Lung cancer remains the leading cause of cancer-related deaths worldwide (1). With the increasing application of low-dose CT for lung cancer screening, more cases of multiple primary lung cancers (MPLC) are being detected. The traditional diagnostic criteria for distinguishing MPLC from pulmonary metastasis are based on the Martini and Melamed (MM) criteria (2) and the Comprehensive Histologic Assessment (CHA) criteria (3), which mainly rely on histological comparison.

RET gene (proto-oncogene tyrosine-protein kinase receptor RET) is a rare genetic mutation in lung cancer, with only 1-2% incidence (4–6). Most MPLC cases show different mutations in different nodules, but some patients’ nodules exhibit the same mutation, necessitating further evaluation to determine whether they are genuinely MPLC.

The case involves a 54-year-old female patient who sought medical evaluation due to the existence of multiple pulmonary nodules in her right lung over six months. A computed tomography (CT) scan performed at our institution identified three nodules within her right lung’s upper, middle, and lower lobes ( Figure 1 ). The postoperative pathological report indicated that the upper lobe nodule was carcinoma in situ with a diameter of 0.6cm. The middle and lower lobe nodules were microinvasive adenocarcinomas measuring 0.5cm in diameter. All three nodules were predominantly lepidic, and two microinvasive adenocarcinoma had acinar components. Postoperative genetic examination confirmed RET mutation in all three nodules. Considering that RET mutation is rare in lung cancer cases, we performed whole exon sequencing (7) on these three nodules to differentiate between multiple primary lung cancers and intrapulmonary metastatic cancer. The results demonstrated varying somatic mutations among the three nodules: 257, 242,105, respectively. Among these mutations, nine were shared, while others were independent. In addition, 6 substitution analysis and 96 substitution analysis showed differences in 3 nodules (t test, p<0.05). The mutation characteristics of the sample and 78 known mutation characteristics were cluster analyzed, and the similarity was found to be low. Based on these findings, we conclude that this patient has multiple primary lung cancers.

Recent years have seen an increase in lung cancer detection due to low-dose CT screening, with over 60% of cases presenting as ground-glass opacities, and up to 10% of patients having multiple ground-glass nodules (8). Most of these cases are considered MPLC (9, 10), although some studies suggest the possibility of metastasis (11). Next-generation sequencing (NGS) is becoming increasingly important in the molecular typing of lung cancer for treatment decisions. Studies have shown that different ground-glass nodules in the same patient often have different gene mutations, and even when nodules exhibit the same gene mutation, their mutation spectra can differ, suggesting the possibility of MPLC. Yanagitani (12) has reported on a 55-year-old woman with advanced lung adenocarcinoma with metastasis in both lungs who showed multiple metastatic ground glass nodules on CT. In recent years, two additional criteria introduced by the American College of Chest Physicians (ACCP) and the International Association for the Study of Lung Cancer (IASLC) emphasize using histological features, immunohistochemistry (IHC), clinical characteristics, and molecular features to differentiate between multiple primary lung cancers and intrapulmonary metastasis (13, 14).

Studies on multiple nodules being primary cancers or lung metastases are limited. Most research is based on targeted genes (9, 10, 15), with few based on genomics (11). Gene targeting for multiple nodules has not found any evidence of intrapulmonary metastasis. Generally, each nodule’s driver gene mutations are highly distinct (9). Next-generation sequencing (NGS) is the most commonly used targeted sequencing technology, allowing for the simultaneous sequencing of millions of DNA fragments and providing sensitive, economical, and high-throughput detection. Therefore, NGS is gaining more and more attention as an auxiliary means in histopathological diagnosis, especially in lung cancer, where molecular typing contributes to treatment selection (16, 17) Research by Park et al. (18) indicated that, by second-generation sequencing of 16 patients with multiple ground-glass nodules, most patients with multiple ground-glass nodules have different gene mutations in each nodule. However, a few patients have the same gene mutation. However, the mutation spectrum is different, suggesting that even if each nodule shows the same gene mutation, the possibility of multiple primary lung cancers still needs to be considered.

Zhou et al. ‘s (19) study showed that whole exon sequencing could distinguish multiple primary tumors from intrapulmonary metastases, including CNV(Copy Number Variation) analysis, somatic mutation, 6 substitution analysis, 96 substitution analysis and other methods. This study suggests that pulmonary metastasis exists in multiple nodules, but the number of multiple nodules is not related to the probability of pulmonary metastasis. Other genomic profiling studies (20–22) have also reported sizeable genetic heterogeneity among multiple lesions of MPLC. Applying genomic analysis may be crucial for accurately managing patients with multiple nodules.

The RET gene is a proto-oncogene located on the long arm of chromosome 10. It encodes for a tyrosine kinase receptor and is involved in the proliferation, apoptosis, and invasion of tumor cells, affecting the development and progression of tumors (4). The formation of tumors is primarily associated with RET gene fusions, which account for only 1-2% of lung cancers. This report presents a unique case where a patient’s CT scan revealed three ground-glass nodules, considered to be multiple primary lung cancers. Postoperative pathology reported that the nodule in the right upper lobe was a 0.6 cm in situ carcinoma, the nodule in the right middle lobe was a 0.5 cm minimally invasive adenocarcinoma, and the nodule in the right lower lobe was also a 0.5 cm minimally invasive adenocarcinoma. However, postoperatively, genetic testing revealed that all three nodules expressed the RET mutation, an occurrence with an extremely low probability. We conducted whole-exome sequencing to assess further whether the patient’s nodules were intrapulmonary metastases. The results of whole exon sequencing showed ( Figure 2 ) that the number of somatic mutations in the three nodules differed, 257,242,105, respectively, and only 9 were common among the three nodules. In contrast, other mutations were independent of each other. However, the mutations in the single nucleic acid showed that the proportion of mutations in each nodule was different, and the mutation spectrum of different nodules in patients was significantly inconsistent at the level of substitution composition (t-test,p<0.05). The mutation features of the three samples were clustered with 78 known mutation features in the COSMIC website, and the three mutation features were not similar to any of the known 78 features (cosine similarity <0.9), the cosine similarity between the three mutation features being less than 0.9.The similarity is not high. Therefore, we conclude that this patient has multiple primary lung cancers.

This case report indicates that for patients with multiple lung cancers, each nodule exhibits independent genetic characteristics, including in cases of rare mutations. The case presented here involved three nodules, all RET mutations, with an incidence of less than one in a hundred thousand. Through whole-exome sequencing analysis, it was shown that despite the presence of the same mutation in all nodules, genetic heterogeneity still exists among them. Genetic testing cannot be used as a basis to distinguish between multiple primary lung cancers and intrapulmonary metastasis, but further differentiation can be achieved through whole-exome sequencing.