Characterization of anticoagulant heparinoids by immunoprofiling

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and–when additional saccharides are present–inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.
Introduction
Glycosaminoglycans (GAGs) are long, unbranched, negatively charged polysaccharide chains. The precursor of heparin and heparan sulfate (HS) is composed of alternating N-acetylglucosamine (GlcNAc) and d-glucuronic acid (GlcA) residues. In chondroitin sulfate (CS) and dermatan sulfate (DS), the aminosugar is a N-acetylgalactosamine (GalNAc) instead of GlcNAc. The polysaccharide chain precursors are modified by a sequential series of enzymatic reactions, including N-deacetylation/N-sulfation, C5-epimerization of GlcA to l-iduronic acid (IdoA), and O-sulfation at various positions [27, 32].
Heparin is widely used as an anticoagulant. A specific pentasaccharide sequence (GlcNAc/NS(6OS)–GlcA–GlcNS(3,6OS)–IdoA(2OS)–GlcNS(6OS)) [5, 23, 37] in heparin binds with high affinity to the enzyme inhibitor antithrombin III, causing a conformational change, resulting in the exposition of its active sites and in inactivation of factor Xa. For the inactivation of factor IIa, an additional site in heparin proximal to the pentasaccharide is necessary. The formation of a ternary complex between antithrombin III, factor IIa and heparin, which requires at least 18 saccharide residues for efficient formation, subsequently results in the inactivation of factor IIa [23]. Next to heparin, other heparinoids are used, including preparations containing other GAGs such as HS and DS, low molecular weight heparins (LMWHs), and fondaparinux, a synthetic pentasaccharide identical in structure to the antithrombin III-binding pentasaccharide. Heparinoids are used to treat patients with venous thrombosis and pulmonary embolism [2, 5, 10, 22]. In addition, they are used to prevent thrombosis in patients that undergo surgery and to prevent clotting in the extracorporeal circulation during hemodialysis [2, 5, 10, 22, 24, 28]. Due to their lower molecular weight, LMWHs have a high anti-factor Xa activity compared to their anti-factor IIa activity resulting in a more refined regulation of coagulation [4, 24]. In addition, the biological half-life of LMWHs/fondaparinux is longer in comparison to heparin, and the incidence of heparin-induced thrombocytopenia (HIT) is substantially reduced [6, 7, 23].
LMWHs are synthesized from heparin by controlled chemical or enzymatic depolymerization processes, resulting in the production of oligosaccharides of various lengths (Table 1). Dalteparin and nadroparin are produced by deamination using nitrous acid, resulting in the formation of an unnatural 2,5-anhydromannitol residue at the reducing end of the resulting oligosaccharides. Enoxaparin is produced by chemical β-elimination at alkaline pH, whereas tinzaparin is produced by enzymatic β-elimination by heparinase. Both processes result in the formation of an unsaturated uronic acid residue at the non-reducing end of the GAG fragments [12, 17].
Table 1Characteristics of therapeutic heparinoidsHeparinoid: generic name (brand name)Composition (and method of heparin modification)Molecular weight, kDaHeparinHeparin5.0–30.0Dalteparin (Fragmin)LMWH (deaminative cleavage with nitrous acid)4.0–6.0Nadroparin (Fraxiparine)LMWH (deaminative cleavage with nitrous acid)2.4–7.2Enoxaparin (Clexane, Lovenox)LMWH (β-eliminative cleavage by alkaline treatment)3.5–5.5Tinzaparin (Innohep, Logiparin)LMWH (β-eliminative cleavage by heparinase)3.4–5.6Sulodexide (Vessel Due F)80% ‘fast-moving’ heparina, 20% DS<8.0Danaparoid (Orgaran)84% HS, 12% DS, 4% CS4.0–10.0Fondaparinux (Arixtra)Pentasaccharide: C31H43N3Na10S81,728All drugs are derived from porcine intestinal mucosa, with the exception of the chemically synthesized pentasaccharide fondaparinux.LMWH Low-molecular weight heparin, HS heparan sulfate, DS dermatan sulfate, CS chondroitin sulfateaHeparin that is less sulfated and has a lower molecular weight [42].
All heparinoids, with the exception of fondaparinux, are heterogeneous mixtures of different GAG chains. Structural analysis of heparinoids is generally done by nuclear magnetic resonance spectroscopy, high pressure liquid chromatography, mass spectrometry, capillary electrophoresis, or polyacrylamide gel electrophoresis [17]. In general, these techniques require sophisticated instrumentation and sometimes extensive sample preparation. Alternative methodologies that are fast and simple are needed. Immunoprofiling of heparinoids could be a fast and simple method to characterize the complex heparinoids. Recently, a number of phage–display-derived antibodies were generated, reactive with various domain structures within heparin. In this study nine antibodies–all defining different heparin epitopes–were applied to study their reactivity with heparinoids.
Materials and methods
Heparinoids
The following heparinoids were included in the study: heparin (Sigma, St. Louis, MO), dalteparin (Pharmacia, Woerden, The Netherlands), nadroparin (Sanofi-Synthélabo, Maassluis, The Netherlands), enoxaparin (Aventis Pharma, Hoevelaken, The Netherlands), tinzaparin (LEO Pharma, Breda, The Netherlands), sulodexide (Alfa Wassermann S.p.A., Bologna, Italy), danaparoid (Organon, Oss, The Netherlands), and fondaparinux (Sanofi-Synthélabo, Maassluis, The Netherlands). Characteristics are given in Table 1.
Quantification of glycosaminoglycans by Farndale assay
A Farndale assay was done to determine the concentration of GAGs in the preparations [13]. To 50 μl of the standard (heparin, 0–100 μg/ml) and heparinoids 1 ml Farndale reagent (40.5 mmol/l glycine, 40.6 mmol/l NaCl, and 0.05 mmol/l 1,9-dimethylmethylene blue, pH 3.0) was added and absorbance was measured directly at 525 nm.
Determination of glycosaminoglycan classes by agarose gel electrophoresis
The different classes of GAGs in the heparinoids were separated by agarose gel electrophoresis, fixed in 0.1% (w/v) azure A in 10 mmol/l MgCl2·6H2O, 50 mmol/l Na–formiate and 0.001% (w/v) Na–azide (pH 3.5), and stained by combined azure A–silver treatment [38].
Enzymatic digestion of heparinoids
Heparinoids were diluted in heparinase I digestion buffer (50 mmol/l NaAc and 50 mmol/l Ca(Ac)2, pH 7.0) and/or chondroitinase ABC digestion buffer (25 mmol/l Tris and 2 mmol/l Mg(Ac)2, pH 8.0) to a final content of 4 mg/ml. The enzymes were added to a final concentration of 0.02 IU/ml for heparinase I (E.C. 4.2.2.7; from Flavobacterium heparinum; Seikagaku, Tokyo, Japan) and 0.04 IU/ml for chondroitinase ABC (E.C. 4.2.2.4; from Proteus vulgaris; Sigma). Heparinase I cleaves the linkages GlcNS±6S–IdoA2S predominantly present in heparin and to a minor extent in HS [11]. Chondroitinase ABC cleaves CS and DS [11]. The solutions were incubated for 16 h at 37°C.
Determination of glycosaminoglycan size by polyacrylamide gel electrophoresis
Heparinoids and their enzymatic digestion products were separated by polyacrylamide gel electrophoresis using a 33% gel [36], fixed in 0.8% (w/v) alcian blue in 2% (v/v) HAc, and stained by combined azure A–silver treatment [38].
Production of anti-heparin antibodies
Antibodies (Table 2) were obtained by biopanning against HS or heparin, using bovine kidney HS (antibodies HS4C3 [41], HS3A8, HS4E4 [8], and LKIV69 [43]), porcine intestinal mucosa heparin (antibodies EW3D10 and EW4G2 [39]), human lung HS (antibody NS4F5; Smits et al., unpublished results), and mouse and human skeletal muscle GAG (antibodies AO4B08 and RB4EA12 [15], respectively). Large scale production of soluble antibodies was performed as described before [8, 41].
Table 2Characteristics of epitope-specific anti-heparin antibodiesAntibodyVH CDR3 sequenceVH familyDP geneGAG used for selectionEssential modificationsInhibitory modificationsHS4C3GRRLKD338Bovine kidney HS [41]N-sulfation 2-O-sulfation (minor importance)6-O-sulfation3-O-sulfationHS3A8GMRPRL338Bovine kidney HS [8]N-sulfation C5-epimerization2-O-sulfation6-O-sulfation (likely)HS4E4HAPLRNTRTNT338Bovine kidney HS [8]N-acetylation2-O-sulfationa 6-O-sulfationN-sulfationC5-epimerizationLKIV69GSRSSR338Bovine kidney HS [43]N-sulfation6-O-sulfationC5-epimerization (likely)2-O-sulfationEW3D10GRTVGRN338Porcine intestinal mucosa heparin [39]Sulfation required, position(s) unknownbEW4G2GKVKLPN338Porcine intestinal mucosa heparin [39]Sulfation required, position(s) unknownbNS4F5SGRKGRMR353Human lung HScN-sulfation C5-epimerization2-O-sulfation (high)6-O-sulfation (high)AO4B08SLRMNGWRAHQ347Mouse skeletal muscle GAG [15]N-sulfation C5-epimerization2-O-sulfationd6-O-sulfation (high)RB4EA12RRYALDY332Human skeletal muscle GAG [15]N-acetylation N-sulfation6-O-sulfationMPB49 (control)WRNDRQ338––Given are the antibody name, amino acid sequence of the VH complementary determining region 3 (CDR3), VH germ line gene family, DP gene number, selection moiety, and relevant modification reactions required for antibody binding. Table derived from Wijnhoven et al. unpublished results.GAG Glycosaminoglycan, HS heparan sulfateaAntibody HS4E4 possibly requires as yet unspecified O-sulfated residues [16].bAlthough the chemical nature of the specific heparin/HS structure recognized is not exactly known, the antibody defines a unique sulfated heparin/HS structure as demonstrated by its specific staining pattern on renal cryosections [8, 39].cSmits et al., unpublished resultsdAntibody AO4B08 requires an internal 2-O-sulfated iduronic acid residue [16].
Enzyme-linked immunosorbent assay
Reactivity of the anti-heparin antibodies with the heparinoids was evaluated by enzyme-linked immunosorbent assay (ELISA). Wells of microtiter plates were coated overnight by incubation with the heparinoids that bound by passive adsorption [41]. After blocking with PBS containing 3% (w/v) BSA and 1% (v/v) Tween-20 for 1 h, anti-heparin antibodies [in PBS containing 1% (w/v) BSA and 0.1% (v/v) Tween-20] were added for 1.5 h. As a negative control, antibody MPB49 was used, which is >95% identical to most antibodies used, but does not bind any GAG. Bound antibodies were detected using mouse IgG anti-VSV tag antibody P5D4 (1:10; Boehringer Mannheim, Mannheim, Germany), followed by incubation with alkaline phosphatase-conjugated rabbit anti-mouse IgG (1:2,000; Dako, Glostrup, Denmark), both for 1 h. Enzyme activity was detected using 100 μl 1 mg p-nitrophenyl phosphate (ICN, Aurora, OH)/ml 1 M diethanolamine/0.5 mM MgCl2 (pH 9.8) as a substrate. Absorbance was measured at 405 nm [43]. Background reactivity, measured with the control antibody MPB49, was substracted. The absorbance with control antibody MPB49 was comparable to the absorbance measured when primary antibody was omitted.
Immunofluorescence staining
Human kidney cryosections (2 μm) were incubated with anti-heparin antibodies in the absence or presence of 100 μg/ml of the heparinoids. Bound antibodies were detected as described [43].
Results
General biochemical analysis of heparinoids
The different classes of GAGs in the heparinoids were separated by agarose gel electrophoresis (Fig. 1). Heparin showed a band at about the same position as the HS standard, whereas the LMWHs dalteparin, nadroparin, enoxaparin, and tinzaparin migrated somewhat further into the gel. Danaparoid displayed bands at the level of HS and DS, whereas the CS band, which only represents 4% of the prepration (Table 1), could not be detected. Sulodexide clearly showed two bands, representing the ‘fast-moving’ heparin (heparin that is less sulfated and has a lower molecular weight [42]) and DS fractions. The chemically synthesized pentasaccharide fondaparinux, which has a much lower molecular weight compared to the other heparinoids, migrated further into the gel.
Fig. 1Agarose gel electrophoresis of heparinoids. The different classes of GAGs in the drugs were visualized by a combined azure A–silver staining procedure. The standard contains 40 ng HS, 20 ng DS, and 20 ng CS
To analyze the molecular range of the heparinoids, gel electrophoresis using 33% polyacrylamide was applied. The LMWHs clearly displayed distinct fragments with a lower molecular weight compared to heparin (Fig. 2a). The smaller fragments of enoxaparin compared to tinzaparin (both prepared by β-elimination) can be explained by a preference of heparinase for 2-O-sulfated iduronic acid in contrast to chemical alkaline treatment, which results in cleavage at both sulfated and non-sulfated iduronic acid residues. Sulodexide and danaparoid showed a broad smear with less distinct fragments, in line with their methods of preparation, which does not include a specific depolymerization step. The pentasaccharide fondaparinux demonstrated primarily one individual band. To further establish the nature of the heparinoids, they were subjected to enzymatic digestion using heparinase I and/or chondroitinase ABC (Fig. 2b for dalteparin, enoxaparin, and sulodexide). Heparin, dalteparin, nadroparin, enoxaparin, tinzaparin, and fondaparinux were only cleaved by heparinase I, in accordance with their derivation from heparin. Sulodexide was cleaved by heparinase I as well as chondroitinase ABC, which is in line with the heparin/DS nature of the preparation. Danaparoid was primarily cleaved by chondroitinase ABC, reflecting the presence of DS/CS, whereas heparinase I was less effective.
Fig. 2Thirty-three percent polyacrylamide gel electrophoresis of undigested (a) and enzymatically digested (b) heparinoids. Heparinoids were visualized by a combined alcian blue-silver staining procedure. Treatment with heparinase I resulted in cleavage of dalteparin and enoxaparin, whereas treatment with chondroitinase ABC did not. Sulodexide was cleaved by heparinase I as well as chondroitinase ABC
Characterization of heparinoids using anti-heparin antibodies
Reactivity of anti-heparin antibodies with immobilized heparinoids was analyzed by ELISA (Table 3). For reference, reactivity of antibodies with heparin was set at 100%. A distinct antibody-binding profile could be observed for all antibodies, although also similar reactivity with a number of antibodies was noticed. For instance, enoxaparin and tinzaparin, both prepared by β-elimination, reacted differently with antibodies LKIV69 and EW4G2, whereas they reacted similarly with the other antibodies. Nadroparin reacted very poorly with all the antibodies tested, which may be in line with its overall low amount of sulfated disaccharides in comparison with other LMWHs [17]. In contrast, sulodexide and danaparoid reacted strongly with antibody HS4E4, which defines a low-sulfated and N-acetylated epitope, in comparison to the LMWHs. This is in line with the presence of low-sulfated heparin (HS) and HS in sulodexide and danaparoid, respectively, and which contain a substantial amount of N-acetylated glucosamine. Compared to heparin, the other heparinoids reacted poorly with antibody NS4F5, which defines a very highly sulfated oligosaccharide.
Table 3Reactivity of anti-heparin antibodies with heparinoids (ELISA) HS4C3HS3A8HS4E4LKIV69EW3D10EW4G2NS4F5AO4B08RB4EA12Heparin100100100100100100100100100Dalteparin75 ± 644 ± 48 ± 53 ± 133 ± 649 ± 80 ± 016 ± 413 ± 2Nadroparin29 ± 35 ± 24 ± 30 ± 03 ± 17 ± 40 ± 01 ± 11 ± 1Enoxaparin80 ± 956 ± 87 ± 310 ± 236 ± 826 ± 71 ± 128 ± 721 ± 6Tinzaparin84 ± 155 ± 28 ± 344 ± 1045 ± 1274 ± 19 ± 220 ± 325 ± 8Sulodexide102 ± 571 ± 11283 ± 2151 ± 743 ± 10100 ± 30 ± 0102 ± 1526 ± 8Danaparoid69 ± 1122 ± 358 ± 172 ± 22 ± 11 ± 10 ± 063 ± 63 ± 1Anti-heparin antibodies were applied to heparinoids immobilized on microtiter plates. Given is the reactivity of the antibodies with the heparinoids relative to unmodified heparin. Values are given as mean ± SEM (n = 4). Each hepainoid shows a distinct immunoprofile.
Heparinoids could be ranked according to their reactivity with antibodies. For instance, reactivity of antibody EW4G2 was highest with heparin and sulodexide, followed by tinzaparin, dalteparin, and enoxaparin. Reactivity with the other heparinoids was very low or absent. Antibody LKIV69 reacted strongly with heparin, followed by sulodexide and tinzaparin, whereas other heparinoids reacted poorly. Reactivity of antibody AO4B08 was highest with heparin and sulodexide, followed by danaparoid and enoxaparin/tinzaparin/dalteparin, whereas reactivity with nadroparin was absent. These data indicate that distinct heparin domain structures are present in different commercially available heparinoids.
The only chemically defined heparinoid studied was the pentasaccharide fondaparinux (GlcNS(6OS)–GlcA–GlcNS(3,6OS)–IdoA(2OS)–GlcNS(6OS)). Due to its small size (1,728 kDa), it was assayed using a competition ELISA (Table 4). Heparin was included as a reference and values obtained for fondaparinux were related to those obtained for heparin. Antibodies HS4E4 and RB4EA12, both requiring N-acetylation in their epitopes, did not react with fondaparinux. Antibody NS4F5, which reacts with an extremely high sulfated oligosaccharide, was also not reactive. These results are in line with the chemical composition of fondaparinux, which contains no N-acetylated glucosamine and a non-sulfated glucuronic acid residue. Reactivity with antibody LKIV69, which reacts poorly with 6-O-sulfated epitopes, was very weak and reflects the high amount of 6-O-sulfation in fondaparinux. Reactivity with antibody AO4B08, which needs C5-epimerization, N-sulfation, high 6-O-sulfation and an internal 2-O-sulfated iduronic acid residue, was only moderate/poor, reflecting the lack of an internal 2-O-sulfated iduronic acid (i.e. flanked by other iduronic acid residues). Reactivity with antibody HS4C3, which requires N-, 3-O-, and 6-O-sulfation, and antibody HS3A8, which requires at least C5-epimerization and N- and 2-O–sulfation, was high, which corresponds with the chemical structure of fondaparinux. Reactivity was also high with antibodies EW3D10 and EW4G2, both defining an as yet undefined, but sulfated oligosaccharide. These data indicate that reactivity with antibodies may provide some chemical information of heparinoids.
Table 4Reactivity of anti-heparin antibodies with heparin and fondaparinux (competition ELISA) HS4C3HS3A8HS4E4LKIV69EW3D10EW4G2NS4F5AO4B08RB4EA12Heparin0.3 ± 0.10.4 ± 0.12.7 ± 0.1 0.2 ± 0.00.1 ± 0.00.1 ± 0.00.1 ± 0.01.2 ± 0.2<0.01Fondaparinux1.7 ± 0.21.3 ± 0.1 >20010.3 ± 0.80.2 ± 0.01.5 ± 0.5 11.7 ± 0.8 12.3 ± 3.821.5 ± 0.5Ratio fondaparinux/heparin5.73.3>7451.521511710.3>2150Given are the amounts of heparinoids (micrograms per milliliter), which result in a 50% inhibition of the binding of the anti-heparin antibodies to immobilized HS/heparin (IC50 values), and the ratio between antibody reactivity with fondaparinux and heparin. Values are given as mean ± SEM (n = 3). Antibody EW3D10 showed a strong reactivity with fondaparinux, followed by antibodies HS4C3, HS3A8, and EW4G2. The other antibodies showed a relatively weak or no reactivity.
Reactivity of the anti-heparin antibodies with immobilized heparinoids was supported by immunofluorescence analysis (Fig. 3). When human renal cryosections were simultaneously incubated with anti-heparin antibodies and heparinoids, staining was completely abolished with those heparinoids, which reacted strongly with the antibodies in ELISA (e.g. heparin and sulodexide for antibodies HS4C3 and HS4E4). An obvious decreased staining was noticed for those heparinoids, which reacted moderately with the antibodies (e.g. nadroparin for antibody HS4C3), whereas no major differences in staining were observed for those heparinoids, which did not bind to antibodies (e.g. nadroparin for antibody HS4E4).
Fig. 3Immunofluorescence staining of human renal cryosections with anti-heparin antibodies HS4C3 and HS4E4 in the absence and presence of heparinoids. Staining was abolished when renal cryosections were incubated with HS4C3 or HS4E4 in the presence of heparin or sulodexide. HS4C3 staining was decreased in the presence of nadroparin, whereas HS4E4 staining was unaffected. Bar represents 50 μm; magnification is identical for each photograph. G Glomerulus, BC Bowman’s capsule, T renal tubule
Discussion
In this study eight heparinoids were characterized by their reactivity with nine different anti-heparin antibodies. All heparinoids showed a distinct antibody-binding profile in line with differences in GAG composition (heparin, HS, CS, DS), molecular weight, and method of preparation. A number of antibodies reacted strongly with certain heparinoids, but not with others. It may therefore be possible to deduce chemical and biological information of a specific heparinoid by virtue of its antibody profile. For instance, antibody NS4F5 showed reactivity with only heparin. This antibody defines a stretch of trisulfated (N-, 2-O-, and 6-O-sulfated) and epimerized disaccharides and therefore defines a highly sulfated heparin domain (Smits et al., unpublished results). The lack of reactivity of the other heparinoids for NS4F5 indicates the absence or the destruction of the domain during their preparation. Absence may hold for sulodexide and danaparoid, which contain HS rather than heparin. Destruction by the depolymerization processes may hold for the LMWHs. In this respect, reactivity with antibody NS4F5 may be indicative for the degree of depolymerization. The antibody profile of the chemically synthesized pentasaccharide fondaparinux is compatible with its chemical structure GlcNS(6OS)–GlcA–GlcNS(3,6OS)–IdoA(2OS)–GlcNS(6OS). Reactivity with antibodies HS4C3 and HS3A8 indicates it to contain iduronic acid and N-, 2-O-, 3-O-, and 6-O-sulfate residues, whereas poor/no reactivity with antibodies HS4E4, LKIV69, AO4B08, and RB4EA12 indicates the lack of internal 2-O-sulfated iduronic acid residues (i.e. flanked by other iduronic acids) and N-acetylated glucosamine residues. Lack of reactivity with antibody NS4F5 indicates that fondaparinux does not contain a stretch of tri-sulfated disaccharides.
Next to information on the chemical properties of the heparinoids, the antibody profile may be indicative for their biological activity, e.g. the anti-Xa/anti-IIa ratio. This ratio is of essential importance since it is associated with a more predictable anticoagulant effect. The anti-Xa/anti-IIa ratio is 1 for unfractionated heparin, 2–4 for LMWHs, 0.5 for sulodexide, and infinite for fondaparinux [2, 23]. Antibodies such as HS4E4 and AO4B08 (Tables 3 and 4), which are highly reactive with heparin and sulodexide, but poorly/not with LMWHs and fondaparinux, likely bind to a heparin domain structure at a site proximal to the pentasaccharide sequence and may be indicative for a high anti-factor IIa activity. These results indicate that when a heparinoid is recognized by antibodies HS4C3, HS3A8, EW3D10, and EW4G2 (which showed a high reactivity with fondaparinux; Table 4), as well as by antibodies HS4E4 and AO4B08, the heparinoid is able to form a ternary complex with antithrombin III and factor IIa, resulting in inactivation of both factors Xa and IIa [23]. On the other hand, when a heparinoid is recognized by antibodies HS4C3, HS3A8, EW3D10, and EW4G2, but not by antibodies HS4E4 and AO4B08, it is does not contain the heparin domain structure at the site proximal to the pentasaccharide required for factor IIa binding. In this case only factor Xa will be inactivated. When a ratio is calculated from reactivity of heparinoids with antibodies HS4C3 and HS4E4 (indicative for 3-O-sulfates and for the presence of domains allowing IIa activity, respectively), and heparin is assigned a ratio of 1, the following numbers are found: dalteparin, 9; nadroparin, 7; enoxaparin, 11; tinzaparin, 11; sulodexide, 0.4; and fondaparinux, >>13 (no antibody HS4E4 reactivity observed). These numbers are in line with the above mentioned anti-Xa/anti-IIa ratios, which were determined by enzymatic assays [2, 23], and indicate that biological characteristics are associated with antibody profiles.
Heparin-induced thrombocytopenia (HIT) is a major complication in patients administered heparin. Heparin binding to platelet factor 4 (PF4) causes a conformational change in the protein, rendering it antigenic. Susceptible patients develop antibodies to the heparin-PF4 complex, and binding of the antibody-PF4-heparin complex to its receptor on platelets results in platelet activation and aggregation. In addition, platelets are removed by activated immune cells, resulting in thrombocytopenia [6]. Highly sulfated domains within heparin/HS are involved in the binding to PF4 [31], and an increase in sulfation and size is associated with an increased risk of HIT [6, 19]. This may explain why the incidence of HIT is lower in patients treated with LMWHs, danaparoid, or fondaparinux compared to unfractionated heparin [7, 23]. Antibody NS4F5, which defines a highly sulfated domain, reacted strongly with heparin, but not with the other heparinoids. Reactivity with this antibody may thus be used to obtain information about the risk of a heparinoid to induce HIT.
Although we have focussed here on the immunoprofiling of heparinoids in the context of anticoagulation, this technique may also be useful to evaluate heparinoids in other settings. Heparin and LMWHs have been used to treat patients with kidney disease since an ameliorating effect on a.o. proteinuria has been found [1, 14, 21, 25, 30, 33, 34, 40]. Likewise, heparinoids are reported to have anti-tumor and anti-metastatic effects [3, 20, 29], the degree of sulfation being of crucial importance. Heparinase III-digested heparin (highly sulfated fragments), inhibited tumor growth, whereas heparinase I-digested heparin (low sulfated fragments) promoted tumor growth [18], underscoring the necessity to characterize the preparation used. In addition to the implementation of these antibodies to characterize the therapeutic potential of heparinoids, they may be used to stage a disease (e.g. kidney disease, cancer) characterized by a specific HS profile in tissue, blood, and/or urine.
Since the antibody profile of the heparinoids used in these studies may be correlated to a specific biological effect, antibodies may be used for the identification and subsequent characterization of the active structures within the heparinoids, which may result in the development of more specific drugs. The antibodies described here may also be used to counteract bleeding in patients administered an overdose of heparinoids. This is generally accomplished by treatment with protamine sulfate [9, 26], but this rather unspecific polycation may cause hypotension, bradycardia, and thrombocytopenia. In addition it may cause other severe side effects including anaphylactic and anaphylactoid reactions, resulting in respiratory distress, circulatory collapse, capillary leak, and pulmonary hypertension [26]. There is a need for safer agents that inhibit the anticoagulant activity of heparinoids, and some antibodies described here may be useful, especially those that also react with fondaparinux, which is difficult to inhibit in vivo. It has already been described that antibodies HS4C3 and EW3D10 strongly inhibited heparin-induced anticoagulation (APTT clotting assay) [35, 39]. Antibody HS4C3 also blocked the anticoagulant activities of heparin and fondaparinux in an anti-factor Xa assay [35]. These studies indicate that anti-heparin antibodies may react with heparinoids thereby neutralizing their anticoagulant effect.
Finally, our antibodies may be used for fast industrial screening of heparinoids. A quality check of heparinoid batches may be performed using the antibodies since each heparinoid shows a specific immunoprofile.
In conclusion, this study presents a novel way of characterizing heparinoids using immunoprofiling. Reactivity with antibodies was found indicative for chemical and biological aspects of the heparinoids, and may therefore be used for a fast and simple screening of industrial batches for defined characteristics.

anticoagulation
heparinoid
immunoprofiling
antibody
heparin-induced thrombocytopenia


Cytogenetic features in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) comprise a group of bone marrow diseases characterized by profound heterogeneity in morphologic presentation, clinical course, and cytogenetic features. Roughly 50% of patients display clonal chromosome abnormalities. In several multicentric studies, the karyotype turned out to be one of the most important prognostic parameters and was incorporated into statistical models aiming for a better prediction of the individual prognosis like the International Prognostic Scoring System. However, due to the profound cytogenetic heterogeneity, the impact of many rare abnormalities as well as combinations of anomalies occurring in a substantial portion of patients with MDS is still unknown and can only be delineated on the basis of large international multicentric cooperations. Recently, the German–Austrian MDS Study Group presented cytogenetic findings in 2,072 patients with MDS, which serve as a basis for the characterization of the cytogenetic subgroups discussed in this article. The availability of new therapeutic options for low- and high-risk MDS targeted against distinct entities characterized by specific chromosome abnormalities, like 5q-deletions, monosomy 7, and complex abnormalities underlines the important role of cytogenetics for the clinical management of MDS. This article thus focuses on the clinical and prognostic relevance, the molecular background, and therapeutic perspectives in these three cytogenetic subgroups.
Introduction
The profound heterogenetity of myelodysplastic syndromes (MDS) is well-known from morphological and clinical studies, which finally lead to the establishment of classification, and prognostic scoring systems. It is becoming increasingly obvious that this heterogeneity also manifests itself against the background of genetic heterogeneity of MDS. In sharp contrast to the situation in chronic myeloid leukemia (CML) where one single cytogenetic abnormality, the translocation t(9;22)(q34;q11), is the genetic hallmark of the disease, there is an enormous variability of cytogenetic abnormalities in MDS hampering not only the prognostic classification but also the delineation of the molecular background of cytogenetic aberrations in MDS. As an example in our recent multicentric cytogenetic analysis of patients with MDS, we observed 684 different types of chromosome abnormalities in a cohort of 1,080 patients with MDS and an abnormal karyotype [1].
The cytogenetic profile of MDS
In general, MDS show a characteristic genetic profile with an overweighing of unbalanced abnormalities. Most frequently, a loss of genetic material in the form of deletions and monosomies can be observed. A gain of genetic material with the appearance of total or partial trisomies is less frequent. Loss or gain of genetic material can also be the result of unbalanced translocations, which are frequently observed in MDS with multiple abnormalities. Taken together, it is thus obvious to assume that a prime molecular mechanism in MDS is the loss or inactivation of tumor suppressor genes, while the activation of oncogenes seems to be less relevant in myelodysplasia.
In contrast to AML, balanced structural abnormalities like translocations and inversions are rare in MDS. Due to the profound genetic heterogeneity, the knowledge about distinct cytogenetic alterations was mainly restricted to the most frequent abnormalities (−5/5q−, −7/7q−, +8, 20q−, and −Y), although, in MDS, rare chromosome abnormalities are present in a substantial portion of patients. In a recent analysis of the German–Austrian MDS Study Group, 59% of all 2,370 abnormalities observed in 1,080 patients with MDS where rare; that is, they occurred with a frequency of less than 2% [1]. In this situation, prognostic knowledge can only be deepened by large-scale multicentric studies with a high enough number of abnormal cases with follow-up data. This development was initiated by the International MDS Risk Assessment Working Group (IMRAW) ending up with the establishment of the International Prognostic Scoring System (IPSS), which was based upon the analysis of 816 patients with de novo MDS of whom 327 had abnormal karyotypes [2]. A next step forward was the study of the Spanish cooperative group with 500 abnormal cases [3] followed by our German–Austrian dataset with 1,080 patients with chromosome abnormalities [1]. The situation is furthermore complicated by the fact that chromosome abnormalities in principle can occur in three different conditions: as isolated abnormality, together with one additional change, and as part of complex abnormalities with at least two additional cytogenetic alterations. Table 1 shows the incidence of the 21 most frequent abnormalities according to the number of accompanying alterations occurring in our German–Austrian patient cohort.
Table 1Incidence of chromosome abnormalities in MDS related to 2,072 patients examined successfullyAnomalytotal, n (% of all cases)Isolated, n (%a)With one additional abnormality, n (%a)As part of complex abnormalities, n (%a)5q−312 (15.1)146 (47)52 (17)114 (36)−7/7q−230 (11.1)86 (37.5)31 (13.5)113 (49)+8173 (8.4)81 (46.8)37 (21.4)55 (31.8)−18/18q−78 (3.8)3 (3.8)2 (2.6)73 (93.6)20q−74 (3.6)36 (48.6)10 (13.5)28 (37.8)−569 (3.3)1 (1.4)4 (5.8)64 (92.8)−Y58 (2.8)41 (70.7)5 (8.6)12 (20.7)+2145 (2.2)5 (11.1)18 (40)22 (48.9)−17/17p−42 (2.0)1 (2.4)1 (2.4)40 (95.2)inv/t(3q)41 (2.0)16 (39)8 (19.5)17 (41.5)−13/13q−40 (1.9)5 (12.5)6 (15)29 (72.5)+1/+1q37 (1.8)3 (8.1)6 (16.2)28 (75.7)−2133 (1.6)3 (9.1)4 (12.1)26 (78.8)+1128 (1.4)6 (21.4)4 (14.3)18 (64.3)−1226 (1.3)02 (7.7)24 (92.3)12p−25 (1.2)7 (28)6 (24)12 (48)t(5q)24 (1.2)6 (25)3 (12.5)15 (62.5)11q−23 (1.1)8 (34.8)4 (17.4)11 (47.8)9q−23 (1.1)8 (34.8)3 (13)12 (52.2)t(7q)22 (1.1)6 (27.3)6 (27.3)10 (45.5)−2022 (1.1)0022 (100)aOf cases with the respective abnormality
Cytogenetic prognosis
The first large-scale cytogenetic studies in patients with MDS encompassing more than 100 patients where published more than 20 years ago [4–7]. In the following years, the patients cohorts increased step by step from nearly 250 patients [8] to some 400 patients [9, 10]. In 1997, Greenberg published a collaborative multicentric international data set of more than 800 patients with de novo MDS, which was the basis for the establishment of the IPSS [2]. This database was further surmounted by the analyses of the Spanish cooperative group with 968 patients examined (Table 2), which represented the largest number of patients with MDS karyotyped so far [3]. Recently, the German–Austrian Study Group published their multicentric analyses of more than 2,100 patients with MDS [1].
Table 2Cytogenetic prognostic findings in publications with greater than 100 patients examinedAuthor, yearNumber of patientsAbnormal (%)FavorableIntermediateUnfavorableKnapp 198517466 (38)Normal (NN)ComplexNowell 1986–198914463 (44)5q−, 20q−+8−7/7q−Billström 198816974 (44)NN, 5q−+8−7/7q−Pierre 1989247106 (43)NNComplexMorel 1993408151 (37)NN, 5q−, −Y, −7/7q−, 20q−+8ComplexToyama 1993401200 (50)+8−7/7q−ComplexWhite 199419875 (38)NN, 5q−12p−, +21Complex, +8, 20q−Greenberg 1997816327 (40)NN, 5q−, 20q−, −YAll othersComplex, abnormal #7Solé 2005968500 (51)NN, 5q−, 20q−, −Y, 11q−, 12p−rea 3q, + 8, +9, t11q, 17p−Complex, −7/7q−, i17qHaase 20072,0721,080 (51)NN, +1/+1q, t(1q), 5q−, t(7q), 9q−, 12p−, abnormal #15, t(17q), 20q−, −21, + 21, −X, −Yrea 3q, −7, 7q−, +8, 11q−, t(11q23), +19, complex (=3)Complex (>3), t(5q)rea Rearranged
What has changed and what has been achieved during these last 25 years of cytogenetic analyses in MDS? If the aberration rate is considered, there is a gradual increase in the portion of abnormal cases from under 40% in the most earlier studies to 50% in the more recent analyses of Toyama, Solé, and Haase [1, 3, 10]. Although the composition of the patient cohorts may play a role for the portion of clonal abnormalities in a given collective, other factors like the improvement of culture conditions possibly due to the use of recombinant myeloid growth factors [11] might have contributed to the increase in the aberration rate in cytogenetic studies. To date, an aberration rate of 50% can be regarded as an international standard.
Prognostic relevance of chromosome abnormalities
The prognostication of patients with MDS has become an important means for the development of therapeutic strategies based on more individualized risk assessment. Even in the most recent prognostic scoring system, the World Health Organization classification-based Prognostic Scoring System, cytogenetics play a decisive role [12]. In general, three to four prognostic cytogenetic categories can be distinguished. In several multicentric investigations, cytogenetics have been proven to be highly relevant independent prognostic parameters ([2, 3, 13], see also Table 2).
Good prognosis
Even in the earliest study considered here, Knapp et al. [4] described the prognostic relevance of cytogenetic findings with a normal karyotype as favorable and complex abnormalities as being associated with bad prognosis. The good prognosis of a normal karyotype was confirmed by nearly all other groups. Thus, in contrary to the situation in AML where patients with a normal karyotype have an intermediate outcome and prognosis is significantly influenced by additional molecular alterations, in MDS, a normal karyotype is undisputedly associated with good prognosis. Further, well-established abnormalities defining good-risk subgroups are deletions of 5q and 20q as well as loss of the Y-chromosome, although the question whether this latter abnormality is age related, or a real clonal marker is unsolved as yet. The Spanish group identified further new cytogenetic abnormalities with a good prognosis (12p− and 11q−) [3].
In the German–Austrian multicentric study, the following abnormalities were associated with a favorable clinical course with a median survival between more than 9 years and 32 months: normal karyotype, t(1q), 5q−, t(7q), 9p−, 12q−, t(15q), t(17q), 20q−, +21, −21, −X, −Y. However, prognosis was only favorable when not more than one additional abnormality was present [1].
Intermediate prognosis
In most studies, patients with trisomy 8 displayed an intermediate clinical course. Until the Spanish group published their results on more than 980 patients, the knowledge of cytogenetic findings with an intermediate prognosis was very limited. In the IPSS, all abnormalities neither belonging to the good-risk group (isolated 5q−, 20q−, and loss of Y-chromosome) nor to the bad risk cohort (complex [greater than or equal to three abnormalities] or any chromosome 7 abnormality) were designated to be of intermediate prognosis not by availability of survival data but by definition [2]. In their large Spanish dataset, Solé et al. delineated new abnormalities associated with an intermediate clinical course (3q abnormalities, trisomy 9, 11q translocations, and 17p deletions) [3]. The results of White et al. [14] and Toyama et al. [10] who identified −7/7q− and 12p− and trisomy 21, respectively, as abnormalities with an intermediate prognosis were not confirmed by other groups. Recently, the multicentric database of the German–Austrian MDS Study Group revealed several infrequent abnormalities with an intermediate prognosis. A median survival time of 23–26 months was observed in patients with trisomy 8 and 11q− (called Intermediate-I). A more worse prognosis with a median survival time between 20 and 14 months was found for 11q23 translocations, chromosome 3q aberrations, trisomy 19, 7q deletions, and complex abnormalities with three different chromosomal alterations and monosomy 7 (called Intermediate-II) [15].
Poor prognosis
There is a great consensus in all publications on cytogenetic prognosis in MDS that complex abnormalities characterize a MDS subgroup with bad prognosis and a median survival time significantly below 1 year, although the threshold at which the number of abnormalities confers bad prognosis is a matter of debate. While in most publications, the term “complex” is used for three or more abnormalities, in the Medical Research Council AML trials, bad prognosis was assigned to cases with five or more abnormalities [16]. Recently, we clearly could show that, in patients with MDS, median survival is significantly reduced only when more than three abnormalities are present, reducing the median survival from 17 months in cases with three abnormalities to less than 9 months in cases with four or more anomalies [1]. Besides complex changes, partial and total monosomy 7 was ranked to the cytogenetic findings with an unfavorable clinical course [3, 5–7]. Within the IPSS, any chromosome 7 abnormality was counted to the bad prognostic findings [2]. As mentioned above, in the German–Austrian dataset, −7/7q− showed a significant better prognosis (14 and 19 months median survival time, respectively) as compared to the complex abnormality group defined by four or more chromosomal changes (8.7 months) and was thus attributed to the intermediate-II group [15]. The findings of Toyama et al. [10] who observed an unfavorable course in patients with trisomy 8 or 20q deletions was not confirmed by any other group. Solé et al. described a median survival of under 12 months in ten patients with an isochromosome of 17q [3]. Since, in the German–Austrian dataset, this abnormality was too infrequent for a statistically meaningful analysis, the Spanish findings are convincing but still need further confirmation.
Prognostic scoring
In the IPSS, three different cytogenetic subgroups were established and weighted against bone marrow blast counts and cytopenias by multivariate analysis [2]. Applying the cytogenetic part of the IPSS to our German–Austrian dataset, 59% of the patients had a good risk karyotype (normal, isolated 5q−, 20q−, or loss of Y-chromosome) with a median survival of 54 months, 19% of the collective had an intermediate prognosis (neither good risk nor bad risk cytogenetics) with a median survival of 31 months, and 22% displayed a dismal prognosis (any chromosome 7 anomaly, complex [≥3] changes) with a median survival of 11 months [1].
The application of the new cytogenetic findings derived from the German–Austrian dataset allows the definition of four different cytogenetic prognostic subgroups (Table 3 and Fig. 1). The low-risk group covers 73% of patients with 14 cytogenetic categories and a median survival of 55 months, the intermediate groups I and II (15.5% of patients) with eight cytogenetic categories and a median survival of 29 (intermediate-I) and 15 months (intermediate-II), and the high-risk group (11.5% of patients) with a median survival of 8 months [15]. In comparison to the IPSS, our new cytogenetic prognostication splits the intermediate group into two distinctly separate subgroups with significantly different median survival. The bad-risk group is more strictly defined with a lower median survival time (8 vs. 11 months). While the IPSS assigned an intermediate risk by exclusion and not by knowledge, our intermediate cytogenetic subgroup was based exclusively on available survival data of patients treated with supportive care only thus reflecting the natural course of the disease. Taking into account only those patients with a known prognosis (in IPSS only good- and bad-risk patients), 81% of our patients could be categorized by the IPSS in comparison to 93% by the German–Austrian prognostic system.
Fig. 1Kaplan–Meier survival curves according to the cytogenetic prognostic classification of the German–Austrian MDS Study Group. Log-rank test: p < 0.0001 (3 degrees of freedom) [15]Table 3New cytogenetic prognostic subgroups of the German–Austrian MDS Study Group in 1,202 patients treated with supportive care onlyCytogenetic riskCytogenetic findingNumber (%)Median survival (months)Good12p−7 (0.6)n.r.9q−6 (0.5)n.r.t(15q)6 (0.5)n.r.15q−5 (0.4)n.r.+2113 (1.1)100.85q−132 (11)77.220q−24 (2)71.0−X6 (0.5)56.4normal karyotype622 (51.7)53.4−Y33 (2.8)39.4t(1q)7 (0.6)34.7t(7q)7 (0.6)34.7t(17q)6 (0.5)32.1−216 (0.5)32.0Intermediate-I11q−11 (0.9)26.1+864 (5.3)23.0Intermediate-IIt(11q23)6 (0.5)20.0Any 3q abnormality16 (1.3)19.9+195 (0.4)19.87q−11 (0.9)19.0Complex (=3 anomalies)32 (2.7)17.0−742 (3.5)14.0PoorComplex (>3 anomalies)134 (11.1)8.7t(5q)7 (0.6)4.4n.r. Median survival not reached
A further element of the IPSS, the weighting of cytogenetics in comparison to other relevant parameters like the bone marrow blast counts, has become questionable. According to the IPSS, 0 scoring points are attributed to good-risk cytogenetics and blasts less than 5%, intermediate cytogenetics as well as 5–10% blasts get 0.5 scoring points, and bad-risk cytogenetic findings are scored with 1.0. Eleven to 20% blasts are scored with 1.5 and 21–30% blasts with 2.0 points. Thus, bad-risk cytogenetics with a median survival of 11 months get less scoring points than patients with 11–20% blasts and a median survival in our cohort of 16.5 months. According to our observations, only patients with 21–30% blasts (11.7 months median survival) had a prognosis comparable to patients with bad-risk cytogenetics. Thus in a revision of the IPSS the weight of bad-risk cytogenetics has to be readjusted [17].
Delineation of the most relevant cytogenetic subgroups 5q−, monosomy 7, and complex abnormalities
5q deletions
Deletions within the long arm of chromosome 5 are the most frequent cytogenetic changes in MDS accounting for roughly 30% of abnormal cases [1, 2, 3]. The deletions can have variable size; however, the common deleted region always spans the chromosome band 5q31. Due to intensive research during the last decade, our knowledge of the molecular background of these abnormalities is increasing. It is generally accepted that not the illegitimate fusion of deoxyribonucleic acid (DNA) sequences at the variable breakpoints of the deletions but loss of genetic information represents the relevant pathomechanism. There could be two different deleted regions in 5q31. One that is located more centromeric is possibly associated with bad prognosis, complex abnormalities, and high-risk, as well as therapy-related, MDS. A second area is located more telomeric in the vicinity of band 5q32 and is supposed to be related to the good-risk 5q− syndrome [18–21. Recently, it has been convincingly shown by the means of sophisticated systematic knockout experiments on 41 candidate genes in the critical 5q− region that the gene for a ribosomal subunit protein, RPS41, seems to be a relevant gene in patients with 5q− syndrome on the basis of haploinsufficiency [22]. The 5q− syndrome has been first described by Van den Berghe in 1974 [23]. It is cytogenetically characterized by an isolated deletion of chromosome 5q. A female preponderance is well documented. The clinical appearance is characterized by a refractory macrocytic anemia, normal or elevated platelets, and mild leukocytopenia. The clinical course is mild and long lasting with a very low risk for leukemic transformation. The bone marrow smears reveal less than 5% blasts, while dysplasia of the erythroid and granulocytic lineages usually are discrete or even absent. Megakaryopoesis shows profound and characteristic dysplasias with either separated multiple nuclei, hypolobulation, microkaryocytes, and most characteristically monolobulated megakaryocytes with a round nucleus.
The prognosis of 5q deletions in MDS is generally favorable if they are not part of complex abnormalities; however, it is significantly modified by single additional cytogenetic changes [1, 24]. Figure 2 shows the Kaplan–Meier survival curves for patients treated with supportive care only with isolated 5q−, 5q− plus one additional abnormality, and 5q− as part of complex abnormalities derived from the German–Austrian database [1].
Fig. 2Median survival according to accompanying abnormalities in patients with 5q deletions. Log-rank test: p ≤ 0.0001 (2 degrees of freedom), p = 0.30 (isolated vs. +1), p ≤ 0.0001 (isolated vs. complex), p = 0.0001 (+1 vs. complex)
It is evident that additional abnormalities negatively influence survival in patients with 5q deletions. However, due to cytogenetic heterogeneity, the additive prognostic impact of distinct single additional abnormalities remains obscure as yet (Table 4).
Table 4Frequencies (in percent of all cases with the respective primary abnormality) of accompanying abnormalities5q− (n = 59)−7/7q− (n = 38)trisomy 8 (n = 44)Additional anomalyPercentAdditional anomalyPercentAdditional anomalyPercent+817+21105q−23+21135q−10+2111−20/20q−8+810der(3q21/q26)7−77inv(3q)5+117der/del(12p)7del(12p)5−75der/del(3p)5t(11q23)5del(12p)5der/t(21q)5iso(17)(q10)5+135t(11q23)3Others50+145Others35+8*5del(1p)5Others22
Over a long period, the therapeutic standard in patients with noncomplex 5q deletions was supportive care only. In clinical trials, retinoic acid turned out to be inefficient, and low-dose cytarabine was more effective but led to a pronounced increase in severe neutropenic infections [25]. A preferential response to other modern therapeutic strategies like immunosuppression and suppression of DNA methyltransferase or histone deacetylase was not reported as yet. However, recently, List et al. reported a remarkably high response rate to the immunomodulating agent lenalidomide, which was especially pronounced in MDS patients with 5q deletions. Nine out of 12 patients with a 5q deletion displayed complete cytogenetic remissions, and 10 out of 12 experienced an erythroid response [26]. Thus, a new promising agent may target cell clones in MDS bearing 5q deletions. These results could recently be confirmed on the basis of a large muliticentric trial [27].
Monosomy 7
Monosomy 7 is the second most frequent distinct chromosome abnormality in MDS occurring in some 25% of abnormal cases. It can present as total or partial monosomy. In the latter case, variable deletions of parts of the long arm lead to loss of genetic material of different size. As yet, no significant differences concerning the prognostic relevance have been observed between total and partial monosomy 7. In the German–Austrian dataset, 36% of monosomy 7 were isolated ones, 14% displayed one additional abnormality, and 50% occurred as part of complex abnormalities [1]. Comparable to the situation in 5q, also in 7q, at least two and maybe more distinct regions of common deletions have been identified: the band 7q22 and the more telomeric regions 7q31–32 and 7q36. It is interesting to note that interstitial as well as terminal deletions might be due to cryptic unbalanced translocations. In single individuals, more than one different deletion has been found in one and the same patient, either within the same copy of chromosome 7 with retention of sequences between the deleted regions or within different cell clones [28, 29]. These observations clearly underline the regional genetic instability of the long arm of chromosome 7, which makes this region especially prone to deletions of different size and localization as well as to structural abnormalities. On the other hand, it can be assumed that there must be a cluster of genes with tumor-suppressive features distributed over several chromosome bands in 7q with the same or very similar consequences when being inactivated by chromosomal abnormalities.
However, in contrast to some presumptions in the 5q deletions, as yet, no prognostic differences have been elaborated between different regions of deletions (reviewed in [20]). Investigations of the molecular background of monosomy 7 MDS are on the way. An association of this cytogentic subgroup with RAS mutations, mutations of AML1, and hypermethylation of p15INK4B have been reported [30, 31]. In an analysis of gene expression profiles in CD34+ cells from MDS patients with monosomy 7, a malignant phenotype with highly proliferative potential was found with an overexpression of HOX9A, PRAME, BMI-1, PLAB, and the DNA repair gene BRCA2. Parallelly, downregulation of the tumor suppressor gene p21, GATA2, and MAP was observed [32]. Clinically, monosomy 7 is characterized by a lower median age of the affected patients as compared to 5q deletions, severe refractory cytopenias, and a proneness to life-threatening infections. Predisposing conditions recurrently observed in cases with monosomy 7 are antecedent aplastic anemia, Fanconi’s anemia, neurofibromatosis type I, cyclic neutropenia, and long-term treatment with granulocyte colony-stimulating factor (G-CSF), as well as Schwachman’s syndrome. It is of special interest that myeloid cells harboring monosomy 7 seem to be preferentially responsive to a stimulation by myeloid growth factors like G-CSF and granulocyte–macrophage CSF in vitro [33, 34] as well as in vivo [35]. Although being obvious, this phenomenon never was exploited therapeutically for a targeted priming strategy for patients with monosomy 7.
In extremely rare cases, for the so-called monosomy 7 syndromes, the abnormality has a familiar background. In a substantial portion of patients, a mutagen exposition (benzene, solvents, irradiation, or radio- and/or chemotherapy) was documented.
In contrast to the situation in 5q deletions, in monosomy 7, additional abnormalities do not have such a profound impact on outcome as seen in 5q deletions, since monosomy 7 even as an isolated abnormality confers a significantly bad prognosis. In our Austrian–German dataset, median survival times were 14 months for the isolated abnormality, 11 months for cases with one additional change, and 8 months for monosomy 7 as part of complex abnormalities (Fig. 3) [1].
Fig. 3Median survival according to accompanying abnormalities in patients with −7/7q−. Log-rank test: p = 0.03 (2 degrees of freedom), p = 0.94 (isolated vs. +1), p = 0.07 (isolated vs. complex), p = 0.43 (+1 vs. complex)
Therapeutic options in the monosomy 7 subgroup are unsatisfying as yet. If age and clinical condition are adequate, patients should be treated with allogeneic stem cell transplantation whenever possible. Conventional intensive chemotherapy bears a high risk of early death and nonresponse. Even if a complete remission can be achieved, this frequently is of only short duration with a high risk of early relapse. In a recent report on 34 patients with MDS or AML treated with 5-azacytidine, the group of Mufti observed a preferentially good response of MDS patients with monosomy 7 to the demethylating agent 5-azacytidine. In this trial, five of the seven (71%) complete responders had isolated chromosome 7 abnormalities and achieved a continuous complete remission (10 months follow-up) in contrast to patients with other karyotype abnormalities like trisomy 8 who relapsed within the first 6 months [36]. Supporting results have been recently published for the use of decitabine by Lübbert et al. [37]. Future multicentric therapy trials are needed to verify this observation in a higher number of patients.
Complex abnormalities
According to the International System for Human Cytogenic Nomenclature criteria, complex chromomosome abnormalities are defined by the simultaneous occurrence of at least three independent abnormalities within one cell clone [38]. This cytogenetic subtype is present in some 15% of all patients with MDS (roughly 30% of all abnormal cases) and thus occurs in a frequency comparable to 5q deletions [1–3]. Complex abnormalities may be the result of a multistep process with sequential accumulation of abnormalities, called karyotype evolution. In some cases, karyotype evolution can be observed if repeated cytogenetic analyses are performed, unraveling the stepwise accumulation of secondary abnormalities with the starting point of a primary abnormality. In a few patients, cytogenetic analysis detects cell clones with primary and others with primary and secondary and possibly further abnormalities at one and the same time point. However, frequently, this process cannot be proven in the individual patient who presents with multiple chromosomal changes at first diagnosis. In most of these cases, complexity is profound with aberration numbers exceeding the threshold of three by far. Frequently, in these cases, cell-to-cell variations occur, and the chromosomal complement presents with a chaotic, mutator-like shape resembling cytogenetics of solid tumors and malignancies in chromosomal breakage syndromes. Thus, it is conceivable that, in these cases, the process leading to complex abnormalities and genetic instability must proceed rapidly on the one hand and must involve mechanisms like DNA repair and cell cycle control comparable to the situation in DNA repair deficiency syndromes. Recent results of gene expression analyses in patients with MDS and complex chromosome abnormalities support these assumptions and are considered below in more detail [39]. In a substantial portion of patients with complex abnormalities, an exposition to therapeutically applied mutagens, like anthracyclines, topoisomerase II inhibitors, alkylating agents, and/or irradiation, can be traced in the medical history [40]. Within the subgroup of patients with complex changes, a broad range of the number of abnormalities with the majority of patients displaying greater than or equal to five chromosomal changes can be observed [39].
There is a strong association with unbalanced structural abnormalities affecting most frequently chromosomes 5q and 7q. Chromosomes less frequently involved in loss of genetic material were chromosomes 3 (p- and q-arms), 12p, 13q, 16q, 17p, 18q, and 20q. Genomic gains were observed for chromosome 8/8q, 11q, and 21q [41]. Mutations of p53 have been observed in up to 90% of patients with AML and complex abnormalities [39]. Matrix comparative genomic hybridization (CGH) analyses in patients with complex abnormalities revealed an association of complex karyotype changes with amplifications in the chromosomal regions 8q24, 9p24,,11q23, 12p13, 13q12, 20q11, and 2q1q22 [42].
Recently, characteristic profiles of complex abnormalities in AML were delineated by gene expression analyses. In this respect, a significant overexpression of RAD21, a double-strand-break DNA repair enzyme, as well as overexpression of other genes related to DNA-repair, apoptotic mechanisms, and cell-cycle control (RAD1, RAD 9A, RAD23B, RAD51AP1, NBS1, MSH6, SUMO1, and PARP2) was observed [39].
Taken together, these data might help to understand complex abnormalities as a maximum manifestation of genetic instability by allowing cells with significant DNA damages to circumvent physiologically protective apoptosis and to escape cytotoxicity of chemotherapy by intensified DNA repair. This model is also compatible with the high extent of chemo-resistance of hematopoietic cells with complex chromosome abnormalities, which is a well-known and severe problem in the clinical management of high-risk MDS. Further clinical associations are the high median age of patients within this cytogenetic subgroup [43] and, related to conventional intensive chemotherapy, short remission durations and a high risk for early relapse [44]. Allogeneic stem cell transplantation, the only curative option for these patients, will only be feasible for a minority of individuals with complex chromosomal changes due to age-related multimorbidity and organ dysfunctions. New therapeutic strategies targeting hypermethylation, deacetylation, and immunomodulation, which have proven their tolerability also in aged patient populations, are now available for the treatment of patients with high-risk MDS. In this respect, two studies have to be mentioned. Cytogenetic responses in a substantial portion of patients with high-risk MDS have been observed in a multicentric study of decitabine, a potent demethylating agent. Major cytogenetic responses (MajCR) were observed in 38% of 50 patients. Remarkably, 10 out of 26 patients (38%) with high-risk cytogenetics displayed a MajCR; five of them had complex chromosome abnormalities. In the patients’ group with MajCR, median survival time was 24 months as compared to the significantly shorter median survival time of 11 months in patients with persisting cytogenetic aberrations [45]. These observations are further supported by observations of our group. Within a cohort of 14 patients with high-risk MDS, we recently observed hematologic and cytogenetic responses to 5-azacytidine according to the modified International Working Group criteria [46] in three out of four patients with complex abnormalities [47].
Another new therapeutic mechanism, immunomodulation, might be effective in cases with complex karyotypic changes too. In this connection, a complete cytogenetic response to the immunomodulatory drug lenalidomide has been reported in patients with high-risk MDS with complex chromosome abnormalities harboring 5q deletions [48]. Further multicentric clinical trials are needed to confirm these observations.
Conclusions
In contrast to CML where the bcr–abl fusion forms the homogenous genetic basis of the disease [49], MDS show a profound heterogeneity, not only on the morphologic and clinical level but also on the genetic presentation. Three cytogenetic subgroups with prognostic relevance have been identified and were incorporated into prognostic scoring by the IPSS. However, in the great majority of cytogenetic changes, the prognostic relevance is still obscure. Genetic heterogeneity in MDS leads to the paradox situation that rare abnormalities can be observed frequently [1] with the consequence that, for a relevant portion of abnormalities, their prognostic impact is still unknown. The only chance to overcome these shortcomings is to establish multicentric cooperations, which are the goals of the German–Austrian MDS Study Group, which is now cooperating with the MD Anderson Cancer Centre as well as the IMRAW group [50–52]. New therapeutic strategies targeting immunomodulation and epigenetic changes proved to be of outstanding effectiveness and tolerability in comparison to established therapies not only for the low-risk 5q syndrome but also for high-risk MDS with as yet deleterious cytogenetic alterations like monosomy 7 and complex abnormalities.
However, what about the subgroup of nearly 50% of patients presenting with a normal karyotype? New techniques will help to further delineate the genetic background in these cases. Thus, very recently, two groups published their results of single nucleotide polymorphism analyses in MDS [53, 54]. The group of Mufti performed high-resolution single-nucleotide polymorphism (SNP) array analyses in 119 low-risk MDS patients of whom 32% displayed clonal chromosome abnormalities to search for cryptic chromosomal aberrations not detectable by chromosomal banding analyses. They found uniparental disomy (UPD), a copy number neutral loss of heterozygosity, in 46% of patients, small deletions in 10%, and amplifications in 8% of the cohort. The changes of copy numbers were acquired while UPDs were constitutional. The former aberrations were associated with a worsened outcome of the patients. The authors speculated that the high frequency of UPD may be indicative for a constitutional genomic instability in these patients [53]. This could be a predisposing factor for MDS. Maciejewski et al. applied 250 K SNP arrays to 94 patients with MDS and observed SNP aberrations in 75% of patients as compared to 59% clonal chromosomal abnormalities detected by metaphase analyses. Previously unknown lesions were found in patients with a normal—as well as in others with an abnormal—karyotype. In this study, UPD occurred in 20% of the MDS patients [54].
New analytical tools like matrix CGH, microarray gene expression analyses, proteomics, and methylation profiling will add substantially to the understanding of pathogenesis, delineation of therapeutic targets, and individualization of therapy in MDS.
Taken together cytogenetics is still the gold standard of genetic diagnosis in MDS providing “labels” like 5q−, monosomy 7, or complex abnormalities for disease entities with a common biological behavior and clinical as well as prognostic impact. In the future, scientific progress will depend on the ability to combine established and newly developed methods to gain a comprehensive understanding of MDS, which will translate into an individualized and highly effective and well-tolerable treatment for all patients with MDS.

cytogenetics
mds
karyotype
prognosis
monosomy 7
complex abnormalities
5q−


A Novel Function of the Receptor for Advanced Glycation End-Products (RAGE) in Association with Tumorigenesis and Tumor Differentiation of HCC

Background The expression of the receptor for advanced glycation end products (RAGE) has an impact on the mechanisms giving rise to characteristic features of various cancer cells. The purpose of this study was to elucidate the clinicopathological relevance of the level of RAGE expression in patients with hepatocellular carcinoma (HCC) and to explore the effect of RAGE expression on the characteristic features of HCC.
Although many cancers arise from chronic inflammation, the relationships between carcinogenesis, cancer promotion, and its molecular characteristics remain poorly understood. Hepatocellular carcinoma (HCC), which typifies an inflammation-related tumor, is one of the most common malignancies in the world, especially in Asia and Africa. Japan has a high incidence of chronic viral hepatitis, cirrhosis, and HCC. The resolution of inflammatory activity at the molecular level may correlate with prevention of hepatocarcinogenesis and cancer promotion.
The receptor for advanced glycation end-products (RAGE) is a multiligand receptor classified as an immunoglobulin superfamily cell surface molecule and acts as a counter-receptor for high-mobility group box 1 (HMGB1),1 advanced glycation end-products (AGEs), S100/calgranulins, and amyloid-β peptides. These interactions trigger the activation of key cell signaling pathways (e.g., p38 and p44/42 MAP kinase,2 NF-κB, cdc42/rac,3 and the generation of reactive oxygen species, and result in the production of proinflammatory cytokines.5 RAGE-mediated proinflammatory processes are now considered to contribute to the progression of many chronic diseases, such as neuropathy, nephropathy,6 macrovascular disease, amyloidoses, inflammatory conditions (e.g., rheumatoid arthritis and inflammatory bowel disease) and sepsis.5,7 In addition to RAGE-mediated proinflammatory events, recent studies have revealed that the interaction of RAGE and its ligands and the resultant signaling play a causative role in the characteristic modulation of cancer cell functions, i.e., increasing tumor invasion and metastasis.8 Furthermore, several clinical studies have demonstrated the (strong) association of RAGE expression with the malignant potential of various cancers such as gastric cancer,9 colon cancer,10,11 common bile duct cancer,12 pancreatic cancer,13 and prostate cancer,14 although one report showed a reverse correlation between RAGE expression and tumor progression.15 Thus, RAGE expression may be expected play have a significant role in the development of HCC, although no data have been reported for this tumor. In terms of HCC, it is known that the pathophysiological conditions or circumstances surrounding tumorigenesis are quite different from those reported for other cancers. For example, small HCC tumors at an early stage are continuously exposed to low oxygen and high glucose as well as the noncancerous hepatic tissues with cirrhosis in which tissue derangement occurs.15 In addition, RAGE is the receptor for AGEs produced from excessive glucose metabolism,16 extracellular HMGB1 released from necrotic cells which could be induced by hypoxia and inflammation, and serum amyloid A (SAA) produced in response to the proinflammatory cytokine IL-6,17 and these RAGE ligands are possibly generated and/or released from inflamed hepatic tissues. Our central hypothesis is thus that RAGE expression may play a particular role in tumorigenesis of HCC, in addition to the role in the invasive and/or metastatic potential of cancer cells. With this background, the objectives of this study were: (1) to clarify the relationship between RAGE expression and the clinico-pathological features of HCC and (2) to investigate the functional role of RAGE expression in HCC development.
MATERIAL AND METHODS
Human samples
From March 2000 to September 2005, 65 patients with primary HCC were treated surgically in the Department of Surgical Oncology and Digestive Surgery, Kagoshima University School of Medicine. Of these 65 patients, 12 who had diabetes mellitus and 6 who underwent preoperative therapy were excluded from the study. RAGE expression for Diabetes patients upregulates at various tissues.18,19,20 A further 11 patients were excluded because their RNA samples were degraded. Samples from the remaining 36 patients (30 men and 6 women with a mean age of 67.1 years) were included in the study. Seven patients (19.4%) were positive for hepatitis B surface antigen and 21 (58.3%) were positive for the antibody to hepatitis C virus. Eight patients (22.2%) were negative for both of these viruses. Twenty-four patients had chronic hepatitis and four had liver cirrhosis. The mean tumor size was 49.7 mm (range, 16–150 mm). The histological grade of each tumor was determined according to the general rules for the clinical and pathological study of primary liver cancer (The Liver Cancer Study Group of Japan, 2000).21 Four tumors (11.1%) were well-differentiated HCC, 28 (77.8%) moderately differentiated HCC, and 4 (11.1%) poorly differentiated HCC. Postoperative tumor recurrence was observed for 11 patients (30.6%) (Table 1). For immunohistochemical study, a further 12 HCC samples (six well- and six poorly differentiated HCC), obtained surgically during the same period at JA Kagoshima Kouseiren Hospital, were added and used to verify the correlation of RAGE expression with tumor differentiation. Finally, a total of 48 HCC nodules (10 well-differentiated HCC, 28 moderately differentiated HCC, and 10 poorly differentiated HCC) were studied. As a control study, six normal liver samples were collected from patients with benign or metastatic liver tumors. Written informed consent, recognized by the ethical committees of Kagoshima University School of Medicine and JA Kagoshima Kouseiren Hospital, was obtained from each patient before tissue acquisition.
TABLE 1.Background of patientsGender  Male30 (83%)  Female6 (17%)Mean age67.2 yearsVirus type  B7 (19.4%)  C21 (58.3%)  None8 (22.2%)Background of livers  Chronic hepatitis24 (60.7%)  Liver cirrhosis4 (11.1%)  Normal liver8 (22.2%)  Mean tumor size 49.7mmHistological grade  Well differentiated4 (11.1%)  Moderately differentiated28 (77.8%)  Poorly differentiated4 (11.1%)Postoperative tumor recurrence 11 (30.6%).
Immunohistochemistry
Consecutive 4-μm sections were cut from each paraffin-embedding block. Sections were immunostained by anti-RAGE antibody (Santa-Cruz, CA, USA) according to the conventional immunoperoxidase technique. Briefly, after peroxidase blocking with 3% H2O2/methanol for 10 min, specimens were blocked with phosphate buffered saline (PBS) containing 5% normal horse serum (Vector Laboratories, Inc., Burlingame, CA, USA). Anti-RAGE antibody was used at 1/200. After overnight incubation at 4°C with the primary antibody, specimens were briefly washed in PBS and incubated at room temperature with the secondary antibody conjugated with peroxidase. The specimens were then washed in PBS and color-developed by diaminobenzidine solution (DAKO). After washing with water, specimens were counterstained with Meyer’s hematoxylin (Sigma Chemical Co., St Louis, MO, USA). Immunostaining of all cases was performed at one time to ensure the same conditions of antibody reaction and DAB exposure. A total of 48 HCC nodules (10 well-differentiated HCC, 28 moderately differentiated HCC, and 10 poorly differentiated HCC) were studied. To evaluate the immunohistochemical staining, ten fields were selected and expression in 1000 tumor cells (100 cell/fields) was evaluated with high-power (×200) microscopy. The immunohistochemical expression of RAGE was defined as positive if distinct staining of the cell membrane was observed in at least 10% of tumor cells.
Cell and cell culture
Hepatoma cell lines HepG2, HuH7, HT17, and Li7 were kindly provided by the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University and Hep3B was obtained from the European Collection of Cell Cultures. HepG2, Hep3B, and HT17 were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) (1000 mg/l) and HuH7 and Li7 were cultured with RPMI. RAGE-transfected Cos7 and its mock-transfectant were kindly provided by Drs. Yamamoto, Department of Biochemistry and Molecular Vascular Biology, Kanazawa University. These transfectants were maintained with DMEM supplemented with 10% Fetal Calf Serum (FCS) in the presence of 650 μg/ml G418. A hypoxic environment was created by placing a cell culture dish with one pouch of Anaero Pack into an airtight jar. (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). This created nearly 0% O2 conditions.
Gene silencing of RAGE with specific siRNA
Cells were seeded in 12-well plates at a density of 1 × 105 cells per well and allowed to adhere overnight. Then siRNAs for the target gene or its control oligoribonucleotide mixed with Dharma FECT2TM transfection reagent (Dharmacon Inc. Chicago, USA) was added to the cells and incubated for 48h at 37°C The efficacy of gene silencing was evaluated using immunoblot analysis.
MTT assay
Cell viability was monitored after incubation for 24, 36, and 48 hours by MTT assay. Briefly, 0.5 mg/mL 3-[4,5]-2,5-diphenyltetrazolium bromide (MTT) in fresh medium was added to each well and the cells were incubated for an additional 3 hours. Afterwards, the blue formazan crystals were dissolved in 1 mL isopropanol and measured spectrophotometrically at 570 nm.
Immunoblot analysis
Whole cell lysates were prepared as per the Santa Cruz protocol. One milliliter of Radio-Immunoprecipitation Assay (RIPA) buffer was added to a 100 mm cell culture plate. The plates were gently rocked for 15 min at 4°C. Adherent cells were scrpaed with a cell scraper, followed by incubation for 30–60 min on ice. The cell lysate was microcentrifuged at 10,000g for 10 min at 4°C. The supernatant fluid was the total cell lysate. The supernatant was transferred to a new microfuge tube and the pellet was discarded. Twelve microgram lysates were subjected to immunoblot analysis using a 12.5% sodium dodecyl sulfate (SDS) -polyacrylamide gel followed by electrotransfer onto nitrocellulose filters. The filters were immunoreacted with anti-RAGE antibody (a gift from TORAY Research Institute, Sagamihara, Japan) or with anti-HMGB1 antibody (BD Biosciences, Tokyo, Japan) and then incubated with peroxidase-conjugated anti-goat IgG (Medical and Biological Laboratories, Nagoya, Japan). The immune complex was visualized using the Enhanced Chemiluminescence (ECL) Western blot detection system (PIERCE, Rockford, IL, USA). The amount of B-actin as an internal control was also examined using a specific antibody (Cytoskelton Inc., Denver, CO, USA and Santa Cruz, CA, USA). At least three independent experiments were performed.
Quantitative RT-PCR
For reverse-transcription PCR (RT-PCR) and real-time quantitative PCR, total RNA was extracted from 30 mg frozen tissue using Total RNA Mini (VIOGENE, CA, USA). For cDNA synthesis, the RNA samples (1 μg) were converted into cDNA by reverse transcription (RT) using random primers (TAKARA, Siga, Japan) according to the manufacturer’s instructions. To estimate the mRNA expression levels of several genes quantitatively, polymerase chain reaction (PCR) amplification was performed using a Light-Cycler system (Roche, Mannheim, Germany) and the Light-Cycler Fast Start DNA Master SYBER green I kit (Roche). Primers were as follows: RAGE: 5′-AAA CAT CAC AGC CCG GAT TG-3′ and 5′-TCC GGC CTG TGT TCA GTT TC-3′, HMGB1: 5′-GCT CAG AGA GGT GGA AGA CCA-3′ and 5′-GGT GCA TTG GGA TCC TTG AA-3′ (14) , GAPDH: 5′-TTG GTA TCG TGG AAG GAC TCA-3′ and 5′-TGT CAT CAT ATT TGG CAG GTT T-3′. Amplification was carried out in 20 μL reactions containing 4 mM MgCl2, 2 μL of primer, 2 μL of Light-Cycler-FastStart DNA Master SYBR green I reagent, and 2 μL of cDNA. Reaction conditions were an initial incubation at 95°C for 10 min, followed by 50 cycles at 95°C for 10 s for denaturation, 64°C for 10 s for annealing of the RAGE primers, 54°C for 10 s for annealing of the HMGB1 primers and 60°C for 10 s for annealing of the GAPDH primers and 72°C for 10 s for extension. Melting curves were obtained according to the protocol under the following conditions: 0 s denaturation period at 95°C, starting temperature of 65°C, end temperatures of 95°C, and rate of temperatures increase of 0.1°C/s. The quantitative value of the target gene (RAGE mRNA) in each sample was normalized using GAPDH expression as an internal control. The quantitative RT-PCR assay was carried out twice and the mean value was calculated. Finally, the mRNA expression ratio of cancerous (C) to noncancerous (N) tissues was calculated using the following formula: R=log{target gene (C)/GAPDH (C)}, R=log{target gene (N)/GAPDH (N)}. These experiments were carried out twice to confirm reproducibility.
Statistical analysis
Statistical analysis was performed using the JMP IN version 5.1.2 software system (SAS institute Inc., Cary, NC, USA). Each value of mRNA expression was log transformed before statistical analysis. Gene expression was compared among normal liver, hepatitis, and HCC using Student’s t-test. The relationships between RAGE-, HMGB1-mRNA expression levels and clinicopathological features were evaluated using Student’s t-test and the Mann–Whitney U test, as appropriate. Immunohistochemical study of RAGE in HCC tissues was evaluated using the χ2 test. A p value of less than 0.05 was considered to be statistically significant.
RESULTS
RAGE and HMGB1 expression in normal liver, hepatitis, and HCC
RAGE antibody yielded a strong band compared to control mouse lung extracts. Using Western blotting and RT-PCR, protein and mRNA expression of RAGE and HMGB1 were examined in both cancer and noncancerous tissues from three cases (Fig. 1a, b). All 3 cases showed co-expression of RAGE and HMGB1 protein and mRNA in these tissues.
FIG. 1.(a) RAGE expression by Western blotting (A) and RT-PCR (B). (b) HMGB1 expression by Western blotting (A) and RT-PCR (B). Abbreviations: normal, normal liver; CH, chronic hepatitis.
Quantitative RAGE mRNA expression in HCC and noncancerous lesions
Comparing the quantitative expression of RAGE mRNA in paired cancer and noncancerous tissues (i.e., chronic hepatitis or liver cirrhosis) of 36 cases, HCC tissues showed significantly higher expression than noncancerous tissues (p < 0.01, Fig. 2a). Moreover, the mean values of RAGE mRNA expression in cancer and noncancerous tissues were higher (p < 0.01 and p = 0.08) than that in normal liver tissues (i.e., non-inflamed liver from benign or metastatic liver tumor patients) (Fig. 2b).
FIG. 2.(a) Quantitative RAGE mRNA expression in paired cancerous (HCC) and noncancerous tissues from 36 cases. (b) Quantitative RAGE mRNA expression in normal liver (n = 6) and noncancerous and cancerous tissues (n = 36).
Relationship between RAGE mRNA expression and clinicopathological features
To elucidate the biological significance of RAGE expression in HCC, we compared the levels of RAGE mRNA expression with the clinico-pathological features of 36 patients. As shown in Table 2, we noted significant differences in RAGE mRNA expression in association with gender, age, the levels of protein induced by vitamin K absence or antagonist (PIVKA-II), and postoperative recurrence. In terms of gender and age, the results are in accordance with a previous report.23 The expression levels of RAGE mRNA tended to be lower in poorly differentiated tumors compared to well- or moderately differentiated tumors (p = 0.06). Moreover, the levels of RAGE mRNA showed a negative correlation with PIVKA-II levels and the presence or absence of recurrence. There were no significant differences regarding tumor size, intrahepatic metastasis, and vascular invasion. On the other hand, it should be noted that a significant difference in the levels of RAGE expression was observed between hepatitis virus-positive and virus--egative tissues (p < 0.01) in the study of noncancerous inflammatory liver tissues (i.e., chronic hepatitis or liver cirrhosis) (data not shown).
TABLE 2.Relationship between tumor RAGE expression and clinicopathological featuresFactorsTumor RAGE mRNA expressionp valuenMean ± SDGender  Male302.267 ± 0.3410.03  female62.777 ± 0.619Age  ≥65 years262.468 ± 0.5680.03  <65 years102.052 ± 0.638Tumor size (mm)  ≥30112.395 ± 0.6130.39  <30252.333 ± 0.620Portal invasion  Absent222.358 ± 0.6550.52  Present142.348 ± 0.594Venous invasion  Absent262.339 ± 0.5770.47  Present102.357 ± 0.633Vascular invasion  Absent192.362 ± 0.6330.46  Present172.340 ± 0.601Intrahepatic metastasis  Absent272.378 ± 0.6620.33  Present92.273 ± 0.435Gross classification  Localized type212.448 ± 0.6360.13  Invasive type152.217 ± 0.564Differentiation  Well42.365 ± 0.5660.43  Moderately282.420 ± 0.5800.08  Poorly41.860 ± 0.783Stage  I,II162.367 ± 0.6430.55  III,IV202.340 ± 0.597PIVKA‡U  Normal92.704 ± 0.385  High252.186 ± 0.6300.03AFP  Normal122.557 ± 0.5860.16  High242.250 ± 0.607Virus  B72.237 ± 0.606  C212.382 ± 0.569  None82.374 ± 0.779Recurrence  Absent252.485 ± 0.6280.04  Present112.050 ± 0.456
RAGE expression and tumor differentiation in HCC
To verify the correlation of RAGE expression with HCC differentiation, we carried out an immunohistochemical examination. (Fig. 3a–c) Well- and moderately differentiated HCC showed a high percentage of RAGE positivity (70% and 64%, respectively); only 2 of 10 (20%) were positive in poorly differentiated HCC (Fig. 3d). Statistically, there were significant differences in the rates of positivity between well- and moderately differentiated HCC and poorly differentiated HCC.
FIG. 3.RAGE expression by immunohistochemical staining: (a) well-differentiated HCC, (b) moderately (mod.) differentiated HCC, (c) poorly differentiated HCC, (e) the number of RAGE positive or negative cases according to tumor differentiation.
RAGE expression in hepatoma cell lines
RAGE antibody yielded a strong band on Western blot analysis of the control RAGE transfected Cos7 cells (Fig. 4a, lane 1). In this experimental setting, the well-differentiated HCC cell line HuH7 (lane 6) expressed RAGE protein at a modest level. The poorly differentiated HCC cell line HT17 (lane 7) expressed RAGE protein at a low level. Hepatoma cell lines Hep3B and Li7 (lane 4 and 5), derived from tumors of unknown state of differentiation, expressed RAGE protein at a modest level. HepG2 (lane 3) did not express RAGE protein at all. These results were very similar to RAGE mRNA expression and depend on the pathological differentiation of HCC.
FIG. 4.(a) RAGE expression in hepatoma cell lines by Western blotting. (b) RAGE expression in hepatoma cell lines under hypoxic conditions by Western blotting. N, normoxic conditions; A, anaerobic conditions.
Enhanced RAGE expression under hypoxic conditions
Early carcinogenesis occurs in an avascular environment and under conditions of hypoxia.15,23,24 In order to clarify the role of RAGE expression under hypoxic conditions, all hepatoma cell lines were cultured in an anaerobic environment. Li7 could survive for 48 h, Hep3B for 12 h, and HuH7 for 24 h under such conditions, up to the appearance of signs of cell death. HepG2 and HT17 rapidly died under anaerobic conditions. The RAGE-positive cell lines (Li7, Hep3B and HuH7) were more tolerant of anaerobic conditions than RAGE-negative or weakly expressing cell lines (HepG2 and HT17). Moreover, RAGE expression in the cell lines that survived was clearly upregulated in response to the hypoxic conditions (Fig. 4b).
Enhancement of cell survival in anaerobic conditions following RAGE transfection
These findings led to the hypothesis that RAGE expression could confer tolerance to hypoxia in HCC. Therefore, we investigated the survival of RAGE-transfected and mock-transfected cells under hypoxic conditions. In continuous incubation under hypoxic conditions, as expected, RAGE-transfected Cos7 cells clearly survived longer than mock-transfected Cos7 cells (observed at 36 h and 48 h) (Fig. 5).
FIG. 5.Comparison of cell survival of RAGE-transfected Cos7 and mock-transfected Cos7 cells under anaerobic conditions. Cell survival of both groups was estimated from six dishes at each time point using the MTT assay. *p < 0.01 by Student’s t-test.
Decline of cell survival in hypoxic conditions following RAGE reduced by siRNA
Consistent with the previous result, reduction of RAGE expression with anti-RAGE siRNA re-increased susceptibility to hypoxia-induced injury in RAGE-overexpressed Cos7 transfectant (Fig. 6a,b), suggesting that RAGE expression might play an important role in the acquirement of hypoxia-resistant cellular phenotype.
FIG. 6.RAGE level in RAGE-transfected Cos7 cells were reduced by siRNA RAGE-transfected Cos7 cells reduced RAGE level by siRNA decreased more than the cells mixed with only transfection reagent. *p < 0.01 by Student’s t-test.
DISCUSSION
Hepatocellular carcinogenesis and associated tumorigenesis occur in a stringent and restrictive environment, such as the cirrhotic liver in which tissue oxygen supply is insufficient. Moreover, in the liver, with a dual blood supply from the hepatic artery and portal vein, liver-specific oxygen and glucose supply are available dependent upon the degree of liver disorder. The multiligand receptor RAGE is particularly relevant in this context. The ligands for RAGE may be produced abundantly during HCC development. For example, HMGB1 released from necrotic cells and AGE generated in the process of higher hepatic glucose metabolism are considered to be major candidates (i.e., the presence of HMGB1 and carboxymethyl lysine in HCC; data not shown). Hepatic SAA production in response to IL-6 may also be included.19 In addition, RAGE expression and its downstream signaling are now considered to play a significant role in the progression of various cancers as well as the development of inflammatory lesions.
Another aspect of the biology of RAGE was suggested by the features of retinoic acid-induced neuroblastoma differentiation, in which RAGE expression played a more important role in cellular survival than in neurite outgrowth.25 In this model, inhibition of RAGE function partially blocked the increase in levels of the anti-apoptotic protein Bcl-2 in the process of neuronal differentiation,25 indicating that RAGE and its signaling also might contribute to the survival of certain cancer cell types undergoing “differentiation”. Furthermore, although indirectly, our previous study demonstrated that NF-κB, currently considered as a causative transcriptional factor for various inflammatory events and also known as a major RAGE-mediated signal, played an important role in the survival of neuronal tumor cells26,27 and in experimentally developed HCC.28
The present study demonstrated that increased RAGE expression was highly associated with the status of pathological “differentiation” in HCC, which played a significant role in acquisition of the hypoxia-resistant phenotype of tumor cells. This conclusion is supported by several lines of experimental evidence. First, the level of RAGE expression was higher in well- and moderately differentiated HCC, while it diminished as the tumors dedifferentiated to poorly differentiated HCC. This was consistent with the evidence that a negative correlation was observed between the level of RAGE mRNA expression and either the level of PIVKA II or the incidence of postoperative recurrence. Second, the analysis of five HCC lines revealed that three of these (Li7, Hep3B, and HuH7) that are resistant to hypoxic stress characteristically showed higher levels of RAGE expression compared to the two hypoxia-intolerant cell lines HepG2 and HT17. Third, sublethal hypoxia exposure induced significantly increased RAGE expression in hypoxia-resistant HCC lines. In the analysis of the association between the level of RAGE expression and the “differentiation status” of HCC lines, the level of RAGE expression was higher in “possibly differentiated” lines (i.e., HCC with low Alpha-Fet protein (AFP) production), consistent with the results from clinical samples. Finally, cells overexpressing RAGE exogenously showed prolonged survival under hypoxic conditions compared to control mock-transfected cells, and siRNA experiments demonstrated similar results.
Our conclusion also provides a new hypothetical concept that hepatic RAGE expression may be relevant to the stage or severity of inflammation and the incidence of carcinogenesis and early tumorigenesis of HCC (Fig. 7). In the process of the development of an HCC lesion, increased RAGE expression by inflamed hepatocytes may confer adaptation to an advanced hypoxic environment during remodeling and carcinogenesis with accelerated cell proliferation. In this context, an HCC lesion which is highly associated with inflammation caused by either hepatitis viruses or drugs is developed through a process of multistage carcinogenesis, i.e., from inflammatory lesions (e.g., hepatitis, cirrhosis and precancerous lesion) to adenomatous hyperplasia, and eventually into HCC. Once cancer is established, HCC dedifferentiates step-by-step to a more malignant histology, from well- and moderately to poorly differentiated HCC. It has also been pointed out that the vascular supply changes significantly between each stage of tumorigenesis. According to the evidence from several clinical studies,15,29,30 both adenomatous hyperplasia and well-differentiated HCC are hypovascular tumors primarily fed by the portal vein system, while moderately and poorly differentiated HCC are hypervascular tumors primarily fed by arterial blood. In addition, early carcinogenesis and the development of the malignant phenotype generally occur in an avascular environment.31–33 Increased RAGE expression, at least in part, may thus play an important role in the mechanism of early HCC development from precancerous inflammatory lesions. In addition, about the expression of RAGE being lower in poorly differentiated HCC when compared to well and moderately differentiated HCC, gastric and colon cancers have been showed that RAGE and other ligands interacts act on tumor promotion in clinical materials.9,11 However, prostate cancer was demonstrated to show no correlation between RAGE expression and tumor differentiation and depth.14 Moreover, non-small-cell lung cancers have been showed a reverse correlation between RAGE expression and tumor stage.34 This result is partially similar to our results. RAGE expression of each clinical samples is various.
FIG. 7Scheme of the change of RAGE expression according to the sequential change of liver tissue: normal → chronic hepatitis (CH) → cirrhosis → HCC. The value indicates the quantitative RAGE mRNA expression.
Some reports have shown that RAGE-expressed cells have invasion and migration potential. Our data from clinical samples did no’ relate to the potential. In vivo, invasive and metastatic potential are reflected by many factors, which may have caused our results.
Our results raise many questions concerning mechanistic and practical processes. It is important to know whether RAGE can bind HMGB1 secreted from activated macrophages in hepatic inflammation, or if occupancy of RAGE by inhibitors would obviate binding of the stimulatory ligands. The functional role of downstream signaling of the RAGE would also be important to determine the cytoprotective mechanism under hypoxia.
Our findings have provided the first evidence of the clinical relevance and function of RAGE in HCC, namely differentiation-associated RAGE expression that confers a hypoxia-resistant phenotype. Although other mechanisms may also be important, our data also introduce the concept that RAGE and its functions may be possible candidates for therapeutic targets in the treatment of HCC.

receptor for advanced glycation end-products ( rage )

hypoxia-reverse-transcription polymerase chain reaction ( rt-pcr )