The aim of this study is to evaluate the prognostic impact of the presence of circulating tumour cells Circulating Tumour Cells (CTCs), Spread Through Air Space (STAS) and lymph node micrometastases in patients undergoing radical surgery for non-small cell lung neoplasia

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths worldwide. Patients diagnosed with early-stage NSCLC are candidates for radical surgical resection with curative intent. However, disease recurrence after surgery is common, with 5-year recurrence rates ranging from about 20% in patients with stage I disease to about 50% in those with stage III disease. In detail, in patients operated with radical resection (R0), the loco-regional recurrence rate varies from 4.6 to 24%, with a significantly higher incidence in the first two years after surgery.

To date, only a few risk factors have been correlated with an increased propensity to develop recurrences, namely stage and some loco regional aggressiveness (e.g. pleural invasion, high-grade growth pattern, lymphovascular invasion), but there are still no certain elements that allow us to understand in advance the biology of the early spread of the disease and thus identify patients at high risk of relapse.

of relapse; the presence of these elements would have the potential advantage of playing a role both in recommending new therapeutic approaches for adjuvant treatment and in acting as biomarkers to anticipate the diagnosis or recurrence of lung cancer.

Until 2022, lobectomy was considered the standard of care for any type of lung neoplasm that was a candidate for surgery. Recent studies, however, have shown that even sublobar resections may allow adequate oncological radicality for tumours peripheral tumours of small size (<2 cm) . Moreover, in the last decade, technological development has allowed a gradual replacement of traditional open techniques with mini-invasive techniques (videothoracoscopy, Video Assisted Thoracic Surgery (VATS), or robotic, Robotic Assisted Thoracic Surgery (RATS)).

Circulating tumour cells (CTCs) are considered to be the site of origin for possible disease recurrence in several cancers, showing a higher rate of disease recurrence in the case of high CTC levels. Despite this, their specific clinical role in lung cancer has not yet been clarified. CTCs from the peripheral blood of patients with breast cancer, melanoma, NSCLC and small cell lung cancer can form tumours in mice immunocompromised mice, confirming their carcinogenic potential. Furthermore, the presence of CTCs has also been correlated with manipulation of the lung during surgery, which would seem to favour dissemination, although the clinical significance is uncertain.

Recent studies have highlighted the importance of the molecular profile of neoplasms lung neoplasms that could change the standard of care with regard to neoadjuvant or adjuvant therapies. Molecular assessment is usually performed on biopsies or on the operative specimen of the primary tumour, but could potentially be performed on circulating tumour cells. Weak evidence on the clinical role of CTCs also includes their role in defining the molecular profile of the tumour.

In addition to haematogenous spread of which CTCs could be a warning sign, NSCLC can potentially spread via the airway and lymphatic route. Spread via the airway and through the alveolar spaces has recently been observed, especially in the adenocarcinoma histotype, and codified by the World Health Organisation (WHO 2021) as Spread Through Air Space (STAS); as for lymphatic invasion lymphatic invasion, in addition to intrapulmonary lymphatic vessel invasion, it is manifested by the presence of lymph node metastases or micrometastases.

In detail, STAS are defined as tumour cells existing within alveolar spaces in the lung parenchyma beyond the edge of the main neoplasm, in close proximity to the edge of the main neoplasm. STAS can be found mainly, but not only, in lung adenocarcinomas and in about 35-40% of early stage clinical NSCLC; some studies have shown their correlation with a worse prognosis than in patients without STAS .

Micrometastases, in other body districts such as breast and axillary locoregional lymph node metastases, on the other hand, are defined as tumour aggregates between between 0.2 and 2.0 mm. In the literature, data on the incidence of lymph node micrometastases in NSCLC are inconsistent and range from 10-80%. Compared to patients with macroscopic lymph node involvement, micrometastases seem to be related to a better prognosis, but, on the other hand, outcomes seem to be worse than in N0 pathological patients.

The objectives of the study are to :

1. Check whether the presence and/or levels of CTC is associated with disease-free survival (DFI)
2. To assess the pattern of recurrence in patients with CTC, STAS and lymph node micrometastases

Secondary objectives

1. Quantify the difference in intraoperative release of CTC into the bloodstream based on the intraoperative variables:

   • Resection sequence of the hilar structure (first vein or artery)
   • Lung management according to different surgical techniques (open, VATS, RATS)
   • Type of resection: segmentectomy, lobectomy, pneumonectomy

2. Check for association between CTC, presence of STAS and presence of lymph node micrometastases

3. Check whether the presence and/or levels of CTC, STAS and lymph node micrometastases are associated with long-term outcomes (DFI, OS)

4. Compare the molecular profile of CTCs and the primary tumour

Inclusion Criteria:

• obtaining voluntary written informed consent
• diagnosis of NSCLC
• clinical stage I-IIA cN0
• Candidates for surgical resection

Exclusion Criteria:

• Pregnancy
• Induction therapy for pulmonary neoplasia
• Patients who have received systemic treatment for previous cancers in the last three years prior to lung surgery
• Any previous surgery for lung cancer
• Patients with other active neoplasms
• Infiltration of the chest wall

The study is low-risk, human-tissue, prospective, single-center experimental study conducted on patients undergoing surgery for removal of stage I-IIA NSCLC.