Immun Checkpoint Washout in Patients With Invasive Ductal Breast Cancer

The most common cancer in women worldwide and the leading cause of death among those aged 20-59 is breast cancer. Invasive breast cancers are primarily classified as ductal and lobular. However, special types of invasive breast cancers, which constitute 10% of total cases, have been included in the current histological classification.

Invasive ductal carcinoma is the most frequently observed type among invasive breast cancers (80%) and is also defined as invasive ductal carcinoma of no special type. Macroscopic or microscopic axillary lymph node metastases have been detected in up to 25% of screening-diagnosed cases and up to 60% of symptomatic cases of invasive ductal carcinoma.

The clinical staging of breast cancer is primarily determined by the physical examination of the breast skin, breast tissue, and regional lymph nodes (axillary, supraclavicular, and internal mammary). However, the accuracy of clinically determining axillary lymph node metastases is only 33%. Proper lymph node staging in breast cancer patients is crucial for both prognosis and treatment.

Ultrasound is significantly more sensitive than physical examination alone in determining axillary lymph node involvement in breast cancer staging. For lymph nodes that remain indeterminate or suspicious on ultrasound, fine-needle aspiration biopsy or core biopsy provides a definitive diagnosis. The most commonly used staging system is the TNM staging system. The American Joint Committee on Cancer (AJCC) recently modified the TNM system. According to the latest TNM staging, the presence of macrometastases in ipsilateral level 1 and level 2 axillary lymph nodes classifies breast cancer as stage 2A or higher. Although biopsy sampling has a high diagnostic accuracy, it requires experienced pathologists, and the evaluation process can take weeks. Therefore, there is a need for faster and more cost-effective diagnostic methods for detecting axillary metastases.

The lymph node washout technique is used to determine lymph node metastasis in malignancies such as papillary thyroid carcinoma, the most common type of thyroid cancer. Papillary thyroid carcinomas generally do not exhibit aggressive clinical behavior and have favorable long-term prognoses. However, at the time of diagnosis, cervical and mediastinal lymph node involvement ranges from 27% to 46%, and the postoperative recurrence rate is between 3% and 30%. Thus, determining preoperative lymph node involvement and distinguishing it from benign reactive lymphadenitis is essential. For this purpose, fine-needle aspiration with thyroglobulin washout sampling has recently become widely used worldwide. In patients with papillary thyroid carcinoma, fine-needle aspiration of cervical lymph nodes and testing for thyroglobulin, a protein normally present in thyroid tissue, is considered one of the most reliable methods for detecting lymph node metastasis. A large-scale study by Moon et al. found that fine-needle aspiration thyroglobulin washout had a sensitivity of 93.2% and a specificity of 95.9% in detecting lymph node metastases in papillary thyroid carcinoma.

Immune checkpoints have gained attention, particularly after being awarded the Nobel Prize in 2018, as they play a crucial role in understanding the relationship between cancer and the immune system. PD-1 and its ligand PD-L1 function as immune checkpoints by inhibiting T-cell receptor signaling and co-stimulatory signals. T-cell immunoglobulin and mucin domain 3 (TIM-3) is expressed on interferon-γ-producing T cells, regulatory T cells (Tregs), dendritic cells, B cells, macrophages, natural killer (NK) cells, and mast cells. Dysregulation of TIM-3 expression has been linked to autoimmune diseases. High TIM-3 expression is associated with T-cell exhaustion and suppression of T-cell responses, particularly in chronic viral infections and tumor development. The clinical success of immune checkpoint inhibitors such as ipilimumab and nivolumab in melanoma and lung cancer has further increased interest in immune checkpoints.

Numerous studies in the literature have investigated the expression of immune checkpoints such as PD-1, PD-L1, and CTLA-4 on the cell surface in breast cancer patients. One study evaluating the genetic expression of immune checkpoints in breast cancer found a negative correlation between B7-H3 mRNA expression and survival, while a positive correlation was observed between survival and the expression of CTLA-4 and tyrosine-based inhibitory motif domain (TIGIT). Another study reported decreased Lymphocyte-activation gene 3 (LAG-3) expression in triple-negative breast cancer and human epidermal growth factor receptor 2 (HER2) positive breast cancers.

Natural soluble forms of immune checkpoint receptors and ligands also exist in body fluids and play a role in immune regulation, although their exact mechanisms remain unclear. Many studies are investigating the role of immune checkpoints in cancer patients. One study evaluated both tumor-associated and soluble levels of PD-L1 and CTLA-4 in the blood, finding that elevated levels of PD-L1 and CTLA-4 were associated with poor prognosis. There are five studies in the literature, including one report, evaluating soluble immune checkpoints in breast cancer patients.

Since immune checkpoints are present on the cell surface and in soluble form in blood and body fluids, they are also highly likely to be found in tumor-metastasized regions. In the literature there is no study using these immunological markers to determine metastasis in patients with invasive ductal carcinoma and metastatic axillary lymph nodes.

In this study, the investigators will evaluate the soluble levels of immune checkpoints commonly associated with cancer-sCD25 (IL-2Ra), 4-1BB, B7.2 (CD86), Free Active Transforming growth factor (TGF)-β1, CTLA-4, PD-L1, PD-1, Tim-3, LAG-3, and Galectin-9-in washout fluid samples obtained from metastatic axillary lymph nodes and benign lymph nodes in patients with invasive ductal breast carcinoma. The effectiveness of immune checkpoints and the washout technique in diagnosing lymphatic metastasis will be assessed as a rapid diagnostic method.