Case D (Case I in Table 2 and Figure 6)

A 74-year-old man, described as case C in the previous section, presented with paratracheal and mediastinal lymph node metastases 1 year after left upper lobectomy (Figure 5F). Although it was not possible to pathologically identify the metastasizing primary lesion (Figure 5G), the mutation profile of the metastatic lymph node was genetically consistent with that of the larger cancer. The genetic diagnosis was lymph node metastasis of the larger cancer (Figure 6).

#### Case E (Case II in Table 2 and Figure 6)

A 77-year-old man with lung cancer underwent left lower lobectomy (Figure 7A). One year later, a nodule appeared in the middle lobe (Figure 7B). Middle lobectomy was performed based on the assumption that the lesion was a double primary tumor. However, after 1 year, subcarinal lymph node metastasis occurred (Figure 7C). Pathologically, all three lesions were of squamous cell carcinoma type and it was impossible to determine which primary lesion had metastasized (Figure 7D–F). Given the tumor size, the tumor in the left lobe was clinically more likely to have metastasized. However, mutation analysis revealed that the two lung lesions had different mutation profiles; therefore, they were diagnosed as double primary lung cancers. Furthermore, the mutation profiles were consistent between the middle lobe lung cancer and the metastatic lymph node. Thus, lymph node metastasis of the middle lobe lung cancer was determined (Figure 6). Programmed death-ligand 1 (PD-L1) staining of tumor cells was 0% and 90% in the left lower lobe and middle lobe tumors, respectively. Treatment with an anti-PD-1 antibody (nivolumab) was administered and a complete response has been maintained for 1 year since the recurrence in the lymph node.

### Case F (Case III in Table 2 and Figure 6)

A 72-year-old man presented with two tumors in the right lower lobe. Imaging findings suggested double primary lung cancers and right lower lobectomy was performed (Figure 7G,H). Postoperative pathological examination revealed metastases in the interlobar and subcarinal lymph nodes. All four lesions, including the double primary lesions and two metastatic lymph nodes, were pathologically similar squamous cell carcinomas. Therefore, it was impossible to determine which primary lesion had metastasized to the lymph nodes (Figure 7I–L). Clinically, the larger segment 9 tumor was likely to have metastasized to the two lymph nodes. However, both segment 6 and 9 tumors, which had different mutation profiles, were genetically identified as double primary lung cancers. In addition, it was found that the larger segment 9 tumor had metastasized to the interlobar lymph node, whereas the smaller segment 6 tumor had metastasized to the subcarinal lymph node (Figure 6). PD-L1 staining of tumor cells was 0% and 70% in the segment 9 and segment 6 tumors, respectively. Despite the administration of an anti-PD-1 antibody (nivolumab), the patient did not respond to the treatment and died of progression of the cancer at 17 months postoperatively.

**Figure 7.** Radiological and histopathological findings in cases E and F. (**A**–**F**) Findings in case E. (**A**) Primary lesion in the left lower lobe. (**B**) Primary lesion in the middle lobe. (**C**) Subcarinal lymph node metastasis. (**D**–**F**) The three lesions displayed a similar histology of squamous cell carcinoma. (**G**–**L**) Findings in case F. (**G**) Primary lesion in right segment 6. (**H**) Primary lesion in right segment 9. (**I**) Histology of the primary lesion in segment 6. (**J**) Histology of the primary lesion in segment 9. (**K**) Histology of the subcarinal lymph node. (**L**) Histology of the interlobar lymph node. Histologically, the four lesions displayed a similar histology of squamous cell carcinoma. Each scale bar indicates 100 μm.
