BACKGROUND: Profiling studies in small-cell lung cancer (SCLC) have mainly focused on primary tumors, omitting the potential molecular changes that might occur during lymphatic metastasis formation. Here, we assessed the molecular discordance between primary SCLCs and corresponding lymph node (LN) metastases in the light of subtype distribution and expression of clinically relevant proteins. METHODS: Comparative profiling of 32 surgically resected primary SCLCs and their LN metastases was achieved by RNA expression analysis and immunohistochemistry (IHC). In addition to subtype markers (ASCL1, NEUROD1, POU2F3, and YAP1), the expression of nine cancer-specific proteins was evaluated. RESULTS: The selected clinically relevant molecules showed no significant differences in their RNA expression profile when assessing the primary tumors and their corresponding LN metastases. Nevertheless, IHC analyses revealed significantly higher DLL3 expression in the primary tumors than in the LN metastases (P = 0.008). In contrast, NEUROD1 expression was significantly lower in the primary tumors (versus LN metastases, P < 0.001). No statistically significant difference was found by IHC analysis in the case of other clinically relevant proteins. Concerning SCLC molecular subtypes, a change in subtype distribution was detected in 21 cases. Phenotype switching from neuroendocrine (NE) subtypes toward non-NE lesions and from non-NE landscape toward NE subtypes were both detected. CONCLUSIONS: Although the molecular landscape of SCLC LN metastases largely resembles that of the tumor of origin, key differences exist in terms of DLL3 and NEUROD1 expression, and in subtype distribution. These diagnostic pitfalls should be considered when establishing the tumors' molecular profile for future clinical trials solely based on LN biopsies.

Department of Pathology and Experimental Cancer Research Budapest Hungary

Department of Pathology University Hospital Ostrava Ostrava Czech Republic; Faculty of Medicine University of Ostrava Ostrava Czech Republic

Department of Physics of Complex Systems Eotvos Lorand University Budapest Hungary

Department of Thoracic Surgery Comprehensive Cancer Center Medical University of Vienna Vienna Austria

Department of Thoracic Surgery Comprehensive Cancer Center Medical University of Vienna Vienna Austria; Division of Pulmonology Department of Medicine 2 Medical University of Vienna Vienna Austria

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary; Department of Obstetrics and Gynecology South Buda Central Hospital Saint Emeric University Teaching Hospital Budapest Hungary

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary; Department of Thoracic and Abdominal Tumors and Clinical Pharmacology National Institute of Oncology Budapest Hungary

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary; Multidisciplinary Centre of Head and Neck Tumors National Institute of Oncology Budapest Hungary

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary; National Koranyi Institute of Pulmonology Budapest Hungary

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary; National Koranyi Institute of Pulmonology Budapest Hungary; Department of Thoracic Surgery Comprehensive Cancer Center Medical University of Vienna Vienna Austria

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary; National Koranyi Institute of Pulmonology Budapest Hungary; Department of Thoracic Surgery Comprehensive Cancer Center Medical University of Vienna Vienna Austria; Department of Translational Medicine Lund University Lund Sweden

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary; National Koranyi Institute of Pulmonology Budapest Hungary; National Institute of Oncology and National Tumor Biology Laboratory Budapest Hungary

Institute of Clinical and Molecular Pathology Medical Faculty Palacký University Olomouc Olomouc Czech Republic; Department of Pathology University Hospital Ostrava Ostrava Czech Republic; Faculty of Medicine University of Ostrava Ostrava Czech Republic

National Koranyi Institute of Pulmonology Budapest Hungary

National Koranyi Institute of Pulmonology Budapest Hungary; Department of Thoracic Surgery Comprehensive Cancer Center Medical University of Vienna Vienna Austria

Torokbalint County Institute of Pulmonology Torokbalint Hungary

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Siegel R.L., Miller K.D., Fuchs H.E., Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72:7–33. PubMed

Gazdar A.F., Bunn P.A., Minna J.D. Small-cell lung cancer: what we know, what we need to know and the path forward. Nat Rev Cancer. 2017;17(12):725–737. PubMed

George J., Lim J.S., Jang S.J., et al. Comprehensive genomic profiles of small cell lung cancer. Nature. 2015;524:47–53. PubMed PMC

Schwendenwein A., Megyesfalvi Z., Barany N., et al. Molecular profiles of small cell lung cancer subtypes: therapeutic implications. Mol Ther Oncolytics. 2021;20:470–483. PubMed PMC

Khuder S.A. Effect of cigarette smoking on major histological types of lung cancer: a meta-analysis. Lung Cancer. 2001;31(2-3):139–148. PubMed

Pleasance E.D., Stephens P.J., O'Meara S., et al. A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature. 2010;463(7278):184–190. PubMed PMC

Megyesfalvi Z., Bárány N., Valkó Z., et al. [Heterogeneity of small cell lung cancer: biological and clinicopathological implications] Magy Onkol. 2020;64(3):243–255. PubMed

Drapkin B.J., Rudin C.M. Advances in small-cell lung cancer (SCLC) translational research. Cold Spring Harb Perspect Med. 2021;11(4):a038240. PubMed PMC

Chan J.M., Quintanal-Villalonga Á., Gao V.R., et al. Signatures of plasticity, metastasis, and immunosuppression in an atlas of human small cell lung cancer. Cancer Cell. 2021;39:1479–1496.e18. PubMed PMC

Evans W.K., Shepherd F.A., Feld R., Osoba D., Dang P., Deboer G. VP-16 and cisplatin as first-line therapy for small-cell lung cancer. J Clin Oncol. 1985;3:1471–1477. PubMed

Paz-Ares L., Dvorkin M., Chen Y., et al. Durvalumab plus platinum-etoposide versus platinum-etoposide in first-line treatment of extensive-stage small-cell lung cancer (CASPIAN): a randomised, controlled, open-label, phase 3 trial. Lancet. 2019;394(10212):1929–1939. PubMed

Ragavan M., Das M. Systemic therapy of extensive stage small cell lung cancer in the era of immunotherapy. Curr Treat Options Oncol. 2020;21(8):64. PubMed

Horn L., Mansfield A.S., Szczęsna A., et al. First-line atezolizumab plus chemotherapy in extensive-stage small-cell lung cancer. N Engl J Med. 2018;379(23):2220–2229. PubMed

Megyesfalvi Z., Gay C.M., Popper H., et al. Clinical insights into small cell lung cancer: tumor heterogeneity, diagnosis, therapy, and future directions. CA Cancer J Clin. 2023;73(6):620–652. PubMed

Rudin C.M., Poirier J.T., Byers L.A., et al. Molecular subtypes of small cell lung cancer: a synthesis of human and mouse model data. Nat Rev Cancer. 2019;19(5):289–297. PubMed PMC

Gay C.M., Stewart C.A., Park E.M., et al. Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities. Cancer Cell. 2021;39(3):346–360.e7. PubMed PMC

Baine M.K., Hsieh M.S., Lai W.V., et al. SCLC subtypes defined by ASCL1, NEUROD1, POU2F3, and YAP1: a comprehensive immunohistochemical and histopathologic characterization. J Thorac Oncol. 2020;15(12):1823–1835. PubMed PMC

Megyesfalvi Z., Barany N., Lantos A., et al. Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study. J Pathol. 2022;257(5):674–686. PubMed PMC

Qi J., Zhang J., Liu N., Zhao L., Xu B. Prognostic implications of molecular subtypes in primary small cell lung cancer and their correlation with cancer immunity. Front Oncol. 2022;12 PubMed PMC

Furuta M., Sakakibara-Konishi J., Kikuchi H., et al. Analysis of DLL3 and ASCL1 in surgically resected small cell lung cancer (HOT1702) Oncologist. 2019;24(11):e1172–e1179. PubMed PMC

Kosari F., Ida C.M., Aubry M.C., et al. ASCL1 and RET expression defines a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation. Oncogene. 2014;33(29):3776–3783. PubMed PMC

Huang Y.H., Klingbeil O., He X.Y., et al. POU2F3 is a master regulator of a tuft cell-like variant of small cell lung cancer. Genes Dev. 2018;32(13-14):915–928. PubMed PMC

Lohinai Z., Megyesfalvi Z., Suda K., et al. Comparative expression analysis in small cell lung carcinoma reveals neuroendocrine pattern change in primary tumor versus lymph node metastases. Transl Lung Cancer Res. 2019;8(6):938–950. PubMed PMC

Stewart C.A., Gay C.M., Xi Y., et al. Single-cell analyses reveal increased intratumoral heterogeneity after the onset of therapy resistance in small-cell lung cancer. Nat Cancer. 2020;1:423–436. PubMed PMC

Ganti A.K.P., Loo B.W., Bassetti M., et al. Small Cell Lung Cancer, Version 2.2022, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2021;19(12):1441–1464. PubMed PMC

Battifora H. The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest. 1986;55(2):244–248. PubMed

Thul P.J., Lindskog C. The human protein atlas: a spatial map of the human proteome. Protein Sci. 2018;27(1):233–244. PubMed PMC

Grillo F., Bruzzone M., Pigozzi S., et al. Immunohistochemistry on old archival paraffin blocks: is there an expiry date? J Clin Pathol. 2017;70:988–993. PubMed

Kim S.W., Roh J., Park C.S. Immunohistochemistry for pathologists: protocols, pitfalls, and tips. J Pathol Transl Med. 2016;50:411–418. PubMed PMC

Früh M., De Ruysscher D., Popat S., Crinò L., Peters S., Felip E., ESMO Guidelines Working Group Small-cell lung cancer (SCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2013;24(suppl 6):vi99–vi105. PubMed

Zhu Y., Ren W., Li S., et al. Heterogeneity of molecular subtyping and therapy-related marker expression in primary tumors and paired lymph node metastases of small cell lung cancer. Virchows Arch. 2025;486:243–255. PubMed

Nesline M.K., Previs R.A., Dy G.K., et al. PD-L1 expression by RNA-sequencing in non-small cell lung cancer: concordance with immunohistochemistry and associations with pembrolizumab treatment outcomes. Cancers (Basel) 2023;15(19):4789. PubMed PMC

Bergethon K., Shaw A.T., Ou S.H., et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol. 2012;30:863–870. PubMed PMC

Takeuchi K., Soda M., Togashi Y., et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med. 2012;18:378–381. PubMed

Davies K.D., Le A.T., Theodoro M.F., et al. Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res. 2012;18(17):4570–4579. PubMed PMC

Sorokin M., Ignatev K., Poddubskaya E., et al. RNA sequencing in comparison to immunohistochemistry for measuring cancer biomarkers in breast cancer and lung cancer specimens. Biomedicines. 2020;8(5):114. PubMed PMC

Saunders L.R., Bankovich A.J., Anderson W.C., et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med. 2015;7(302) PubMed PMC

Solta A., Ernhofer B., Boettiger K., et al. Small cells - big issues: biological implications and preclinical advancements in small cell lung cancer. Mol Cancer. 2024;23(1):41. PubMed PMC

Huang J., Cao D., Sha J., Zhu X., Han S. DLL3 is regulated by LIN28B and miR-518d-5p and regulates cell proliferation, migration and chemotherapy response in advanced small cell lung cancer. Biochem Biophys Res Commun. 2019;514(3):853–860. PubMed

Zhang Y., Tacheva-Grigorova S.K., Sutton J., et al. Allogeneic CAR T cells targeting DLL3 are efficacious and safe in preclinical models of small cell lung cancer. Clin Cancer Res. 2023;29(5):971–985. PubMed

US FDA . News Release; 2024. FDA Grants Accelerated Approval to Tarlatamab-dlle for Extensive Stage Small Cell Lung Cancer.https://tinyurl.com/bddwjpk2 Available at.

Ahn M.J., Cho B.C., Felip E., et al. Tarlatamab for patients with previously treated small-cell lung cancer. N Engl J Med. 2023;389(22):2063–2075. PubMed

Gong J., Salgia R. Managing patients with relapsed small-cell lung cancer. J Oncol Pract. 2018;14(6):359–366. PubMed PMC

Morgensztern D., Besse B., Greillier L., et al. Efficacy and safety of rovalpituzumab tesirine in third-line and beyond patients with DLL3-expressing, relapsed/refractory small-cell lung cancer: results from the phase II TRINITY study. Clin Cancer Res. 2019;25(23):6958–6966. PubMed PMC

Yang S., Zhang Z., Wang Q. Emerging therapies for small cell lung cancer. J Hematol Oncol. 2019;12(1):47. PubMed PMC

Park S., Hong T.H., Hwang S., et al. Comprehensive analysis of transcription factor-based molecular subtypes and their correlation to clinical outcomes in small-cell lung cancer. eBioMedicine. 2024;102 PubMed PMC

Chiang C.L., Huang H.C., Luo Y.H., et al. Clinical utility of immunohistochemical subtyping in patients with small cell lung cancer. Lung Cancer. 2024;188 PubMed

Caeser R., Egger J.V., Chavan S., et al. Genomic and transcriptomic analysis of a library of small cell lung cancer patient-derived xenografts. Nat Commun. 2022;13(1):2144. PubMed PMC

Chen P., Sun C., Wang H., et al. YAP1 expression is associated with survival and immunosuppression in small cell lung cancer. Cell Death Dis. 2023;14(9):636. PubMed PMC

Redin E., Quintanal-Villalonga Á., Rudin C.M. Small cell lung cancer profiling: an updated synthesis of subtypes, vulnerabilities, and plasticity. Trends Cancer. 2024;10:935–946. PubMed

Ng J., Cai L., Girard L., et al. Molecular and pathologic characterization of YAP1-expressing small cell lung cancer cell lines leads to reclassification as SMARCA4-deficient malignancies. Clin Cancer Res. 2024;30:1846–1858. PubMed PMC

Borromeo M.D., Savage T.K., Kollipara R.K., et al. ASCL1 and NEUROD1 reveal heterogeneity in pulmonary neuroendocrine tumors and regulate distinct genetic programs. Cell Rep. 2016;16:1259–1272. PubMed PMC

Mollaoglu G., Guthrie M.R., Böhm S., et al. MYC drives progression of small cell lung cancer to a variant neuroendocrine subtype with vulnerability to Aurora kinase inhibition. Cancer Cell. 2017;31(2):270–285. PubMed PMC

Lim J.S., Ibaseta A., Fischer M.M., et al. Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer. Nature. 2017;545:360–364. PubMed PMC

Meder L., König K., Ozretić L., et al. NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas. Int J Cancer. 2016;138(4):927–938. PubMed PMC