PMC10008903_01
The clinical course of the patient (A) Timeline showing the clinical course; (B) Change of the tumor lesion revealed by CT scan; (C) Hematoxylin and Eosin (H&E) stain of biopsied and surgical samples; (D) PD-L1 expression assessed by immunohistochemistry (IHC) assay; (E) Results of metagenomic next-generation sequencing (mNGS) with patient's bronchoalveolar lavage fluid (BALF) and biopsied sample. TP1: initial relapse; TP2: 1 month after relapse; TP3: 2 months after relapse.
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A 55-year-old male with a smoking history of 40 pack/year visited our clinic in April 2020 due to a mass (46x22 mm) in the left upper lobe revealed by a chest CT scan ( Figures 1A, B ). An endobronchial ultrasound (EBUS) guided transbronchial biopsy was performed, and the histopathological examination revealed lung keratinizing squamous cell carcinoma ( Figure 1C ) with strong positive PD-L1(22C3) expression (70%) ( Figure 1D ). After neoadjuvant immunotherapy, a chest CT showed that the mediastinal and left hilar lymph nodes shrank ( Figure 1B ), so the patient underwent a left upper lobectomy in August 2020. No viable tumor cells were found in the resection (necrotic tissue 65%; inflammatory cells and fibrous tissue 35%) ( Figure 1C ) and no cancer cells were detected in lymph nodes. Postoperatively, the patient received two cycles of nivolumab (200mg, q3w) plus paclitaxel (300mg, q3w), followed by three cycles of maintenance therapy with nivolumab monotherapy (200mg, q3w), and remained disease-free ( Figure 1B ). However, seven months after the completion of maintenance therapy, a follow-up CT scan revealed an enlarged subcarinal lymph node measuring 2.2 cm ( Figure 1B ). Lymph node biopsy via bronchoscopy showed squamous cell carcinoma ( Figure 1C ). After one month, a repeated chest CT scan showed fusion, further enlargement of the subcarinal lymph node (measuring 10.6cm), and circular low-density shadow with a small cavity, indicating lymph node recurrence but with partial tumor necrosis. ( Figure 1B ). Only a few atypical cells were found in the biopsied sample of the lymph node ( Figure 1C ). Meanwhile, high copies of respiratory syncytial viruses (RSV), EB virus, Streptococcus, Corynebacterium striatum, and other microorganisms were detected in the patient's bronchoalveolar lavage fluid by the metagenomic next-generation sequencing (mNGS) technique ( Figure 1E ) using the MAPMI  panel on BioelectronSeq 4000 (CapialBio, Beijing, China). One month later, a chest CT revealed marked shrinkage of the enlarged lymph node (2.8cm in diameter) ( Figure 1B ). Only a small amount of moderate atypical hyperplasia of squamous epithelial cells and granulation tissue were observed ( Figure 1C ). mNGS test with the biopsied sample was negative for pathogenic microorganisms ( Figure 1E ).
The dynamic change of tumor microenvironment. (A) Proportions of various infiltrated immune cells estimated by CIBERSORT based on whole transcriptome sequencing (WTS) data; (B) Densities of different multiplex immunofluorescence (mIF) markers assessed by inForm image analysis software (PerkinElmer, Waltham, Massachusetts, US); (C) Multiple immunoflorescent stain captured by Mantra System (PerkinElmer, Waltham, Massachusetts, US), composite five-color images showing the expression of PD-1(green), PD-L1(yellow), CD3 (red), CD8 (white), and panCK (cyan) DAPI(blue) and images showing CD8, DAPI, and panCK markers; (D) KEGG pathway enrichment. TP1: initial relapse; TP2: 1 month after relapse; TP3: 2 months after relapse.
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CIBERSORT analysis based on the WTS data revealed that the proportion of CD8+ T cells was the highest at TP1, decreased at TP2, and increased again at TP3 ( Figure 2A ). Table S1 ). The results showed an increase of CD8+ T cell density in the tumoral compartment from TP1 (14 no./mm2) to TP2 (43 no./mm2) ( Figures 2B, C ) ( Table S2 ). Comparatively, in the stromal compartment, CD8+ T cell density decreased from TP1 (127 no./mm2) to TP2 (46 no./mm2) and then remained unchanged at TP3 (44 no./mm2). The densities for CD3+ and Besides, enrichments of NF-kappa B and TNF signaling pathways were noted at TP2 ( Figure 2D ).