Proteomic profiling of FFPE specimens: Discovery of HNRNPA2/B1 and STT3B as biomarkers for determining formalin fixation durations.

Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long durations. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-duration acid formalin(AF)-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-duration neutral buffered formalin-fixed liver and lung tissues and granular staining in all long-duration AF-fixed FFPE tissue types. Similar trends were observed in the long-duration fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. SIGNIFICANCE: The quality of FFPE samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using LC/MS and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation duration as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin fixation duration for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics.

A dose-dependent decrease in the fraction of cases harboring M6P/IGF2R mutations in hepatocellular carcinomas from the atomic bomb survivors.

The risk for hepatocellular carcinoma (HCC) development is significantly heightened in the atomic bomb survivors, but the mechanism is unclear. We have previously reported finding a radiation dose-dependent increase in HCCs with TP53 mutations from the survivors. We now show that, in the same HCC samples, the frequency of 3'-untranslated region (3'UTR) mutations in M6P/IGF2R, a candidate HCC tumor suppressor gene, decreases with dose (P = 0.0091), implying a radiation dose-dependent negative selection of cells harboring such mutations. The fact that they were in the 3'UTR implicates changes in transcript stability rather than in protein function as the mechanism. Moreover, these M6P/IGF2R 3'UTR mutations and the TP53 mutations detected previously were mutually exclusive in most of the tumors, suggesting two independent pathways to HCC development, with the TP53 pathway being more favored with increasing radiation dose than the M6P/IGF2R pathway. These results suggest that tumors attributable to radiation may be genotypically different from tumors of other etiologies and hence may provide a way of distinguishing radiation-induced cancers from "background" cancers--a shift from the current paradigm.