Microvascular invasion (MiVI) is a major risk factor in postoperative tumor recurrence and mortality in hepatocellular carcinoma (HCC). Unfortunately, this histological feature is usually missed by liver biopsy because of limited sampling, and MiVI is commonly detected only after surgery and examination of the full resected specimen. To date, there exists no reliable tool for identifying MiVI prior to surgical procedures. This study aimed to compare the proteome of HCC with and without MiVI in order to identify surrogate biomarkers of MiVI. A training cohort comprising surgically resected primary HCC with MiVI (n 5 30) and without MiVI (n 5 26) was subjected to matrixassisted laser desorption ionization imaging mass spectrometry (MALDI IMS). Comparative analysis of acquired mass spectra of the two groups yielded 30 differential protein peaks, among which 28 were more strongly expressed in HCC with MiVI. Among these, two peaks were identified as N-term acetylated histone H4 dimethylated at lysine (K) 20, and N-term acetylated histone H4 dimethylated at K20 and acetylated at K16. Both peaks were validated in the training cohort and in an independent validation cohort (n 5 23) by immunohistochemistry and western blot. Conclusion: These results demonstrate the potential of MALDI IMS for uncovering new relevant biomarkers of MiVI in HCC, and highlight the role of epigenetic modifications in the prognosis of HCC. Preoperative detection of modified forms of histone H4 expression in tumor biopsies would be helpful in management of patients with HCC. (HEPATOLOGY 2013;58:983-994) H epatocellular carcinoma (HCC) is the main primary malignancy of the liver, and ranks as the third most common cause of cancerrelated death worldwide, despite significant efforts made in follow-up of cirrhosis patients and screening of HCC.1 HCC prognosis remains poor, mainly related to a high rate of tumor recurrence following surgical treatment that attains 70% at 5 years after liver resection and 15%-30% after liver transplantation (LT).2 Microvascular invasion (MiVI) is a major risk factor for recurrence and mortality in HCC.3-6 Importantly, MiVI, defined as malignant cells in peritumoral vessels, can only be assessed after careful histological assessment of the whole surgical specimen. Thus, there Abbreviations: ACN, acetonitrile; H4K16ac, histone H4 acetylated at lysine 16; H4K20me2, histone H4 dimethylated at lysine 20; HCC, hepatocellular carcinoma; HCC/MiVI1, hepatocellular carcinoma with microvascular invasion; HCC/MiVI2, hepatocellular carcinoma without microvascular invasion; LT, liver transplantation; LTQ, linear trap quadrupole; K, lysine; MALDI IMS, matrix-assisted laser desorption ionization imaging mass spectrometry; MiVI, microvascular invasion; MS, mass spectrometry; MS/MS, tandem mass spectrometry; nano-LC-ESI-MS/MS, nano-liquid chromatography, electrospray ionization, tandem mass spectrometry; PTMs, posttranslational modifications; RP-micro-LC, reverse-phase micro-liquid chromatography; SA, sinapinic acid; TFA, trifluoroa...

Severe dental troubles are associated with X-linked hypophosphatemic rickets and are mainly related to impaired dentin mineralization. In dentin matrix, matrix extracellular phosphoglycoprotein (MEPE) may be protected from proteolysis by a specific interaction with PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome). The objective of our work was to determine whether PHEX impairment induces MEPE cleavage in dentin and the subsequent release of the C-terminal acidic serine-and aspartate-rich motif (ASARM) peptide, which is known to inhibit mineralization. By Western blot analysis, we explored dentin extracts from seven hypophosphatemic patients with mutations of the PHEX gene. A proteomic approach combining immunoprecipitation, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry and matrix-assisted laser desorption ionizationtime of flight analysis of the samples completed this exploration. This study shows a 4.1-kDa peptide containing the MEPE-derived ASARM peptide in hypophosphatemic samples. The presence of ASARM was less marked in patients treated with 1-hydroxylated vitamin D and phosphate during growth. Moreover, recombinant ASARM implanted in a rat pulp injury model disturbed the formation of the reparative dentin bridge. These results suggest that abnormal MEPE cleavage occurs when PHEX activity is deficient in humans , the ASARM peptide may be involved in the mineralization defects and the PHEX-MEPE interaction may be indirect, as ensuring a better phosphate and vitamin D environment to the mineralizing dentin prevents MEPE cleavage. (Am J Pathol