Imaging Mass Spectrometry Reveals Modified Forms of Histone H4 As New Biomarkers of Microvascular Invasion in Hepatocellular Carcinomas

Abstract

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...

A computed tomography radiogenomic biomarker predicts microvascular invasion and clinical outcomes in hepatocellular carcinoma

“…Similarly, nuclear grade, which is a known independent predictor of prognosis, also relies on tissue procurement and, when assessed on preoperative biopsy specimens, is equally susceptible to sampling error . The same is true of more recent proteomic and genomic markers of MVI, which require invasive tissue acquisition . Previous attempts to noninvasively predict MVI have utilized demographic criteria, serum proteins, and imaging features .…”

JARID2 promotes invasion and metastasis of hepatocellular carcinoma by facilitating epithelial-mesenchymal transition through PTEN/AKT signaling

“…Data of conventional clinical and pathological variables were also collected for analysis, including age, gender, hepatitis B status, liver cirrhosis, Edmondson-Steiner grade, capsular formation, size of the tumor, number of tumor nodes, microvascular invasion, Child-Pugh classification, TNM stage and BCLC stage. Microvascular invasion was defined as tumor cells forming a thrombus in peritumoral vessels, can only be assessed after careful histological assessment of the whole surgical specimen [38, 39]. The follow-up status and any recurrence were regularly updated in the database for each patient.…”

Discussion point: reporting guidelines for mass spectrometry imaging

“…1 is often only the starting point for further evaluation and analysis of the acquired data. Figure 2 shows examples of the basic data analysis workflow of recently published clinical MSI investigations of proteins, which can be summarized as diagnostic biomarker discovery [28] (A), prognostic biomarker discovery [29] (B), and MSI-based molecular cartography [30] (C). In all examples the MSI data sets are registered to the histological images (the histological images can be recorded from the same section after MSI data acquisition or from adjacent sections).…”