Data independent acquisition mass spectrometry (DIA-MS) analysis of FFPE rectal cancer samples offers in depth proteomics characterization of response to neoadjuvant chemoradiotherapy

Background Understanding the molecular features associated with response to neoadjuvant chemoradiotherapy is an unmet clinical need in locally advanced rectal cancer (LARC). The aim of the study was to apply a high-sensitivity proteomic approach for in-depth characterization of the LARC proteome in search of patients who might have a good response to preoperative treatment and potentially be followed by a watch-and-wait strategy, rather than having immediate surgery, maximizing the therapeutic effect and quality of life. Methods A total of 97 LARC patients treated at the Institute for Oncology and Radiology of Serbia in the period of 2018-2019 were included in the study. Patients were treated with long-course chemoradiotherapy (CRT): Radiotherapy (RT) was delivered with a total dose of 50.4 Gy in 28 fractions; concomitant chemotherapy (5-FU, 350 mg/m 2 daily) and Leucovorin (25 mg/m 2 daily) was administered during the first and the fifth week of RT. Patients were evaluated in week 6-8 after treatment completion with pelvic MRI scan and rigid proctoscopy. Pathohistological response after surgery was assessed according to tumor regression grading (TRG) categories by Mandard. Twenty biopsy samples taken at diagnosis were used for proteomic analysis, 9 responders (R, TRG 1-2), and 11 non-responders (NR, TRG 3-5), to achieve the maximum range of different molecular features potentially associated with response. Formalin-fixed paraffin-embedded (FFPE) biopsies were processed, and isolated proteins were digested with trypsin. The resulting peptides were analyzed by liquid chromatography coupled to a Q Exactive HF-X mass spectrometer operated in data independent mode (DIA-MS). Data analysis was performed with DIA-NN and Perseus. Data are available via ProteomeXchange with the identifier PXD040451. Results The use of DIA-MS allowed the identification and quantification of more than 3,000 proteins per sample in general, a significant increase when compared to the 1,000 proteins previously identified by Data Dependent Acquisition-MS (DDA-MS) in LARC FFPE samples. In total, 4,849 proteins were identified in 20 rectal cancer FFPE samples. Principal Component Analysis (PCA) indicated that responders had a significantly different proteomic profile than non-responders. Statistical analysis of the two groups resulted in the identification of 915 differentially expressed proteins (DEPs) (215 in responders and 700 in non-responders, p<0.05), and 384 with more stringent criteria (p<0.01). Results indicate that some of the leading signaling pathways that correlate with response include the metabolism of RNA, MYC targets, neutrophil degranulation, cellular transport, and response to stimuli. Conclusions The DIA-MS approach offered unprecedented proteome coverage for FFPE samples. The differentially expressed proteins and biological processes constitute interesting findings that hold the potential for improving LARC patient management.