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Imprinted H19 Oncofetal RNA Is a Candidate Tumour Marker for Hepatocellular Carcinoma

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Imprinted H19 Oncofetal RNA Is a Candidate Tumour Marker for Hepatocellular Carcinoma J Clin Pathol:Mol Pathol 1998;51:21–25 21 Imprinted H19 oncofetal RNA is a candidate tumour marker for hepatocellular carcinoma I Ariel, H-Q Miao, X-R Ji, T Schneider, D Roll, N de Groot, A Hochberg, S Ayesh Abstract This observation was confirmed recently by Li Aims/Background—To study the expres- et al, who found parallel expression of H19 and sion of the H19 gene in hepatocellular car- insulin-like growth factor 2 (IGF II).15 cinoma. H19 is an imprinted, maternally H19 is one of the few genes that functions as expressed gene, which is tightly linked, RNA molecules,16 similar to Xist,17 the im- both physically and functionally, to the printed gene in the Prader-Willi syndrome paternally expressed insulin-like growth region18 and the 3' untranslated region of á factor 2 (IGF II). IGF II is known to be tropomyosin.19 In these cases, a protein prod- involved in liver carcinogenesis. H19 was uct does not exist, and gene expression can, first discovered in the fetal mouse liver to therefore, be determined only by the presence be under the same regulatory genes as á of RNA transcripts in the tissue. We performed fetoprotein (áFP), a widely used tumour radioactive and non-radioactive in situ hybridi- marker for hepatocellular carcinoma. sation (ISH) for H19 RNA on paraYnwax Methods—Using both radioactive and sections of 18 hepatocellular carcinomas and non-radioactive in situ hybridisation, the examined its expression in both the tumour expression of the H19 gene was compared and adjacent liver tissue (when present in the with the presence of áFP,as demonstrated section). by immunohistochemistry, in 18 cases of Alpha fetoprotein (áFP) is a widely used hepatocellular carcinoma. tumour marker in hepatocellular carcinoma.20 21 Results—H19 expression was present in 13 The expression of áFP mRNA in mice is Department of of 18 cases, whereas staining for áFP was determined by at least two transacting genes, Pathology, Perinatal positive in only nine of 18 cases. Concord- raf and Rif. The former determines the basal Pathology and ance was found in 12 of 18 tumours levels of áFP, while the latter induces its Quantitative Molecular 14 Pathology Unit, (66.7%). In general, the staining pattern expression during regeneration. The H19 Hadassah University for H19 was more diVuse than the immu- gene was first characterised as a locus unlinked Hospital Mount nohistochemical staining for áFP. to áFP, which is also regulated by raf and Rif in Scopus, PO Box 24035, Conclusions—The addition of a non- the liver, but not in other tissues.14 il-91240 Jerusalem, radioactive in situ hybridisation assay for We examined the presence of áFP by immu- Israel H19 RNA to the panel of tumour markers nohistochemical staining, and correlated our I Ariel D Roll used for the histopathological and cyto- findings with the expression of H19 in these logical diagnosis of hepatocellular carci- samples. We found that H19 expression is con- Department of noma might be useful. cordant with the presence or absence of áFP in Biological Chemistry, (J Clin Pathol:Mol Pathol 1998;51:21–25) the tumour in two thirds of the cases, and the Institute for Life ISH for H19 was found to be positive in more Sciences, Hebrew Keywords: hepatocellular carcinoma; H19; cases, although the diVerence was not found to University of á fetoprotein Jerusalem, Jerusalem, be significant in this small series. We suggest Israel that ISH for H19 RNA should be examined T Schneider further, as this molecule might be a useful N de Groot Imprinted genes—genes that are expressed additional tumour marker for the histopatho- A Hochberg from only one allele, depending on the parental logical and cytological diagnosis of hepatocel- S Ayesh origin—are implicated in playing an important lular carcinoma. role in the tumorigenesis of certain human Department of neoplasms.1–4 To date, the H19 gene is one of Oncology, Tumor Methods and materials Biology Research Unit, the few genes proved to be imprinted in SAMPLES 56 Hadassah University humans. Although it has been suggested by ParaYn wax blocks of 18 patients with hepato- Hospital, Kiryat other investigators that H19 acts as a tumour cellular carcinoma from the department of Hadassah, Jerusalem, suppressor gene,7 we have shown that it exhib- pathology, Qingdao University School of Israel its oncofetal characteristics, and is re-expressed H-Q Miao Medicine in Qingdao, China were submitted in tumours arising from tissues that express the for the study. The medical charts were 8–11 Department of gene in fetal life. One of the organs that searched for clinical and laboratory data Pathology, Qingdao expresses H19 abundantly from the early including tumour size, serum áFP, and hepati- University School of stages of embryogenesis is the liver.12 13 Expres- tis B serum antigen (HBsAg). Medicine, Qingdao, sion is confined to hepatocytes and mesenchy- We performed radioactive and non- China mal tissues, and is undetectable in haemapoi- X-R Ji radioactive ISH studies for H19 RNA and an etic tissue, one of the main tissue components immunohistochemical study for áFP according 10 13 Correspondence to: in the developing liver. H19 expression is to the following protocols. Dr Ariel. downregulated in most adult tissues, including email: [email protected] the liver, but may reappear in the liver during PREPARATION OF H19 TRANSCRIPTION VECTOR 14 Accepted for publication regeneration. We were the first to document Human H19 gene (800 base pairs) was 4 December 1997 H19 expression in hepatocellular carcinoma.10 subcloned at EcoRI site into Bluescript II KS 22 Ariel, Miao, Ji, et al Table 1 Clinical data of 18 patients with hepatocellular carcinoma and the results of the RADIOACTIVE IN SITU HYBRIDISATION in situ hybridisation for H19 RNA and immunohistochemistry for á fetoprotein The procedure was performed on paraYnwax sections as described in detail previously.10 The Case Sex Age Tumour size HBsAg sáFP áFP H19 slides were examined and photographed using 1 M 47 Multicentric + + + f + d an Axioplan 2 (Carl Zeiss Jena GmbH, Göttin- 2 NANANA NANA+f+d 3 M 49 Biopsy − − + d + d gen, Germany) microscope under bright and 4 M 63 Multicentric + + + f + d dark field illumination. 5 F 44 Multicentric + − − + f The negative controls in this study included 6F644×4×4cm + − − +f 7 NANANA NANA+f+d hybridisation with sense RNA probe and 8 M 45 Multicentric + + − − RNase prehybridisation treatment. Negligible 9 M 59 10 × 10 × 10 cm + + − − signal was observed in all the controls. Positive 10 F 48 Multicentric + + − + f 11 F 49 10 × 10 × 10 cm + + + d − controls consisted of sections of fetal renal and 12 M 55 Multicentric − − − + f liver tissue. 13 M 41 Multicentric + + + d + d 14 M 67 Multicentric − + − + f × × 15 M 40 9 8 6cm + + +d +d PREPARATION OF RIBOPROBE FOR 16 M 63 7 × 7 × 7cm + + − − 17 M 52 22 × 14 × 8cm − − +d +d NON-RADIOACTIVE IN SITU HYBRIDISATION 18 M 49 7 × 7 × 7cm + + − − Digoxigenin labelled in vitro RNA transcripts were produced by labelling with DIG-11-UTP HBsAg, hepatitis B surface antigen; sáFP, serum á fetoprotein; f, focal; d, diVuse; NA, data not available. by SP6, T3, or T7 RNA polymerase in an in vitro transcription reaction (Boehringer Mann- plasmid (Stratagene) behind the T7 and T3 heim). RNA polymerase binding sites. In vitro RNA transcription with T7 RNA polymerase was used to produce antisense H19 cRNA from NON-RADIOACTIVE IN SITU HYBRIDISATION linearised plasmid DNA. Sense H19 RNA pre- Sections were prepared as for radioactive ISH, pared with T3 polymerase was used for and were rehydrated through a series of control. alcohols, followed by a wash in 0.9% NaCl and then a wash in phosphate buVered saline (PBS). Basic proteins were removed by incuba- PREPARATION OF RIBOPROBE FOR RADIOACTIVE IN tion in 0.1 N HCl at room temperature for 15 SITU HYBRIDISATION minutes. After a further wash in distilled water, 35S-labelled in vitro RNA transcripts (107 cpm/ sections were treated with 10 µg/ml proteinase µg) were produced using the Amersham RPN K (Sigma, Poole, Dorset, UK) in 50 mM Tris, 2006 Kit and RNA polymerases from Boe- 5 mM EDTA for 30 minutes at 37°C, rinsed in hringer Mannhein (Mannheim, Germany). PBS, and then refixed for five minutes in 4% Linearised plasmids were prepared by diges- paraformaldehyde/PBS. Thereafter, the sec- tion with HindIII (antisense) and EcoRI tions were rinsed in PBS and acetylated for 10 (sense). The fragments were separated from minutes in fresh acetic anhydride diluted 1/400 unincorporated nucleotidases by ethanolic in 0.1 M triethanolamine (Sigma) at pH 8.0. precipitation. The slides were then rinsed in PBS for five Figure 1 (A) Immunohistochemical staining for á fetoprotein in hepatocellular carcinoma. Positive staining is present in single tumour cells (arrows). (B) H19 expression in the same tumour, as demonstrated by digoxigenin labelled in situ hybridisation. Expression is evident in most tumour cells. H19 in hepatocellular carcinoma 23 Figure 2 H19 expression in hepatocellular carcinoma. (A) Bright and (B) dark field. The expression is most prominent at the boundary of the tumour nodule. (In situ hybridisation of 35S-labelled antisense of H19 with haematoxylin and eosin as a counterstain.) minutes and in 0.9% NaCl for another five probe; and hybridisation with hybridisation minutes, dehydrated, and air dried. buVer without the probe. A section of fetal kid- The hybridisation buVer contained 50% ney, which expresses H19 in the metanephric deionised formamide, 0.3 M NaCl, 20 mM blastema, was used as a positive control. Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM × NaH2PO4 (pH 8.0), 10% dextran sulphate, 1 Denhardt’s solution, and 0.5 µg/ml yeast IMMUNOHISTOCHEMISTRY A polyclonal rabbit primary antibody was used tRNA.
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