receptor 2 ADC antibody drug conjugate mPFS median progression-free survival FDA Food and Drug Administration RNA-seq RNA sequencing ORR Overall Response Rate FFPE formalin-fixed paraffin-embedded CNVs Copy number variations SVs Structural variants ILD interstitial lung disease IHC Immunohistochemistry GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes PD1 Programmed Cell Death Protein 1 BFB breakage–fusion–bridge Declarations Funding This work was supported by the following research grants: the Talent Innovation Capacity Building Program of Army Medical Center (Grant No. ZXYZZKY07); the National Natural Science Foundation of China (NSFC) (Grant No. 82472748); and the Science-Health Joint Research Project of Chongqing (Grant No. 2024MSXM035). Author Contribution J.L. and M.L.: conceptualization, methodology, investigation, validation, formal analysis, data curation, and writing – original draft. X.L.: methodology, investigation, formal analysis, and data curation. Y.X. and M.L.: conceptualization, resources, supervision, funding acquisition. Y.X., X.L., S.Y., Z.H., and M.L.: writing – review & editing. R.H.: formal analysis. X.D. and X.K.: methodology and validation. W.G.: investigation and resources. All authors reviewed and approved the final manuscript. Data Availability The datasets generated and analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1426580. The raw sequencing data can be accessed via the following SRA run accessions: SRR37420559–SRR37420570. The corresponding BioSample accessions are SAMN55876066–SAMN55876074. Disclosure Statement Ethical approval for this study was obtained from the Institutional Review Board of the Army Medical Center of the PLA, Army Medical University. Written informed consent was obtained from all participating patients. Clinical trial registration number: not applicable. All authors have reviewed and approved the final version of the manuscript. The authors declare no conflicts of interest. References Reinhorn, D., Moskovitz, M., Tap, W. D. & Li, B. T. Targeting her2 in lung cancers: Evolving treatment landscape and drug development strategies. Cancer 131 (Suppl 1), e35780 (2025). Zhang, S. et al. Chinese expert consensus on the diagnosis and treatment of her2-altered non-small cell lung cancer. Thorac. Cancer . 14 , 91–104 (2023). Nützinger, J. et al. Management of her2 alterations in non-small cell lung cancer - the past, present, and future. Lung Cancer . 186 , 107385 (2023). Yu, Y., Yang, Y., Li, H. & Fan, Y. 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(D) Visualization of Chromothripsis on chr12 before and after treatment. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Apr, 2026 Reviews received at journal 26 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviewers agreed at journal 16 Apr, 2026 Reviewers agreed at journal 15 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 13 Apr, 2026 Editor invited by journal 04 Mar, 2026 Submission checks completed at journal 02 Mar, 2026 First submitted to journal 02 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8854444","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":624534051,"identity":"90e8e201-9389-40ea-b7d4-1e59fc8def1e","order_by":0,"name":"Jiangyang Li","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Jiangyang","middleName":"","lastName":"Li","suffix":""},{"id":624534052,"identity":"39de505c-4fec-435e-94af-a876a44224a2","order_by":1,"name":"Xianfeng Lu","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Xianfeng","middleName":"","lastName":"Lu","suffix":""},{"id":624534053,"identity":"51adb04b-1d08-437e-ad60-0c046a23245d","order_by":2,"name":"Shuai Yue","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Yue","suffix":""},{"id":624534054,"identity":"59b865eb-e359-4dd4-be3c-f684c5ac5a99","order_by":3,"name":"Ruyi Hang","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Ruyi","middleName":"","lastName":"Hang","suffix":""},{"id":624534055,"identity":"bd63eca2-695f-4f65-a073-5fef8988ab59","order_by":4,"name":"Xiaoyan Dai","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Dai","suffix":""},{"id":624534056,"identity":"9781c573-c1a0-466f-b9db-aad17502795d","order_by":5,"name":"Xunjie Kuang","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Xunjie","middleName":"","lastName":"Kuang","suffix":""},{"id":624534057,"identity":"67e18510-150b-4104-89a6-d174329abaaa","order_by":6,"name":"Wei Guo","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Guo","suffix":""},{"id":624534058,"identity":"9cb720fd-d9a1-4058-a9d7-035ec340ea26","order_by":7,"name":"Zhou Huang","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Huang","suffix":""},{"id":624534059,"identity":"c8d8c3ec-8aff-486f-bf87-1c062f780f5a","order_by":8,"name":"Yanli Xiong","email":"","orcid":"","institution":"Army medical university","correspondingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Xiong","suffix":""},{"id":624534060,"identity":"36a95d1b-0189-43ef-9d1b-c7813d8ed2dc","order_by":9,"name":"Mengxia Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBACPmYIzcPA3gAVOkBACxtcC89hYrXAWRLJxGph5058XPDrsAz/zPdHN91sY5Dju5HA+LkAr8N4NxvP7EvjkbidzHY7t43BWPJGArP0DPxatknz9tjwMEC1JG64kcDGzINfy/bfvD0SPPI3D4O11BOjZRszzw8bHoMbzGAtCQZEaNkszduQxmN4Jtnsds45CcOZZx42S+PTws9/duNnnj+H7eWOH3x2O6fMRp7vePLBz/i0gAFjG5wpAeI2ENIABH+IUDMKRsEoGAUjFwAAuL5E1FcBGB8AAAAASUVORK5CYII=","orcid":"","institution":"Army medical university","correspondingAuthor":true,"prefix":"","firstName":"Mengxia","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-02-11 17:29:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8854444/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8854444/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107519670,"identity":"06266543-da97-4987-9218-7ac55cada3f7","added_by":"auto","created_at":"2026-04-22 08:57:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3512105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal Clinical Course During Neoadjuvant T-DXd Therapy. \u003c/strong\u003eTreatment timeline annotated with serum CEA levels (carcinoembryonic antigen; normal \u0026lt;5 ng/mL).Radiological evaluation through two cycles of neoadjuvant T-DXd. Schematic representation (lower-right): Tissue sampling regions for multiplex immunofluorescence (mIF), whole-genome sequencing (WGS), and transcriptome profiling (RNA-seq). CEA, carcinoembryonic antigen; LN, lymph node; PT, primary tumor; Adjacent lung (AL); Distal lung (DL).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8854444/v1/a093c8bafc55da20fa6a6db9.png"},{"id":107519689,"identity":"4159ceb6-8b20-46cc-ac41-bfdc074ae167","added_by":"auto","created_at":"2026-04-22 08:57:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10499142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeoadjuvant T-DXd-Mediated Remodeling of the Immune Microenvironment\u003c/strong\u003e. (A) Pre/Post neoadjuvant T-DXd pathological analysis: H\u0026amp;E staining (25×) of FFPE samples; mIF showing post-treatment CD8\u0026lt;sup\u0026gt;+\u0026lt;/sup\u0026gt; T-cell infiltration into tumor core; IHC demonstrating elevated PD1 expression. (B) Pre/Post chemotherapy pathological analysis: H\u0026amp;E staining (25×) of FFPE samples; mIF indicating CD8\u0026lt;sup\u0026gt;+\u0026lt;/sup\u0026gt; T-cells retained at tumor periphery (pre/post-treatment); IHC revealing no significant change in PD1 expression. (C) Quantitative analysis of CD117 CD8\u0026lt;sup\u0026gt;+\u0026lt;/sup\u0026gt; T-cells count. (D) Quantitative analysis of PD1 expression levels. H\u0026amp;E, hematoxylin and eosin; mIF, multiplex immunofluorescence; IHC, immunohistochemistry; Scale: 50mm/100 mm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8854444/v1/4ace98c919cb032f0654bcf2.png"},{"id":107519929,"identity":"401ceb82-cf73-4d26-80f8-14e3a960e8f3","added_by":"auto","created_at":"2026-04-22 08:58:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1650754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic Heterogeneity Regression During T-DXd Therapy\u003c/strong\u003e. (A) Subclonal copy number alterations pre/post Neoadjuvant T-DXd therapy are illustrated by the profile, with the two variants displaying distinct representations of subclonal copy number alterations. (B) ecDNA structural dynamics, pre-therapy AmpliconArchitect reconstruction revealed a complex hybrid ecDNA amplicon on chr12 with high-level amplification. Post-T-DXd therapy showed ecDNA disassembly into residual focal amplification. (C) Clonal evolution analysis: Near-complete eradication of tumor clones post-T-DXd.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8854444/v1/d11f286c9dd459616d27a36c.png"},{"id":107519686,"identity":"ac45bf75-435b-40a3-96e1-336aa89bb28e","added_by":"auto","created_at":"2026-04-22 08:57:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4710037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdverse effects by T-DXd treatment\u003c/strong\u003e. (A) Representative chest CT imaging demonstrating the emergence of pulmonary fibrosis in the right lower lobe after two cycles of T-DXd treatment. (B) Transcriptome-based immune cell infiltration analysis comparing the tumor microenvironment across distinct anatomical compartments: primary tumor lesions, adjacent lung regions(3cm from margin), and distal lung tissues post-treatment (15cm from margin). (C) Histopathological evaluation by H\u0026amp;E staining revealing inflammatory cell infiltration within the stromal compartment, accompanied by immunohistochemical profiling of CD117 expression across the three tissue regions. (D) Quantitative analysis of CD117 expression levels. Scale bars: 50mm. PT, primary tumor; Adjacent lung (AL); Distal lung (DL).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8854444/v1/f6ecfe8cfff6b8b513d9c7f0.png"},{"id":107520196,"identity":"adcfd44b-01ca-41aa-bb78-e50e559f84f0","added_by":"auto","created_at":"2026-04-22 08:59:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18828321,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8854444/v1/e06ed4cb-a23b-4d66-ba8e-d1599aa9c9dd.pdf"},{"id":107519759,"identity":"63dc8110-ec89-4186-9108-614e926880f7","added_by":"auto","created_at":"2026-04-22 08:57:50","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":749597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1:\u003c/strong\u003e \u003cstrong\u003eGenomic Stability Assessment Before and After Neoadjuvant T-DXd Therapy. \u003c/strong\u003e(A) Comprehensive analysis of genome-wide variant relationships presented through a Circos plot. (B) Kategis landscape on chr12 before and after treatment. (C) Changes in single-base substitutions (SBS) before and after treatment. (D) Visualization of Chromothripsis on chr12 before and after treatment.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8854444/v1/aad8cc7a541c6ac2af1ec66d.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor Genome and Microenvironment Alteration by Trastuzumab Deruxtecan as Neoadjuvant Therapy for HER2 Mutant NSCLC","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eHER2, encoded by ERBB2 gene, exon 20 insertion (HER2 20ins), predominantly Y772_A775dupYVMA, is the most common HER2 gene mutation in non-small cell lung cancer (NSCLC), with the incidence of 2\u0026ndash;4% in non-squamous NSCLC.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Although the standard of care for patients with advanced disease carrying HER2 mutations in first line is chemotherapy, they usually have superior responses to the treatment than patients with or without other actionable driver mutations.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Meanwhile, traditional HER2-targeted or pan HER-targeted therapies, including monoclonal antibodies and small molecules, failed to show robust efficacies and acceptable toxicities in this cohort of NSCLC patients.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTrastuzumab deruxtecan (T-DXd, 8201) is the first FDA-approved HER2 mutation-targeted antibody drug conjugate (ADC) for NSCLC in second-line setting, based on the results of DESTINY-Lung02 study which demonstrated an ORR of 54.9% and a median progression-free survival (mPFS) of 8.2 months.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The DESTINY-Lung05 study further confirmed comparable benefits and toxicities in the Chinese population (ORR 58.3%, mPFS 10.8 months).\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e However, the efficacy and safety of T-DXd in locally advanced patients, particularly those who are unable to tolerate chemotherapy as a neoadjuvant treatment, are yet to be established.\u003c/p\u003e \u003cp\u003eThis study presents the first reported case of an elderly patient with stage IIIA HER2 20ins NSCLC who achieved significant tumor regression and underwent successful surgical resection after two cycles of neoadjuvant T-DXd therapy. More importantly, comprehensive profiling of the tumor genome and microenvironment was performed using WGS, RNA-seq, and multiplex immunofluorescence. This approach demonstrated the promising efficacy of neoadjuvant T-DXd and provided insights into alterations in the tumor genome and microenvironment following treatment. These findings may facilitate the future development of combination therapies with PD-1 inhibitors.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Clinical samples collection\u003c/h2\u003e \u003cp\u003eAll human sample collection procedures were conducted in accordance with the principles of the Declaration of Helsinki and were granted exemption from ethical review by the Ethics Committee of the Army Medical Center of the PLA. All specimens were sourced from the Thoracic Surgery Department, Daping Hospital \u0026amp; Army Medical Center of PLA, Army Medical University. For the study subject, biopsy samples were collected prior to T-DXd treatment, while surgical specimens including tumor tissue, proximal peritumor tissue (within 3 cm of the tumor margin), and distal peritumor tissue (beyond 15 cm from the tumor margin) were obtained following T-DXd therapy from the same patient. Additionally, paired pre- and post-treatment FFPE samples were collected from control patients who underwent conventional chemotherapy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 DNA Extraction and Whole-Genome Sequencing\u003c/h2\u003e \u003cp\u003eTotal DNA was extracted from both formalin-fixed paraffin-embedded (FFPE) biopsy samples and fresh frozen (FF) surgical specimens, including tumor tissues and matched adjacent non-tumor tissues(3 cm from tumor margin). Adjacent samples were used to filter out germline variants. High-quality genomic DNA was extracted using a commercial DNA extraction kit (Qiagen) following the manufacturer\u0026rsquo;s protocol. Libraries were constructed with the TruSeq Nano DNA Library Prep Kit (Illumina) and subjected to paired-end sequencing (2 \u0026times; 150 bp) on the Illumina HiSeq X platform. High-quality reads were aligned to the UCSC human reference genome (hg38) using Burrows\u0026minus;Wheeler Aligner (BWA).\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e The resulting alignments were sorted, converted to BAM format, and indexed using Samtools.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e All downstream preprocessing (duplicate marking and base quality recalibration) and somatic variant calling were executed using GATK4.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Functional annotation of somatic variants was performed with ANNOVAR.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Copy number variations (CNVs) were analyzed using CNVkit to identify amplified and deleted genomic regions.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Structural variants (SVs) were detected with Delly.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Complex amplicon structures such as extrachromosomal DNA (ecDNA) and breakage\u0026ndash;fusion\u0026ndash;bridge (BFB) cycles were identified with AmpliconArchitect.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Clonal evolution analysis was performed using PhyloWGS.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The genomic landscape of somatic mutations was visualized using Circos.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Kataegis hypermutation foci were identified and analyzed with Maftools R package.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Chromothripsis events were identified using ShatterSeek with SV and CN data.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Mutational signatures were extracted and deconvoluted with MutationalPatterns R package.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e All statistical analyses were conducted in R v4.3.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 RNA Isolation and RNA-Sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from post-treatment surgical specimens, including tumor tissue, proximal peritumor tissue (within 3 cm of the tumor margin), and distal peritumor tissue (beyond 15 cm from the tumor margin). High-quality total RNA was isolated using a commercial RNA extraction kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions. RNA sequencing was performed on the Illumina HiSeq X Ten platform to generate high-throughput paired-end sequencing data. Gene expression quantification with FeatureCounts\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e was followed by differential expression analysis via DESeq2.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Functional enrichment of differentially expressed genes was performed for Gene Ontology (GO)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e trems and Kyoto Encyclopedia of Genes and Genomes (KEGG)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e pathways. Immune cell infiltration in the samples was further evaluated using CIBERSORTx to characterize alterations in the immune microenvironment.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunohistochemical Analysis\u003c/h2\u003e \u003cp\u003eImmunohistochemical (IHC) staining was performed on formalin-fixed, paraffin-embedded (FFPE) tissue 4 \u0026micro;m-thick sections. The sections were incubated with the following primary antibodies: anti-PD-1 antibody (Zhongshan Goldenbridge Biotechnology; dilution 1:200) and anti-c-Kit antibody (Selleck Chemicals; dilution 1:400). Antigen retrieval was conducted using citrate buffer (pH 6.0) under high-temperature and high-pressure conditions. After blocking endogenous peroxidase activity with 3% hydrogen peroxide, the sections were treated with non-immune serum to reduce nonspecific binding. The antibody-antigen complexes were visualized using a horseradish peroxidase (HRP)-labeled polymer detection system and diaminobenzidine (DAB) as the chromogen. Finally, the sections were counterstained with hematoxylin, dehydrated, cleared, and mounted. All stained slides were independently evaluated by two pathologists to ensure consistency and accuracy. Analysis was performed on randomly chosen immunohistochemical images of equal dimensions utilizing the ImageJ software package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Multiplex Immunofluorescence Analysis\u003c/h2\u003e \u003cp\u003eAfter deparaffinization and antigen retrieval, sections were blocked with 3% BSA for 30min. The following primary antibodies were sequentially applied and incubated overnight at 4℃: anti-CD8(Abcam, ab178089), anti-pan Cytokeratin (panCK) (Abcam, ab215838), anti-CD4(Abcam, ab133616), anti-CD20(Abcam, ab64088), anti-FoxP3(Abcam, ab20034), and anti-CD68(Abcam, ab192847). After each primary antibody incubation, slides were incubated with HRP-conjugated secondary antibodies at room temperature for 30 min, followed by a 5 min incubation with fluorophore-conjugated tyramide signal amplification reagent. Each staining cycle was followed by PBS washing and 100 ℃heat-induced antibody stripping. Finally, slides were counterstained with DAPI, and images were acquired using the AKOYA Vectra Polaris system. Quantification of positively stained cells in multiplex immunofluorescence was performed utilizing the digital pathology platforms Q-path and Phenochart within the analysis workflow.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 H\u0026amp;E staining\u003c/h2\u003e \u003cp\u003eConventional hematoxylin and eosin (H\u0026amp;E) staining was performed on paraffin-embedded tissue sections. Briefly, sections were first baked at 65\u0026deg;C for 2 hours, followed by deparaffinization and hydration. Subsequently, the nuclei were stained with hematoxylin and the cytoplasm was counterstained with eosin. After dehydration, the sections were mounted with neutral balsam and imaged under an inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with GraphPad Prism 8.0. All values are presented as means\u0026plusmn;standard deviation (SD). Student\u0026rsquo;s t-test was used to determine statistical differences between two groups. And P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (*:P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **: P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***: P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; NS: no significance).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Clinical Courses\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCase 1\u003c/strong\u003e \u003cp\u003eA 74-year-old male, heavy smoker, was hospitalized due to accidentally finding of an nodule in the right lower lung during preoperative examination for \"vocal cord cyst\". Enhanced computed tomography scan revealed a 4.0\u0026times;2.1 cm lesion in the dorsal segment of the right lower lobe, with enlarged mediastinal lymph nodes in station 4L and 7. Needle biopsy at lung lesion was then validated by pathological evaluation of lung adenocarcinoma with clinical stage as cT2aN2M0, stage IIIA. The driver genes status was then revealed HER2 exon 20 insertion mutation (YVMA) by ARMS methodology using quantitative PCR. In regard of a complex medical history of sick sinus syndrome, coronary atherosclerotic heart disease, hypertension (grade 3, very high risk), syphilis, and chronic hepatitis B, multidisciplinary team (MDT) consultant was suggested the lesions are potentially resectable with effective preoperative systemic treatment. After discussion with patients and family members, patient refused to receive standard of care immuno-chemotherapy with concerns about the age and multiple comorbidities. After acquiring informed consent from the patient, we offered trastuzumab deruxtecan (T-DXd) amonotherapy at dose of 5.4 mg/kg, administered once every 3 weeks, for a total of 2 cycles as neoadjuvant therapy. Only grade 1 constipation was observed during the neoadjuvant treatment.The first tumor evaluation revealed a partial response was achieved through neoadjuvant T-DXd with a tumor shrinkage of 47%, but the tumor was still in very active status with SUVmax of 13.99 by positron emission tomography scan. The second round of MDT were conducted and definitive surgery was recommended. Right lower lobectomy and hilar/mediastinal lymph node dissection was successfully performed 1 week after the second dose of T-DXd. Moderate tissue adhesion is observed during operation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Pathological evaluation revealed after induction of T-DXd, the TNM stage was mildly downstaged as ypT1N2M0 with residual tumor cell was 60% which categorized to pathological regression grade 3 (CAP/NCCN) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After 9 cycles of adjuvant T-DXd therapy, patient discontinued receiving further doses due to the economic reason and followed up every 3 months. As of the latest follow-up at October 2025 (16 months disease-free), no signs of recurrence or metastasis have been observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 2\u003c/strong\u003e \u003cp\u003e46-year-old female presented with headache and was diagnosed with left upper lung adenocarcinoma (clinical stage cT3N2M1b, IVB) carrying HER2 exon 20 insertion mutation, complicated by left hilar, mediastinal lymph node, and right occipital lobe (brain) metastases. Initial conversion therapy (bevacizumab\u0026thinsp;+\u0026thinsp;pemetrexed/cisplatin, 1 cycle) achieved stable disease (SD). She then underwent navigational microscope-assisted right parieto-occipital craniotomy for right parietal lobe brain metastasis resection under general anesthesia. One month later, video-assisted thoracoscopic surgery (VATS) was performed, involving left upper lobectomy, systematic lymph node dissection, and pleural adhesion lysis. Postoperatively, 7 cycles of adjuvant therapy were completed, with stable disease control at follow-up.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Cancer genome profiling before and after T-DXd treatment\u003c/h2\u003e \u003cp\u003eWGS analysis of paired samples from a T-DXd-treated patient revealed genomic instability in the pre-treatment sample \u003cb\u003e(Figure S1A)\u003c/b\u003e. In particular, ERBB2 gene copy number gain (CN\u0026thinsp;=\u0026thinsp;13.34) and multiple known driver gene alterations were detected, such as EP300, KMT2B, KDM5C, ABL1, and MED12. After T-DXd treatment, the tumor underwent significant genomic remodeling, including a decrease in ploidy from 2.94 to 2.22, normalization of the ERBB2 copy number (CN\u0026thinsp;=\u0026thinsp;2.1), and clearance of the vast majority of driver gene alterations. This indicates that the tumor underwent rapid evolution under the pressure of T-DXd treatment, further implying its robust capability to eradicate tumor clones with unstable genomes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. WGS data revealed the full landscape of genomic rearrangements, including structural variations (SVs), somatic copy-number alterations (SCNAs), extrachromosomal DNA (ecDNA), and genome-wide catastrophic events (kataegis and chromothripsis). A substantial burden of SVs (Deletion: 8444, Duplication: 1800, Inversion: 92232, Insertion: 1366, Breakend: 1971) was identified in pre-treatment tumor tissues. In contrast, post-treatment samples exhibited a pronounced reduction in SVs (Deletion: 9099, Duplication: 1761, Inversion: 1051, Insertion: 1582, Breakend: 2252). Our analysis of single base substitution (SBS) mutational signatures revealed that SBS1, SBS5, and SBS46 present prior to treatment were significantly reduced post-treatment, with only a small number of SBS5 mutations remaining \u003cb\u003e(Figure S1C)\u003c/b\u003e. This suggests that tumor subclones harboring SBS1 and SBS46 were selectively eradicated through drug-mediated clearance during treatment. Furthermore, analysis of CNAs revealed a markedly higher degree of intratumoral copy number heterogeneity in pre-treatment samples compared to post-treatment samples, indicative of an effective cytotoxic response to T-DXd \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eeccDNA (extrachromosomal circular DNA) is a form of circular DNA that originates from chromosomal DNA but exists independently of it, whereas ecDNA (extrachromosomal DNA) specifically refers to larger circular DNA found in tumors, which has been further analyzed due to its reported roles in amplifying oncogenes and enabling accelerated tumor evolution during tumorigenesis and therapeutic resistance.\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Due to the absence of centromeres, ecDNA is randomly distributed during cell division, resulting in easier copy number gain or loss of ecDNA under survival stress.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Analysis of ecDNA amplification dynamics revealed that the tumor exhibited both ecDNA-mediated amplification and complex non-circular amplification events (including CDK12/ERBB2/MIEN1) prior to treatment. Following treatment, only a non-circular amplification structure retaining AGAP2, CDK4, and MDM2 genes was detected \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Notably, this structure essentially originated from the evolution of pre-existing chr12 ecDNA, and prominent chromothripsis was observed in the same genomic region both before and after treatment. Assessment of clustered alterations detected kataegis and chromothripsis \u003cb\u003e(Figure S1B,D)\u003c/b\u003e, predominantly on chromosome 12 (chr12), in both pre-treatment and post-treatment samples. A concerted reduction in the burden of kataegis and the genomic extent of chromothripsis after T-DXd treatment indicates that the underlying subclones bearing these catastrophic events were effectively eliminated, likely due to their high drug sensitivity. Transcriptomic analysis showed sustained abnormal activation of the PI3K-AKT pathway after treatment.\u003c/p\u003e \u003cp\u003e \u003cdiv description=\"Figure3\" class=\"Drawing\" id=\"3\" name=\"图片 3\"\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Tumor Microenvironment Analysis\u003c/h2\u003e \u003cp\u003eTissue samples were collected both pre- and post-treatment to analyze the effects of T-DXd. Multiplex immunofluorescence revealed a significant increase in CD8\u0026thinsp;+\u0026thinsp;T cell infiltration post-treatment. Similarly, immunohistochemistry showed a substantial upregulation of PD-1 expression in the tumor core (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,C,D). Transcriptomic immune infiltration analysis of post-treatment samples also revealed this phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, a sample from a comparable HER2 exon 20 insertion mutant patient who received conventional chemotherapy exhibited minimal CD8\u0026thinsp;+\u0026thinsp;T cell infiltration and no significant change in PD-1 expression after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,C,D). This stark difference suggests that T-DXd, unlike chemotherapy, can remodel the tumor immune microenvironment and potentially sensitize tumors to PD-1 checkpoint inhibitors. Furthermore, transcriptome analysis revealed that many effector cells (NK cells, CD4\u0026thinsp;+\u0026thinsp;T cells CD8\u0026thinsp;+\u0026thinsp;T cells and NKT cells) are infiltrated into tumor core after T-DXd treatment. Aligned with mIF results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), these results collectively indicate immune-cold tumor could turn into immune-hot tumor by T-DXd treatment. In addition, neutrophil extracellular traps (NETs) have been documented to promote tumorigenesis and metastasis and dampen immunotherapy efficacy.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e As expected, we further found neutrophils appeared to be significantly more enriched in peritumoral and distant regions instead of tumor core, reflecting the more activated immune environment and may synergize with immunotherapy. Taken together, these results suggest that T-DXd treatment can induce a highly activated TME, which may hold significant potential for improving immunotherapy outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Mechanism of adverse effects by T-DXd treatment\u003c/h2\u003e \u003cp\u003ePatients receiving T-DXd treatment are at risk of pulmonary adverse events, particularly interstitial lung disease (ILD), with a predominant incidence of interstitial pneumonitis.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e This patient also displayed a interstitial changes in right lower lobe according to CT scan after 2 cycles of T-DXd, without any symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In this regards, distal and peritumor normal lung tissues were also under further investigation using RNA-seq and pathological analysis. A study has shown that alveolar macrophages residing in the perivascular microenvironment play a key role in the development of T-DXd-related ILD, with SPP1 (osteopontin) identified as a critical mediator in this process.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Through transcriptome analysis, we found that SPP1 expression in cancer tissues was significantly higher than in the adjacent normal lung tissues, both adjacent (\u0026gt;\u0026thinsp;3 cm) and distal (\u0026gt;\u0026thinsp;15 cm) to the tumor. Pathological analysis revealed substantial infiltration of inflammatory cells in all three distinct regions(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Immune microenviroment analysis by RNA-seq further suggested that mast cell infiltration was more pronounced in the distal cancer-adjacent tissues compared to the proximal tissues, a phenomenon potentially associated with the development of ILD. Immunohistochemical results corroborated the increased mast cell population in the distal cancer-adjacent tissues(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D). These findings suggest that mast cells may contribute to the development of T-DXd-related ILD by promoting fibrosis in the distal cancer-adjacent tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eWhile T-DXd has demonstrated efficacy in advanced HER2-mutant NSCLC, its role in neoadjuvant therapy for locally advanced disease remains underexplored. To our knowledge, this is the first report to describe a case of locally advanced (cT2aN2M0 IIIA) HER2 20ins mutation (A775_G776insYVMA) carrying NSCLC patient who successfully underwent surgical resection after receiving neoadjuvant T-DXd treatment and achieving partial response. This case demonstrates that T-DXd has significant efficacy with acceptable safety. WGS results show that dominant tumor clones with highly unstable genome and enriched ecDNA were cleared. More importantly, T-DXd treatment induced significant CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration in the tumor core area and upregulated PD1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting that sequential or concurrent addition of PD-1 inhibitors may further enhance therapeutic efficacy. Recent reports have shown that T-DXd combined with PD-1 inhibitors successfully induced pathological complete response (pCR) and achieved R0 resection in stage IIIB HER2-mutant NSCLC patients (PD-L1 50%).\u003c/p\u003e \u003cp\u003eAlthough the patient's tumor exhibited a typical \"cold\" immune phenotype prior to treatment, neoadjuvant T-DXd therapy significantly remodeled the tumor microenvironment, leading to enhanced infiltration of CD8\u0026thinsp;+\u0026thinsp;T cells. Furthermore, multiplex immunofluorescence analysis revealed that PD-1 expression followed a pattern similar to that of CD8\u0026thinsp;+\u0026thinsp;T cell infiltration, with both showing significant upregulation after treatment. These findings suggest that T-DXd may exert its antitumor effects by remodeling the immune microenvironment, thereby providing a rationale for its combination with PD-1 inhibitors.\u003c/p\u003e \u003cp\u003eDespite its significant efficacy, T-DXd therapy increases the risk of interstitial lung disease (ILD), which is notably higher in lung cancer patients compared to those with breast or gastric cancer.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e The development of T-DXd-related ILD/pneumonitis may be attributed to HER2 protein expression in the bronchial and bronchiolar epithelium of the lung or a direct cytotoxic effect of T-DXd on lung tissue.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Prior studies using mouse models have revealed that alveolar macrophages (AMs) serve as the primary target cells of T-DXd in the pulmonary microenvironment and contribute to the development of T-DXd-related interstitial lung disease.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e RNA-seq analysis and immunohistochemical profiling of CD117 revealed a higher level of mast cell infiltration in distal lung tissue (\u0026gt;\u0026thinsp;15 cm) compared to adjacent lung tissue (\u0026gt;\u0026thinsp;3 cm)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,C). This spatial distribution suggests a potential role for mast cells in ILD pathogenesis. Recent studies have indicated that mast cells, as a potential source of fibrotic factors such as TGF-β, play an important role in the fibrosis process. The tryptase they secrete can stimulate the proliferation of pulmonary fibroblasts and enhance extracellular matrix (ECM) synthesis, thereby promoting the pathological progression of fibrosis.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Pathological examination of resected specimens revealed a pro-fibrotic response, characterized by significant chronic inflammatory cell infiltration, multinucleated giant cell reactions, and calcified fibrous proliferative changes in adjacent lung tissue. These findings suggest that T-DXd can induce fibrotic responses, underscoring the need for heightened vigilance regarding ILD during treatment and surgery.\u003c/p\u003e \u003cp\u003eThe primary limitation of this study is its single-case design. While providing valuable mechanistic insights, the findings require validation in larger prospective cohorts to determine the generalizability of the immune responses observed. Our findings specifically support trials investigating combination therapy with PD-1 inhibitors to enhance efficacy. Additionally, the identified fibrotic response and mast cell infiltration highlight the need for translational research focused on predicting and preventing T-DXd-related ILD.\u003c/p\u003e \u003cp\u003eIn conclusion, this report highlights the potent efficacy and manageable toxicity of T-DXd in the neoadjuvant treatment of HER2-mutant NSCLC, while also demonstrating its immune-modulating effects. Additionally, mast cell-mediated fibrotic responses were identified as a potential mechanism for T-DXd-related ILD, providing a foundation for future studies aimed at optimizing the therapeutic potential of T-DXd and mitigating its associated risks.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSCLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-small cell lung cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT-DXd\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrastuzumab Deruxtecan\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewhole-genome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eecDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextrachromosomal DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eeccDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextrachromosomal circluar DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHER2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Epidermal Growth Factor Receptor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eERBB2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpidermal Growth Factor Receptor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eantibody drug conjugate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emPFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emedian progression-free survival\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFood and Drug Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNA-seq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eORR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOverall Response Rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFFPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eformalin-fixed paraffin-embedded\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCNVs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCopy number variations\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSVs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStructural variants\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eILD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterstitial lung disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProgrammed Cell Death Protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBFB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebreakage\u0026ndash;fusion\u0026ndash;bridge\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the following research grants: the Talent Innovation Capacity Building Program of Army Medical Center (Grant No. ZXYZZKY07); the National Natural Science Foundation of China (NSFC) (Grant No. 82472748); and the Science-Health Joint Research Project of Chongqing (Grant No. 2024MSXM035).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.L. and M.L.: conceptualization, methodology, investigation, validation, formal analysis, data curation, and writing \u0026ndash; original draft. X.L.: methodology, investigation, formal analysis, and data curation. Y.X. and M.L.: conceptualization, resources, supervision, funding acquisition. Y.X., X.L., S.Y., Z.H., and M.L.: writing \u0026ndash; review \u0026amp; editing. R.H.: formal analysis. X.D. and X.K.: methodology and validation. W.G.: investigation and resources. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1426580. The raw sequencing data can be accessed via the following SRA run accessions: SRR37420559\u0026ndash;SRR37420570. The corresponding BioSample accessions are SAMN55876066\u0026ndash;SAMN55876074.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from the Institutional Review Board of the Army Medical Center of the PLA, Army Medical University. Written informed consent was obtained from all participating patients. Clinical trial registration number: not applicable. All authors have reviewed and approved the final version of the manuscript. The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eReinhorn, D., Moskovitz, M., Tap, W. D. \u0026amp; Li, B. T. 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Biol.\u003c/em\u003e \u003cb\u003e2117\u003c/b\u003e, 135\u0026ndash;157 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HER2 (ERBB2), NSCLC, Deruxtecan (T-DXd, DS-8201), Genomic instability, Immune microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-8854444/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8854444/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNon-small cell lung cancer (NSCLC) patients carrying HER2 20ins exon 20 insertion (HER2 20ins) are poorly respond to conventional therapies and pan HER inhibitors. Trastuzumab Deruxtecan (T-DXd) has been approved in second line treatment in advanced NSCLC, but its efficacy in operable patients remains unclear. Here, we report a case of a patient with operable, HER2 20ins NSCLC who received neoadjuvant T-DXd. We utilized whole-genome sequencing (WGS) and immune microenvironment analysis to investigate the treatment's impact. WGS analysis revealed significant reduction in tumor clones with genome instability and extrachromosomal DNA (ecDNA) following T-DXd treatment. Meanwhile, immune profiling demonstrated substantially increased CD8\u0026thinsp;+\u0026thinsp;T-cell infiltration in tumor cores with elevated PD1 expression. In contrast, conventional chemotherapy did not significantly alter CD8\u0026thinsp;+\u0026thinsp;T cell infiltration or PD1 expression in another matched HER2 20ins patient. These findings suggest that neoadjuvant T-DXd may offer a well-tolerated and efficient therapeutic strategy for locally advanced NSCLC with HER2 20ins mutations.\u003c/p\u003e","manuscriptTitle":"Tumor Genome and Microenvironment Alteration by Trastuzumab Deruxtecan as Neoadjuvant Therapy for HER2 Mutant NSCLC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-22 08:55:30","doi":"10.21203/rs.3.rs-8854444/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T07:23:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T17:31:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T20:01:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47285266620766681148853425198082781092","date":"2026-04-16T12:39:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74075073468139762602128306618730176635","date":"2026-04-16T00:18:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-14T02:22:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T14:56:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-04T10:50:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-02T21:49:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-02T13:48:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e9be7f9-0403-4f8d-bafe-e8c26c289579","owner":[],"postedDate":"April 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66474817,"name":"Biological sciences/Cancer"},{"id":66474818,"name":"Biological sciences/Immunology"},{"id":66474819,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-14T06:24:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-22 08:55:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8854444","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8854444","identity":"rs-8854444","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}