The probability of implantation metastasis after peripheral lung cancer biopsy

Introduction

Lung cancer is one of the most common malignant tumors of the respiratory system. In China, the incidence and mortality of lung cancer rank first in both men and women (1), seriously threatening people’s lives and health. Definitive diagnosis is the key to the treatment of lung cancer, central lung cancer can be clearly diagnosed by fiberoptic bronchoscope or endobronchial ultrasound-guided transbronchial needle aspiration, while surgery is the preferred method for the diagnosis of peripheral lung cancer (2,3). Surgery can not only confirm the diagnosis, but also treat peripheral lung cancer, however, some patients have undergone lung biopsy before surgery. Previous studies have shown that lung biopsy can cause pleural cavity and needle tract implantation metastasis (4,5), but this phenomenon has not yet attracted the attention of medical oncologist. In the present study, we simulated lung biopsy. We punctured the tumor body on the isolated lung lobe, obtained the pleural pinhole and needle tip flushing fluid, and inoculated them subcutaneously in nude mice. According to the tumorigenesis rate of nude mice, we indirectly obtained the probability of implantation metastasis in pleural cavity and needle tract after peripheral lung cancer biopsy. Through this study, we believe that patients with lung nodules suspected of being early stage cancer and who are fit for surgical resection may benefit from forgoing lung biopsies to avoid pleural cavity or needle tract seeding with tumor cells. We present this article in accordance with the ARRIVE reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-529/rc).

Methods

Clinical tissue samples

The 30 intraoperative isolated completely collapsed fresh intact human lung lobes were obtained from patients with peripheral lung cancer who had not received anti-tumor treatment in The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital. All cases were diagnosed with lung cancer by two associate chief physicians of the pathology department of our hospital. Clinical information was collected from the patient medical records and is reported in Table S1. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Hunan Cancer Hospital (No. KYJJ-2021-242) and informed consent was taken from all the patients.

Preparation of inoculum

After fresh lung lobes were isolated, the tumor was punctured three times with a puncture needle (Semiautomatic biopsy needle 18GL-160 mm, TSK Co., Ltd, Fukuroi City, Japan) in a sterile environment. After each puncture, 10 mL normal saline (NS) was used to rinse the pleural pinhole and needle tip five times, respectively. The flushing fluid of the pleural pinhole group and the needle tip group was collected, centrifuged at 800 rpm for 5 min, then the supernatant was discarded. Use 200 µL NS to resuspend the cells to prepare the inoculum, and smear the inoculum for microscopic examination.

Subcutaneous inoculation of nude mice

A total of 60 BALB/c nude mice with innate cell-free immunity (female, 4 weeks) were purchased from Hunan SJA Laboratory Animals (Changsha, China), they were housed in specific pathogen free level feeding rooms and divided into pleural pinhole group (n=30) and needle tip group (n=30). Inoculum was subcutaneously injected into the right posterior neck of the nude mice. After keeping the nude mice for 30 days, they were euthanized, the tumors were excised. The collected tumor tissues were fixed for pathological examination, through steps of gradient dehydration, sectioning, and embedding in paraffin. Experiments were performed under a project license (No. 2021-113) granted by the Ethics Committee of Hunan Cancer Hospital, in compliance with Chinese national or institutional guidelines for the care and use of animals.

Hematoxylin-eosin (H&E) staining

The paraffin-embedded tumor tissue samples were heated at 65 ℃ for 2 h for dewaxing, followed by multiple steps of hydration. Alcohol was used to fix the cancer cells from the inoculum onto the slide. The nuclei and cytoplasm of the tumor cells were stained with hematoxylin (AiFang Biological, Changsha, China) and eosin (AiFang), respectively. The slices were dried and preserved with neutral resin (AiFang) for further pathological examination. The sections were reviewed by two associate chief physicians of the pathology department of our hospital.

Results

We performed smear microscopic examination of inoculum of two groups and found that cancer cells were indeed present in each inoculum (Figure 1A,1B). After 30 days of continuous feeding, one nude mouse in pleural pinhole group, the inoculum was obtained from patient 9, and one nude mouse in needle tip group, the inoculum was obtained from patient 18, formed tumors, the tumorigenesis rate in both groups were 3.3% (Figure 1C-1F, Table S1). The xenografts of the nude mice were examined by H&E staining (Figure 1G,1H).

Figure 1 Microscopic examination of the inoculant and observation of subcutaneous tumorigenesis in nude mice. (A) Smear microscopic examination of inoculum in pleural pinhole group (hematoxylin-eosin staining, 200×, scale bar: 50 μm). (B) Smear microscopic examination of inoculum in needle tip group (200×, scale bar: 50 µm). (C,D) Tumorigenic nude mouse in pleural pinhole group and needle tip group (the arrows indicate the tumor). (E,F) Subcutaneous tumor of nude mouse in pleural pinhole group (the arrow indicates the tumor) and needle tip group. (G,H) Images of hematoxylin-eosin staining in tissue samples of subcutaneous tumors (200×, scale bar: 50 µm).

Discussion

Surgery is the preferred treatment for peripheral lung space occupying lesions which are not clearly diagnosed. At surgery, diagnosis can be confirmed by frozen section, and after the diagnosis is confirmed, corresponding radical resection of lung cancer can be carried out. Surgical resection is more consistent with the principle of no tumor (3). In previous reports, some scholars suggested that after lung biopsy, cancer cells could leak through the pleural pinhole, and the leaked cancer cells had the possibility to seed in pleural cavity (6). Other scholars reported case of needle tract implantation metastasis after lung biopsy, and considered that the implantation metastasis after lung biopsy mainly occurred in the pleural cavity and needle tract (4,5,7), but so far no one has proposed a possible probability of occurrence. In our study, we found that after lung biopsy, both the probability of cancer cells overflowing through the pleural pinhole and carried out by the puncture needle tip planted in the pleural cavity and needle tract was 3.3%. This study indirectly provides the probability of pleural cavity or needle tract implantation metastasis after lung cancer biopsy by nude mice tumorigenesis experiment.

Conclusions

Above all, we believe that patients with lung nodules suspected of being early stage cancer and who are fit for surgical resection may benefit from forgoing lung biopsies to avoid pleural cavity or needle tract seeding with tumor cells.

Acknowledgments

We sincerely thank Prof. Gaoming Xiao for his kind support to all members of our research group.

Funding: This study was supported by grants from the Natural Science Foundation of Hunan Province (No. 2022JJ40244) and the Science and Technology Innovation program of Hunan Province (No. 2023SK2058).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-529/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-529/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-529/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-529/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. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Hunan Cancer Hospital (No. KYJJ-2021-242) and informed consent was taken from all the patients. Experiments were performed under a project license (No. 2021-113) granted by the Ethics Committee of Hunan Cancer Hospital, in compliance with Chinese national or institutional guidelines for the care and use of animals.

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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