Decoding CRC progression: impact of patient individuality on the proteomic phenotype

Colorectal cancer (CRC) is one of the most prevalent malignant tumors and the second most common cause of cancer-related mortality worldwide. Overall, the prognosis for CRC with liver metastasis is inferior and little is known about the patient-individual metastasis. The study aimed to assess significant differences in protein expression using a unique set of paired and unpaired patient samples respecting tumor heterogeneity.

We analysed formalin-fixed paraffin-embedded (FFPE) tissue samples derived from healthy colon mucosa (NM), colorectal carcinoma (T) and liver metastases (LM, n=78 patients). Paired tissue samples (NM-T-LM) were obtained from 18 patients.

For global proteome analysis, we employed DIA-PASEF (data-independent acquisition - Parallel Accumulation-Serial Fragmentation) mass spectrometry coupled with neural-network-based, library-free protein identification. For global proteomic profiling, we employed principal component analyses, differential expression analyses and gene set enrichment analyses.

Protein profiling revealed 6.673 identified proteins after strict raw data processing. Based on the group comparison results, clear separation between the NM from T and LM samples was detected in a principal component analysis. To assess the impact of patient individuality, matched and unmatched T were compared to LM and revealed 298 (matched) and 246 (unmatched) differentially expressed proteins (q-values ≤ 0.05, net log2 fold-change ≥ 1, Figure 1A-B). Interestingly, 94 and 42 proteins were unique for the matched and unmatched test, respectively (Figure 1C).

Our research results highlight the importance of individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between NM, primary T, and LM samples from individuals, which might accelerate implementation of precision oncology in the future.

Figure 1: Differential expression analysis results. A: Volcano plot visualizing the differential expression analysis results of LM samples compared to their T pairs. Significance was considered at q-value ≤ 0.05 and net log2 fold change ≥ 1. B: Volcano plot visualizing the differential expression analysis results of LM compared to unpaired T samples. For group size balancing, 18 T samples were randomly selected. C: UpSet plot visualizing set intersections and unique sets of differentially expressed proteins for paired and unpaired testing setups.