Comprehensive genomic profiling of synchronous invasive adenocarcinoma and squamous cell carcinoma within the same lobe: a case report

Introduction

Synchronous multiple primary lung cancer (sMPLC) involves two or more independent primary tumors occurring simultaneously within the ipsilateral or contralateral lungs (1). The incidence of multiple primary lung cancer (MPLC) ranges from 0.2–15% of all lung cancer cases (2). In most instances, sMPLCs share the same histological type, typically multiple adenocarcinomas, and arise in different lobes (3). The clinical diagnosis of MPLC has traditionally relied on the Martini-Melamed criteria. Recently, the American College of Chest Physicians and the International Association for the Study of Lung Cancer have proposed updated guidelines that incorporate comprehensive histologic assessment and emphasize the role of molecular profiling to enhance diagnostic accuracy (4,5). Synchronous tumors with distinct histologies occurring within the same lobe are exceedingly rare (6). The accurate classification of MPLCs has profound clinical implications for staging, therapeutic decision-making, and prognosis. While traditional histopathological criteria are still valuable, histology alone is often insufficient to determine whether two lesions represent independent primaries or intrapulmonary metastasis. Advances in molecular profiling have enabled more precise clonal assessments, with whole genome sequencing (WGS) providing the most comprehensive approach by characterizing exonic mutations, structural variations, genome-wide copy number changes, and noncoding alterations.

The field cancerization hypothesis proposes that exposure to carcinogens, such as tobacco smoke, generates a “field” of genetically altered epithelium, from which multiple independent tumors may arise (7). While this model explains why smokers are predisposed to multifocal neoplasia, only a subset of individuals develops multiple synchronous cancers, suggesting that genetic vulnerability also plays a pivotal role. DNA repair genes are crucial guardians of genomic stability, and deficiencies in mismatch repair (MMR) or nucleotide excision repair (NER) pathways increase susceptibility to carcinogen-induced mutations (8). Furthermore, epigenetic alterations such as DNA methylation and somatic mosaicism may reinforce clonal divergence, supporting a multidimensional mechanism underlying field cancerization. Alterations in PMS2, a key gene involved in the MMR pathway, and ERCC2, a helicase involved in the NER pathway, have been linked to increased cancer predisposition.

We present a case of synchronous adenocarcinoma and squamous cell carcinoma arising within the same lung lobe, where WGS revealed divergent mutational profiles that confirm independent primaries, as well as alterations in PMS2 and ERCC2, suggesting that deficiencies in DNA repair pathways contributed to tumorigenesis. WGS was employed instead of whole-exome sequencing (WES) to enable comprehensive detection of structural and non-coding variants, which provide critical insights for distinguishing clonally independent tumors. We present this article in accordance with the CARE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-aw-1206/rc).

Case presentation

An 80-year-old man with a medical history of hypertension, diabetes mellitus, chronic obstructive pulmonary disease, and unstable angina, along with a 50-pack-year smoking history (ceased 5 years prior), was evaluated for pulmonary nodules detected on chest imaging. A computed tomography (CT) scan performed in October 2024, with contrast enhancement and a slice thickness of 1 mm, revealed a 19-mm part-solid nodule in the apicoposterior segment of the left upper lobe (LUL) (Figure 1A) and an additional small ground-glass nodule in the same lobe (Figure 1B). A further tiny ground-glass nodule was also observed in the left lower lobe (Figure 1C). Despite a heavy smoking history, preoperative CT demonstrated no definite evidence of interstitial pneumonia or emphysema. Pulmonary function testing revealed a marked obstructive pattern consistent with smoking-related small-airways disease. Endobronchial ultrasound-guided cryobiopsy of the 19-mm LUL lesion demonstrated atypical pneumocytic proliferation, suspicious for adenocarcinoma in situ (Figure 1D), with tumor cells positive for carcinoembryonic antigen (Figure 1E) and thyroid transcription factor-1 (Figure 1F). Although the biopsy findings suggested a noninvasive lesion, the part-solid appearance on CT raised clinical suspicion of invasive adenocarcinoma. On fluorodeoxyglucose positron emission tomography-CT, the lesions were interpreted as lung cancer with possible same-lobe metastasis, and the patient was referred for surgical management. A left upper lobectomy was performed. Postoperative histopathology revealed two distinct invasive carcinomas within the same lobe. The first tumor, corresponding to the previously biopsied lesion, measured 2.1 cm × 1.9 cm and was an adenocarcinoma with acinar- and papillary-predominant patterns (Figure 2A). The second tumor, measuring 1.1 cm × 0.7 cm, was a non-keratinizing squamous cell carcinoma (Figure 2B), with no regional lymph node metastasis (pN0). Additionally, a small atypical adenomatous hyperplasia was identified in the resected left lower lobe specimen (Figure 2C), although tissue was insufficient for genomic analysis. The squamous component showed diffuse p40 positivity (Figure 2D) and was negative for Napsin A (Figure 2E). Initially, the two invasive tumors within the LUL were regarded as intrapulmonary metastasis (clinical T3). However, given their distinct histological features and subsequent WGS confirming clonal independence, they were reclassified as synchronous MPLC. Pathologic staging was therefore assigned separately: pT1cN0M0 (Stage IA3) for the adenocarcinoma and pT1bN0M0 (Stage IA2) for the squamous cell carcinoma. This reclassification from clinical T3 to dual early-stage primaries influenced postoperative management, with no adjuvant chemotherapy indicated for either tumor.

Figure 1 Chest CT and histopathological findings of pulmonary nodules. (A) CT revealed a 19-mm part-solid nodule in the apicoposterior segment of the LUL. (B) An additional small ground-glass nodule was identified in the same lobe. (C) A further tiny ground-glass nodule was also noted in the LLL. (D) Representative tumor section obtained via endobronchial ultrasound-guided cryobiopsy of the 19-mm LUL lesion demonstrated atypical pneumocytic proliferation, suspicious for adenocarcinoma in situ (hematoxylin and eosin staining; ×60; inset: ×600). Scale bar, 500 µm; inset scale bar, 20 µm. (E,F) Immunohistochemistry of the tumor section showed that tumor cells were positive for CEA (×60, E) and TTF-1 (×60, F). Scale bar, 500 µm. CEA, carcinoembryonic antigen; CT, computed tomography; LLL, left lower lobe; LUL, left upper lobe; TTF-1, thyroid transcription factor-1.

Figure 2 Postoperative histopathology demonstrating synchronous distinct invasive carcinomas and atypical adenomatous hyperplasia. (A-C) Hematoxylin and eosin staining showing: (A) invasive adenocarcinoma in the LUL, measuring 2.1 cm × 1.9 cm, with predominantly acinar and papillary patterns, consistent with the biopsy findings (original magnification ×12; inset ×800); (B) a second distinct tumor in the LUL, measuring 1.1 cm × 0.7 cm, was identified as a non-keratinizing squamous cell carcinoma (×24; inset ×400); (C) a small atypical adenomatous hyperplasia was also noted in the left lower lobe (×12; inset ×400). (D,E) Immunohistochemistry demonstrated diffuse p40 positivity (D, ×30; inset ×300) and negative Napsin A expression (E, ×30) in the squamous carcinoma. Scale bars, 2,000 µm in (A) and (C); 1,000 µm in (B,D,E). Inset scale bars, 20 µm in (A); 50 µm in (B) and (C); 100 µm in (D). LUL, left upper lobe.

Comprehensive WGS was performed on adenocarcinoma, squamous carcinoma, and matched normal tissue. WGS was conducted using the Illumina NovaSeq X platform with 151-bp paired-end reads, achieving mean coverage of 27.52× in normal tissue, 22.28× in the adenocarcinoma, and 21.62× in the squamous carcinoma. Raw sequencing reads were processed through a standard GATK-based workflow, including alignment to the GRCh38 reference genome with BWA-MEM and paired tumor-normal somatic variant calling using Mutect2. Variants passing quality filters were annotated with Ensembl VEP and used for comparative genomic analyses. At the gene level, 357 genes were mutated in adenocarcinoma and 170 in squamous carcinoma, with 25 overlapping genes (Figure 3A). Copy number and structural variation analysis revealed largely distinct genomic landscapes between the two tumors (Figure 3B). Tumor mutational burden was moderate in both tumors, with 6.04 mutations per Mb in adenocarcinoma and 5.24 mutations per Mb in squamous carcinoma (Figure 3C). Oncogenic pathway enrichment further underscored divergence, with adenocarcinoma enriched in MET and cell cycle pathways, while squamous carcinoma displayed alternative pathway priorities (Figure 3D). A total of 564 variants were identified across both tumors, of which only six were shared, corresponding to a Jaccard similarity index of 0.022. These shared variants, together with the tumor-specific variants, were used for clonal relatedness analyses. Clonal analysis showed very weak correlations, with variant allele frequency (VAF) correlation at 0.046 and cancer cell fraction correlation at 0.095, strongly supporting independent origins (Figure 3E). Germline variant analysis, performed using blood-derived DNA with rare variants filtered at a population allele frequency of <1% (gnomAD, hg38), further revealed enrichment in biological processes, including transcriptional regulation, DNA repair, DNA damage response, and immune-related pathways, providing additional context for potential host susceptibility factors (Figure 3F).

Figure 3 Comprehensive genomic profiling of synchronous adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) from the same lobe. (A) Gene-level overlap of mutated genes in LUAD and LUSC. A total of 357 mutated genes were identified in LUAD and 170 in LUSC, with 25 genes overlapping (7.0%). (B) Circos plots illustrating distinct copy number alterations and structural variations in LUAD and LUSC, showing largely non-overlapping genomic landscapes. (C) TMB comparison between LUAD and LUSC, demonstrating moderate levels in both tumors (6.04 vs. 5.24 mutations/Mb). (D) Oncogenic pathway enrichment analysis. LUAD was enriched for alterations in MET and cell cycle pathways, while LUSC displayed distinct pathway priorities. Different mutation types are indicated (missense, nonsense, splice-site, frameshift indel, in-frame indel/ins). (E) Clonal relatedness analyses. Left: VAF correlation of shared variants between LUAD and LUSC (n=6, r=0.05). Right: CNV scatter plot comparing LUAD and LUSC (r=0.05). Both analyses revealed minimal correlation, supporting independent clonal origins. (F) Germline variant-associated functional enrichment. GO analysis of high-impact germline variants revealed enrichment in multiple biological processes, including regulation of transcription, DNA repair/damage response, immune-related pathways, and signal transduction. Bar length indicates the number of genes involved in each GO term, and color reflects gene count intensity. CNV, copy number variation; GO, Gene Ontology; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; TMB, tumor mutation burden; VAF, variant allele frequency.

Several gene alterations were identified across both tumors. Commonly mutated genes included ANAPC1, FANK1, and members of the MUC6 family. No major driver alterations were detected in epidermal growth factor receptor (EGFR) or Kirsten rat sarcoma (KRAS), and no canonical anaplastic lymphoma kinase (ALK) fusions were identified in either tumor. In contrast, alterations detected only in lung adenocarcinoma (LUAD) included PMS2, implicating MMR dysfunction, while ERCC2 and ANKRD36C were uniquely observed in lung squamous cell carcinoma (LUSC), suggesting impaired NER. Additional gene alterations are summarized in Table 1. In addition, analysis of non-coding RNA variants identified four alterations in LUAD and three in LUSC. Among these, several lncRNAs such as LINC01410 and LINC01278 have been reported to exert oncogenic roles, whereas LINC01550 exhibits tumour-suppressive activity, and others remain functionally uncharacterized (Table 2).

The patient, an elderly man who initially hesitated to undergo surgery because of his age and comorbidities, ultimately agreed to proceed after thorough counseling regarding the risks and potential benefits. Following a successful curative resection, he expressed great satisfaction with the outcome and relief that no adjuvant chemotherapy was required. He stated that the genomic clarification provided additional reassurance about his prognosis and confidence in the treatment decision.

All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from the patient for 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

This case highlights the importance of confirming independent primaries, as genomic profiling revealed that the synchronous adenocarcinoma and squamous carcinoma within the same lobe were indeed independent primaries. Quantitative comparison showed a Jaccard index of 0.022 and a VAF correlation (r=0.046), indicating minimal variant overlap and genomic independence consistent with sMPLC thresholds (<0.2), thereby providing strong molecular evidence against metastatic spread. WGS provided insights beyond what whole exome sequencing could offer, detecting structural variations, genome-wide copy number changes, and noncoding RNA alterations that solidified the clonal relationships with confidence. The occurrence of synchronous tumors of distinct histological types within the same lobe is exceedingly rare. To our knowledge, only a few such cases involving both adenocarcinoma and squamous carcinoma have been described (6). Our case contributes to the growing body of evidence supporting the importance of comprehensive molecular evaluation in these rare clinical scenarios.

A critical question is why this patient developed two independent primary cancers rather than a single tumor. The field cancerization model offers one potential explanation: prolonged smoking exposure creates a mutagenic “field” in the bronchial epithelium, from which multiple clones can evolve independently. However, while smoking is a key risk factor, not all smokers develop synchronous cancers, indicating the presence of additional susceptibility factors. The identification of somatic PMS2 and ERCC2 alterations in this case further emphasizes the role of DNA repair deficiencies as an acquired genetic vulnerability. PMS2 dysfunction is associated with Lynch syndrome, a hereditary cancer syndrome, while ERCC2 plays a crucial role in repairing bulky DNA adducts caused by tobacco carcinogens. Biologically, PMS2-mediated MMR deficiency typically manifests as COSMIC Signature 6 (occasionally 15 or 26), whereas ERCC2-related NER defects are associated with Signature 5, indicating distinct repair-pathway-driven mutational processes. This combination of smoking-related mutagenic stress, age-related genomic instability, and impaired DNA repair capacity likely created a permissive environment that facilitated the emergence of two independent tumors. However, PMS2 immunohistochemistry could not be performed due to limited remaining tumor tissue, and this should be considered when interpreting the PMS2 alteration.

Clinically, these findings have several implications. First, confirming independent primaries supports the classification as synchronous multiple primaries rather than metastatic disease, directly impacting staging and treatment decisions. In this patient, the initial impression was of intrapulmonary metastasis within the same lobe (clinical T3), which would have suggested more advanced disease and the need for adjuvant therapy. Such misclassification could have resulted in stage migration bias and potential overtreatment. However, genomic confirmation of independence allowed for reclassification into two early-stage primaries (pT1c and pT1b), fundamentally altering both staging and management decisions. Second, while PMS2 and ERCC2 were somatic rather than germline, their presence still raises the possibility that patients with similar profiles may harbor underlying repair vulnerabilities, warranting careful surveillance. Given this context, genetic counseling should be considered in such cases to exclude latent hereditary predisposition, particularly for MMR-associated genes such as PMS2. Third, patients with repair deficiencies may benefit from intensified surveillance, given the elevated risk of additional primaries. Finally, therapeutic opportunities exist, as DNA repair-deficient tumors may be more sensitive to agents such as PARP inhibitors or synthetic lethality approaches.

This case also demonstrates that histology alone is often insufficient to differentiate between multiple primary tumors and metastasis, even when the tumors exhibit distinct morphologies. This highlights the critical role of genomic profiling in such diagnostic contexts. Additionally, the detection of non-coding RNA variants introduces another layer of genomic complexity. However, as a single-case analysis, this study lacks transcriptomic and functional validation, and the pathogenicity of the identified variants was not experimentally confirmed. While the clinical significance of these alterations is still being explored, these preliminary findings emphasize the unique advantage of WGS in capturing genomic variations that extend beyond the protein-coding regions of the genome. Furthermore, germline variant analysis revealed an enrichment of genes associated with key cellular processes such as transcriptional regulation (RNA polymerase II), cell division, DNA damage response, and DNA repair. Although these germline variants alone do not constitute sufficient causative factors, their presence suggests the existence of a permissive genomic background that may have facilitated the development of two distinct primary tumors in this patient, consistent with prior observations that germline alterations in DNA repair genes may influence cancer susceptibility (8). This underscores the potential contribution of inherited susceptibility factors, interacting with smoking-related mutational stress and age-related genomic instability, in the formation of synchronous cancers. These mechanistic insights could inform future biomarker-driven surveillance strategies or trials aimed at early detection in high-risk former smokers.

Conclusions

This case highlights the critical role of WGS in the evaluation of synchronous lung cancers. By confirming the independent origins of the tumors and identifying key DNA repair gene alterations, WGS clarified the diagnosis and provided valuable insights into the underlying tumor biology. The co-occurrence of adenocarcinoma and squamous carcinoma in this patient is best explained by the combination of smoking-induced field cancerization and a predisposition to DNA repair defects. These findings underscore the importance of genomic profiling in challenging oncological scenarios, suggesting that genetic counseling and personalized surveillance strategies are essential for patients harboring DNA repair gene mutations. Moreover, DNA-repair-deficient tumors may exhibit therapeutic vulnerability to agents targeting DNA damage response pathways, such as PARP or ATR inhibitors, meriting further investigation. Clinicians should consider integrating WGS into complex or ambiguous multifocal lung cancer assessments, where feasible.

Acknowledgments

We would like to thank Editage (www.editage.co.kr) for English language editing.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-aw-1206/rc

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-aw-1206/prf

Funding: This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean Government (MSIT) (Nos. 2022R1A2C2010148 and RS-2022-NR071878). This work was also supported by grants from the Korea Health Technology Research & Development (R&D) Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Nos. RS-2020-KH088690, RS-2022-KH130308, and RS-2024-00440671).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-aw-1206/coif). H.Y.K. is employed by NGeneS Inc. The other 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 ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from the patient for 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/.

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