Application of a Targeted Proteomics Strategy for the Development of a Blood Protein Signature for Breast Cancer Stratification and Prognostication

Abstract

Breast cancer (BC) is a common malignancy in women worldwide, affecting roughly 3 million and of these 15 to 20% of cases are triple-negative breast cancer (TNBC). In comparison to other types of breast cancer, TNBC is distinguished by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. This makes it a particularly challenging therapeutic target due to its lack of specific receptor signalling pathways that can be targeted therapeutically. Patients with TNBC often experience systemic relapse and poor prognosis. The traditional treatment guidelines for early TNBC are based on chemotherapy, surgery, and radiation to prevent disease recurrence. Although novel treatments are constantly being introduced for BC, conventional cytotoxic chemotherapy is the primary treatment option for TNBC. Therefore, the approach to treating early TNBC has shifted towards neoadjuvant treatment (NAC), given to the patient before surgery. This approach aims to reduce tumour size, thus improving the chances of surgical intervention and reducing the risk of recurrence. Research has proven that NAC prior to surgery is associated with 30% of patients achieving pathological complete response (pCR), directly linked to improved survival rates. Thus, a novel predictive biomarker of NAC response is paramount to selecting patients with good prognoses, de-escalating systemic treatment to minimize chemotherapy toxicities, and increasing the likelihood of achieving pCR. So, the objective of this study is to apply mass spectrometry-based proteomics to serum samples from TNBC patients (both those who achieved pCR and those who did not) who have received NAC to identify i) biomarkers that might measure response to NAC, ii) biomarkers that might correlate with the extent of residual disease. Serum samples from TNBC patients were provided by the Royal College of Surgeons in Ireland (RCSI), following ethical guidelines and approval. Patient samples included in this study had undergone NAC treatment, anthracyclines and taxanes, for approximately 3-5 months before surgery. Patients were then distinguished based on whether they achieved pCR during surgery or not. In this study, there are two essential proteomic approaches to identify and initially verify biomarkers: discovery and targeted proteomics. Label-free LC-MS/MS based protein discovery was undertaken on depleted serum samples from both patient groups, revealing a total of 19 significant proteins and 124 unique peptides, that could be beneficial for classifying TNBC patients who would achieve residual disease after the NAC. These candidate biomarkers along with those identified in literature reviews, and tissue-based gene expression data (Human Protein Atlas), are being incorporated into the development of targeted proteomics multiple reaction monitoring (MRM) assays. Robust MRM assays were successfully designed and developed for a total of 73 proteins represented by x 84 peptides to facilitate further verification and evaluation of these proteins of interest. MRM measurements of the 73 candidate biomarker proteins were performed on serum samples acquired from the same patients used in the discovery approach, verifying the significant changes in expression between patients with and without disease recurrence. The results identified a subpanel of 6 proteins, with 5 identified in the discovery approach and one from the literature, indicating potential for prognosis assessment. The second part of the study utilized three different TNBC cell lines to assess the robustness of the developed MRM assays. Several detectable proteins were identified across the three cell lines, CAL-5, MDA-MB231 and MDA-MB 436 cells. Moreover, the successful performance of the MRM assays led to the identification of three proteins that were significantly expressed across the three cell lysates, offering insight into potential functions and aiding in understanding the variation observed in TNBC. All protein measurements reported in this study were achieved with a high degree of reproducibility resulting from the technical rigor under which all phases of the experimental process were undertaken. Overall, these studies highlight the utility of mass spectrometry-based proteomics to address key unmet clinical needs in TNBC. Namely, the need for novel serum-based protein biomarkers to distinguish and early prognostication between TNBC patient groups who are being treated with NAC. Besides, the development of a robust MRM method for identifying protein biomarkers associated with NAC responses in both TNBC group may serve as a valuable resource for ongoing research and in the BC research community internationally.