Pathological and molecular characterization of a four-component combined small cell lung carcinoma: a case report

Introduction

According to the World Health Organization Classification of Tumors of the Lung [2021], combined small cell lung carcinoma (cSCLC) is defined as a small cell lung carcinoma (SCLC) containing one or more non-small cell lung cancer (NSCLC) components, such as adenocarcinoma (AD), squamous cell carcinoma (SQ), pleomorphic carcinoma (PPC), or large cell carcinoma (1). The reported incidence of cSCLC ranges from approximately 5% to 30% of all SCLC cases (2-4), although the exact rate varies depending on sampling methods and diagnostic criteria.

Most cases of cSCLC consist of SCLC combined with a single NSCLC subtype—typically AD or SQ—and have been relatively well characterized in the literature. In contrast, cSCLC containing multiple NSCLC components is exceedingly rare, with only a few reported cases involving three or more distinct histologic subtypes to date (5-8). Consequently, the molecular characteristics and clonal relationships among the diverse components of such complex tumors remain largely unexplored.

Here, we describe a surgically resected case of cSCLC composed of four distinct histologic components—SCLC, SQ, PPC, and AD. We performed comprehensive pathological and molecular analyses, including RNA sequencing, to elucidate the molecular background and potential lineage relationships among the different tumor components. To the best of our knowledge, this is the first documented case of cSCLC with four histologic subtypes to undergo detailed molecular characterization, providing novel insights into intratumoral heterogeneity and the possible mechanisms underlying tumor differentiation in cSCLC. We present this article in accordance with the CARE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-1-0144/rc).

Case presentation

A 70-year-old man presented to a local hospital in July 2022 with right shoulder pain. He had a 13-pack-year smoking history, and his past medical history included hypertension and gastritis. A chest X-ray and contrast-enhanced computed tomography (CT) of the chest revealed a soft tissue mass measuring 43 mm in the upper lobe of the right lung (Figure 1A,1B). Lung cancer was suspected, and he was referred to our department for further evaluation. ¹⁸F-fluorodeoxyglucose positron emission tomography/CT showed markedly increased uptake in the S1 nodule, with a maximum standardized uptake value (SUVmax) of 38 (Figure 1C). Serum tumor markers were elevated: carcinoembryonic antigen, 5.8 ng/mL; cytokeratin (CK) 19 fragment, 5.0 ng/mL; SQ antigen, 2.4 ng/mL; and pro-gastrin-releasing peptide, 87.2 pg/mL. Histopathological examination of bronchoscopic biopsy specimens suggested NSCLC. Based on these findings, a clinical diagnosis of stage IIIA NSCLC (cT3N0M0) was made. The patient subsequently underwent right upper lobectomy with en bloc resection of the chest wall and regional lymph node dissection.

Figure 1 Initial imaging findings. (A) Chest X-ray showing a mass shadow in the right lung. (B) Contrast-enhanced CT scan demonstrating a 43-mm subpleural tumor in the right upper lobe. (C) FDG-PET image showing intense uptake by the tumor, with a maximum standardized uptake value of 38. CT, computed tomography; FDG-PET, fluorodeoxyglucose positron emission tomography.

Histopathological examination of the surgically resected specimen revealed four distinct neoplastic components. The predominant component was SQ (approximately 50% of the tumor), characterized by sheet-like aggregates of neoplastic cells and positive immunostaining for p40 (Figure 2A). The second component was PPC (approximately 30%), composed of spindle-shaped and multinucleated giant cells (Figure 2B), which were positive for CK AE1/AE3 and negative for thyroid transcription factor-1 (TTF-1) (data not shown). The third component consisted of AD (approximately 15%) with atypical columnar cells with enlarged oval nuclei, forming complex, branching/anastomosing glands and exhibiting lepidic growth, and displaying positive staining for TTF-1 (Figure 2C). The fourth and final component was SCLC (approximately 5%), consisting of small round to oval cells with finely granular chromatin and positive immunostaining for synaptophysin (SYP; Figure 2D) and chromograninA (CHGA; data not shown). The tumor cells were negative for p40 and showed focal positivity for TTF-1, without evidence of keratinization or intercellular bridges. The final pathological diagnosis was cSCLC containing SQ, AD, and PPC components, corresponding to pT3N0M0, pathological stage IIIA. The postoperative course was uneventful, and the patient did not receive adjuvant chemotherapy. He has remained recurrence-free for more than three years after surgery. The clinical course of the patient is summarized in Table 1.

Figure 2 Representative histopathological and immunohistochemical findings. Hematoxylin and eosin staining of (A) squamous cell carcinoma, (B) pleomorphic carcinoma, (C) adenocarcinoma, and (D) small cell carcinoma (magnification, approximately ×200; scale bar: 100 µm). Small insets in the lower right of each panel show immunohistochemical staining for (A) p40, (C) TTF-1, and (D) synaptophysin (magnification, approximately ×200; scale bar: 100 µm). TTF-1, thyroid transcription factor-1.

To further characterize the biological features and clarify the molecular heterogeneity of this complex tumor, we performed comprehensive pathological and molecular analyses of each histological component. Each histological component was carefully identified by experienced pathologists based on morphological features. For molecular analyses, spatially distinct tumor regions corresponding to each component were selectively sampled using manual macrodissection with an adequate margin to minimize cross-contamination between adjacent components. Although the SCLC component accounted for a relatively small proportion of the tumor, it formed a morphologically and spatially recognizable area. As shown in the gross photograph (Figure S1), this component was visually identifiable, which facilitated targeted sampling. The methodological details are provided in the Appendix 1. Pathogenic or potentially pathogenic single-nucleotide variants (SNVs) and insertions/deletions (indels) detected across the four components are summarized in an Oncoprint (Figure 3A). A TP53 nonsense mutation was observed in all components, whereas other mutations were uniquely detected in individual components. Notably, no pathogenic or potentially pathogenic SNVs or indels, apart from TP53, were identified in the AD component.

Figure 3 Genomic and transcriptomic characterization of the four histological tumor components. (A) Oncoprint summarizing pathogenic or potentially pathogenic SNVs and indels detected across the four histological components—AD, SQ, SCLC, and PPC. Each row corresponds to a gene, and each column represents a histological component, with colored boxes indicating the presence of specific mutations. (B) Clonal phylogenetic tree inferred from tumor mutation profiles. Each node denotes a clone or subclone, with gene symbols indicating pathogenic or potentially pathogenic mutations present within each clone. Branch lengths reflect the number of mutations separating clones. (C) Radar plot depicting the mRNA expression profiles of commonly used biomarkers for histological classification of lung cancer across the four components—LUAD, LUSC, SCLC, and PPC. Each axis represents an individual biomarker, and relative expression levels are shown for each component. (D) Heatmap illustrating pathway and metabolic signature scores across the four histological components—AD, SQ, SCLC, and PPC—calculated using the Hallmark gene sets from the Molecular Signatures Database (MSigDB). Each row represents a pathway signature, and each column denotes a histological component. Color intensity reflects the relative enrichment score of each signature. AD, adenocarcinoma; GL, germline; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PPC, pleomorphic carcinoma; SCLC, small cell lung cancer; SNV, single-nucleotide variant; SQ, squamous cell carcinoma; TMB, tumor mutational burden.

Using the variant allele frequency profiles of somatic alterations, including those classified as pathogenic or potentially pathogenic, we inferred a clonal phylogenetic tree using LICHeE (Figure 3B). This analysis identified a putative founding clone shared among all components, from which multiple subclones diverged, forming a hierarchical branching pattern. Each histological component—AD, SQ, SCLC, and PPC—corresponded to a distinct subclone, which is consistent with a multilineage evolutionary process within the tumor. Specifically, AD and SCLC were derived directly from the founding clone, whereas SQ and PPC arose via intermediate subclones. Most pathogenic mutations identified in the Oncoprint were localized within the subclones corresponding to each component, confirming that the evolutionary branching was associated with the acquisition of component-specific molecular alterations.

Using RNA sequencing (RNA-seq) data, we evaluated the mRNA expression levels of commonly used biomarkers for histological classification of lung cancer. In the AD component, the AD marker NAPSA showed the highest expression, followed by elevated TTF-1. In the SQ component, squamous markers TP63, KRT5, and KRT6A were markedly upregulated. The SCLC component exhibited high expression of neuroendocrine markers, including CHGA, SYP, and NCAM1, as well as elevated TTF-1. In contrast, the PPC component did not display a lineage-specific expression pattern; although TP63 and NAPSA were expressed at relatively high levels, SYP was also moderately expressed (Figure 3C). Comprehensive transcriptomic analyses, including mutation profiling, phylogenetic analysis, biomarker expression, and pathway enrichment, are summarized in Figure 3. Principal component analysis revealed that AD and SCLC were transcriptionally similar, whereas SQ and PPC displayed expression profiles that were markedly distinct from those of the other components (Figure 4A).

Figure 4 Transcriptomic and tumor microenvironment analyses. (A) PCA plot illustrating the transcriptional profiles of the four histological tumor components—AD, SQ, SCLC, and PPC—based on the expression of commonly used biomarkers for histological classification. Each point represents one tumor component, and spatial separation indicates transcriptional similarity or dissimilarity among components. (B) Bar plot showing the estimated proportions of immune cells within each histological component—AD, SQ, SCLC, and PPC—as inferred from TME deconvolution. Each bar represents the relative abundance of individual immune cell types within the respective component. AD, adenocarcinoma; NK, natural killer; PC, principal component; PCA, principal component analysis; PPC, pleomorphic carcinoma; SCLC, small cell lung cancer; SQ, squamous cell carcinoma; TME, tumor microenvironment.

Tumor microenvironment (TME) deconvolution based on RNA-seq data indicated a higher proportion of immune cells in AD and PPC, predominantly lymphocytes. Notably, PPC exhibited a particularly high proportion of CD8-positive T cells (Figure 4B). Finally, pathway and metabolic signature analyses demonstrated distinct transcriptomic profiles across the components. In SCLC, cell cycle- and proliferation-related pathways, including mitotic spindle, E2F targets, and G2M checkpoint, were markedly enriched. In SQ, numerous oncogenic pathways, such as MYC, Wnt/β-catenin, and p53, were highly scored, along with microenvironment-associated pathways including hypoxia and angiogenesis. AD exhibited comparatively modest pathway differences, although the PI3K/AKT/mTOR signaling pathway was relatively enriched. PPC demonstrated prominent inflammatory-related pathways, including interferon signaling, IL-6/JAK/STAT3, and other inflammation-associated signatures, relative to the other components (Figure 3D).

All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board of Oji General Hospital. Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Discussion

To the best of our knowledge, this is the first documented case of cSCLC containing three NSCLC components—SQ, PPC, and AD—analyzed through comprehensive pathological and molecular approaches. Nicholson et al. reported NSCLC components in 28 of 100 resected SCLC cases, with 57% classified as large cell carcinoma, 32% as AD, and 11% as SQ (2). Li et al. identified 223 cSCLC cases among 580 postoperative SCLC cases, with large cell neuroendocrine carcinoma being the most frequent NSCLC component (67%), followed by AD (15%), SQ (13%), and others, including giant or spindle cell carcinoma (1.7%). PPC has been reported only rarely (4,6). Cases of cSCLC containing two NSCLC components are uncommon (5-8), and the coexistence of three distinct components, as in our case, is exceedingly rare.

Prognostically, cSCLC demonstrates outcomes comparable to or worse than those of pure SCLC (2,3,9,10). Babakoohi et al. reported longer overall survival (OS) in cSCLC, including resected patients, compared with SCLC (15 vs. 11 months; P=0.035) (3). He et al. observed an OS of 18 months in stage IA–IB cSCLC compared with 13 months for SCLC (P<0.01) (9). In advanced disease, response rates to carboplatin/cisplatin plus etoposide ranged from 39% to 53% (11,12), suggesting limited efficacy of systemic chemotherapy and a potential survival benefit from surgical resection. Furthermore, molecular profiling carries important clinical implications in cSCLC. Epidermal growth factor receptor (EGFR) mutations have been detected in a small subset of cases—2 of 107 SCLC (4%) and 3 of 15 cSCLC cases (13). Onishi et al. reported a case of EGFR-mutant cSCLC with a squamous component that partially responded to gefitinib (14). These findings underscore the importance of molecular testing in cSCLC, as targeted therapies, such as EGFR tyrosine kinase inhibitors, may represent potential treatment options in EGFR-mutant cases.

The molecular mechanisms underlying the heterogeneity of cSCLC remain incompletely understood. Zhao et al. hypothesized that multiple subclones derived from tumor stem cells may acquire distinct mutations under the influence of the TME, suggesting that different histological components could originate from a common stem cell source (10). Achaete-scute homolog 1 (ASCL1), a basic helix–loop–helix transcription factor, is essential for SCLC initiation and regulates neuroendocrine differentiation and proliferation (15-17). Previous studies have demonstrated shared TP53 and retinoblastoma1 (RB1) mutations between NSCLC and SCLC components, implicating ASCL1 in cSCLC heterogeneity, with lower ASCL1 expression in NSCLC components potentially influencing morphological evolution (18). In the present case, four histological components—AD, SQ, SCLC, and PPC—coexisted within a single tumor, each exhibiting classical histopathological features consistent with RNA-seq marker profiles. Signature analyses revealed component-specific pathway activation: the SCLC component showed elevated cell cycle and proliferation signatures; the AD component exhibited relatively high PI3K/AKT/mTOR pathway activity; the SQ component demonstrated activation of multiple oncogenic pathways, including Notch and p53; and the PPC component displayed prominent inflammatory signatures consistent with the lymphocyte infiltration observed in TME deconvolution. Notably, the IL-6/JAK/STAT3 signaling pathway, known to regulate both inflammation and tumor cell proliferation (19), and the IL-2/STAT5 pathway, associated with T-cell and natural killer cell activation (20), were relatively enriched in the PPC component, suggesting that these pathways may contribute to both immune modulation and tumor progression in this component. These transcriptional features reflect lineage-associated programs—neuroendocrine differentiation in SCLC, squamous differentiation in SQ, epithelial/glandular characteristics in AD, and an inflammatory or less lineage-restricted state in PPC—thereby suggesting that the observed histological heterogeneity may reflect functionally differentiated tumor states, rather than simply representing a coexistence of independent components. In the context of recently proposed molecular subtypes of SCLC, including ASCL1, NEUROD1, POU2F3, and YAP1 associated subtypes, the transcriptional features of the SCLC component in this case may be broadly consistent with established neuroendocrine subtypes. However, precise subtype classification would require further marker-based analysis.

Clonal phylogenetic analysis indicated that all four components were derived from a common ancestral clone, with AD and SCLC arising directly and SQ and PPC developing through intermediate subclones. This phylogenetic structure is suggestive of a non-random, hierarchically organized evolutionary trajectory, rather than purely parallel or stochastic divergence of independent subclones. Integration of phylogenetic relationships with transcriptional profiles further suggests that lineage divergence may be associated with functional specialization. In particular, the phylogenetic relationships, together with the transcriptional differences among components, are consistent with a structured evolutionary process originating from a common ancestral clone. This interpretation may provide a perspective that is a partially distinct from the well-recognized plasticity of SCLC. This distinction is important, as it raises the possibility that the observed heterogeneity may not be fully explained by previously described plasticity alone, but instead may reflect a structured and hierarchically organized evolutionary process. While transdifferentiation between neuroendocrine and non-neuroendocrine states has often been described as dynamic and reversible, the present case may suggest that, in certain contexts, lineage diversification could proceed along a branching evolutionary trajectory associated with relatively stable transcriptional programs. This case offers potential insight into how multi-lineage differentiation may arise from a single clone, suggesting coordinated lineage diversification, potentially influenced by microenvironmental factors. Whole-exome sequencing did not identify known driver mutations, including EGFR, KRAS, ALK, ROS1, BRAF, MET, RET, and ERBB2, and no RB1 mutations were detected. However, RB1 alterations in SCLC may include copy number loss or structural variants that are not always captured by standard mutation analysis and therefore, functional inactivation of the RB pathway may not be fully excluded. Consistent with this, previous studies have reported an association of RB pathway disruption and YAP1 suppression with neuroendocrine differentiation in SCLC (21).

The transcriptional similarity between AD and SCLC components, contrasted with the divergence of SQ and PPC, is consistent with a non-random evolutionary trajectory characterized by biased lineage differentiation. This pattern suggests that transcriptional states may reflect lineage relationships, rather than being solely attributable to independent phenotypic shifts. These observations allow us to propose a possible model in which intrinsic transcriptional programs interact with microenvironmental cues to guide lineage-specific differentiation within a single tumor. In this context, the PPC component may represent a lineage state potentially influenced by microenvironmental influences rather than a purely genetically defined lineage. These observations indicate that tumor evolution in this case may have, at least in part, proceeded along distinct differentiation pathways originating from a shared ancestral clone. These functional differences appear to be aligned with the underlying phylogenetic structure, supporting the notion that lineage divergence may be associated with functional specialization. Overall, this case demonstrates the evolutionary and molecular heterogeneity of cSCLC, illustrating how multiple histological components can emerge from a shared ancestral clone through structured lineage diversification, with potential implications for differential treatment responses.

This study has several limitations. As a single-case analysis, the generalizability of the findings is inherently limited. In addition, spatial relationships and potential transitional zones between histological components were not assessed, which restricts direct inference regarding lineage transitions. Furthermore, treatment responses could not be evaluated for each component, limiting the ability to directly link lineage-specific features with therapeutic sensitivity. Future studies incorporating spatially resolved analyses or multi-region sampling will be required to validate the proposed evolutionary and differentiation framework.

Conclusions

cSCLC with three distinct NSCLC components, as presented in this case, is exceedingly rare and appears to have a prognosis comparable to or worse than that of pure SCLC. Although our comprehensive pathological and molecular analyses revealed marked molecular heterogeneity, further accumulation and integrative molecular characterization of similar cases are needed to optimize therapeutic strategies for this rare subtype.

Acknowledgments

We sincerely thank the patient who provided consent for the publication of this case report. We also thank Dr. Yasushi Ishii for his/her valuable assistance with the additional pathological analyses.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-1-0144/rc

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-1-0144/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-1-0144/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board of Oji General Hospital. Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Nicholson AG, Tsao MS, Beasley MB, et al. The 2021 WHO Classification of Lung Tumors: Impact of Advances Since 2015. J Thorac Oncol 2022;17:362-87. [Crossref] [PubMed]
2. Nicholson SA, Beasley MB, Brambilla E, et al. Small cell lung carcinoma (SCLC): a clinicopathologic study of 100 cases with surgical specimens. Am J Surg Pathol 2002;26:1184-97. [Crossref] [PubMed]
3. Babakoohi S, Fu P, Yang M, et al. Combined SCLC clinical and pathologic characteristics. Clin Lung Cancer 2013;14:113-9. [Crossref] [PubMed]
4. Li Y, Wang Y, Zhou W, et al. Different clinical characteristics and survival between surgically resected pure and combined small cell lung cancer. Thorac Cancer 2022;13:2711-22. [Crossref] [PubMed]
5. Zhang C, Yang H, Zhao H, et al. Clinical outcomes of surgically resected combined small cell lung cancer: a two-institutional experience. J Thorac Dis 2017;9:151-8. [Crossref] [PubMed]
6. Asahina M, Fukumura Y, Mamat O, et al. A case of combined small cell lung carcinoma with unique morphology: Investigation of tumorigenesis. Pathol Int 2018;68:618-23. [Crossref] [PubMed]
7. Gotoh M, Yamamoto Y, Huang CL, et al. A combined small cell carcinoma of the lung containing three components: small cell, spindle cell and squamous cell carcinoma. Eur J Cardiothorac Surg 2004;26:1047-9. [Crossref] [PubMed]
8. Shimanuki Y, Isobe K, Kaburagi K, et al. A resected case of combined cell carcinoma containing components of squamous cell carcinoma, and adenocarcinoma. Japanese Journal of Lung Cancer 2021;61:225-9.
9. He J, Xu S, Pan H, et al. Treatments for combined small cell lung cancer patients. Transl Lung Cancer Res 2020;9:1785-94. [Crossref] [PubMed]
10. Zhao X, McCutcheon JN, Kallakury B, et al. Combined Small Cell Carcinoma of the Lung: Is It a Single Entity? J Thorac Oncol 2018;13:237-45. [Crossref] [PubMed]
11. Luo J, Wu FY, Li AW, et al. Comparison of vinorelbine, ifosfamide and cisplatin (NIP) and etoposide and cisplatin (EP) for treatment of advanced combined small cell lung cancer (cSCLC) patients: a retrospective study. Asian Pac J Cancer Prev 2012;13:4703-6. [Crossref] [PubMed]
12. Li YY, Zhou C, Yang DX, et al. Paclitaxel-etoposide-carboplatin/cisplatin versus etoposide-carboplatin/cisplatin as first-line treatment for combined small-cell lung cancer: a retrospective analysis of 62 cases. Cancer Biol Med 2015;12:117-25. [Crossref] [PubMed]
13. Tatematsu K, Shimizu J, Murakami Y, et al. Epidermal Growth Factor Receptor Mutations in Small Cell Lung Cancer. Clin Cancer Res 2008;14:6092-6.
14. Onishi Y, Kawamura T, Mimura R, et al. An autopsy case of EGFR mutation-positive combined small-cell lung carcinoma with squamous cell carcinoma. Annals of the Japanese Respiratory Society Ann JPN Respir Society 2019;8:405-9.
15. Jiang T, Collins BJ, Jin N, et al. Achaete-scute complex homologue 1 regulates tumor-initiating capacity in human small cell lung cancer. Cancer Res 2009;69:845-54. [Crossref] [PubMed]
16. Ishii J, Sato H, Yazawa T, et al. Class III/IV POU transcription factors expressed in small cell lung cancer cells are involved in proneural/neuroendocrine differentiation. Pathol Int 2014;64:415-22. [Crossref] [PubMed]
17. Osada H, Tomida S, Yatabe Y, et al. Roles of achaete-scute homologue 1 in DKK1 and E-cadherin repression and neuroendocrine differentiation in lung cancer. Cancer Res 2008;68:1647-55. [Crossref] [PubMed]
18. Iida Y, Nakanishi Y, Shimizu T, et al. Comprehensive genetic analysis of histological components of combined small cell carcinoma. Thorac Cancer 2022;13:2362-70. [Crossref] [PubMed]
19. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer 2009;9:798-809. [Crossref] [PubMed]
20. Ross SH, Cantrell DA. Signaling and Function of Interleukin-2 in T Lymphocytes. Annu Rev Immunol 2018;36:411-33. [Crossref] [PubMed]
21. Rudin CM, Poirier JT, Byers LA, et al. Molecular subtypes of small cell lung cancer: a synthesis of human and mouse model data. Nat Rev Cancer 2019;19:289-97. [Crossref] [PubMed]