CD10)-positive Myofibroblasts—Precursors of Angiogenesis in Chorioangiomas?

Placenta (2003), 24, 758–766 doi:10.1016/S0143-4004(03)00107-3

VEGF-, KIT Protein- and Neutral Endopeptidase (NEP/CD10)-positive Myofibroblasts—Precursors of Angiogenesis in Chorioangiomas? F. Noack a,d, K. Sotlar b, C. Thorns a, J. Smrcek c, K. Diedrich c, A.C. Feller a and H.-P. Horny a a

Department of Pathology, University Hospital Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany; b Department of Pathology, University of Tu¨bingen, Germany; c Department of Obstetrics and Gynecology, University Hospital Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany Paper accepted 23 April 2003

Chorioangiomas are benign angiomatous tumours of the placenta occurring with a frequency of approximately one per cent of all examined placentae. Hypoxia and genetic factors are discussed to be predisposing factors for chorioangiomas. However, not much is known about the tumorigenesis of these benign tumours. Screening with various antibodies in a rare case of chorangiomatosis, we found disseminated spindle cells coexpressing vascular epithelial growth factor (VEGF), neutral endopeptidase 24.11 (NEP/CD10), and KIT protein (CD117) within the tumour stroma. A possible involvement of such factors in angiogenesis and tumorigenesis of chorioangiomas/chorangiomatosis has not been studied so far. Seven placentae with chorioangiomas (n=6) or chorangiomatosis (n=1), six normal placentae, and four cutaneous haemangiomas were analysed immunohistochemically (ABC and APAAP methods) using antibodies against VEGF, NEP, KIT protein, as well as endothelial markers like PECAM-1 (CD31), CD34, v. Willebrand factor (factor VIII), and ulex europaeus. In addition, analysis of c-kit ‘gain of function’ mutation Asp 816 to Val by means of Hinfl digestion and direct sequencing of semi-nested polymerase chain reaction products was performed. All chorioangiomas and haemangiomas strongly expressed the endothelial markers CD34, CD31, and FVIII, while only weak expression of ulex lectin was noted. Disseminated groups of VEGF-, NEP-, and KIT protein-positive spindle cells, which coexpressed vimentin and smooth-muscle actin were identified as myofibroblasts in the stroma of four chorioangiomas. These spindle cells were quantified as numerous in two and as rare in two other cases. No VEGF-positive myofibroblasts, however, were detected in the villous stroma of normal control placentae and haemangiomas. Only scattered perivascular myofibroblasts expressing KIT protein and NEP were detected in early gestational placenta controls. In all chorioangiomas and chorangiomatosis PCR analysis failed to unveil c-kit ‘gain of function’ mutation Asp 816 to Val in KIT protein-positive spindle cells. Moreover, a significant increase in mast cells was observed only in the haemangiomas. As expected, endothelial origin of chorioangiomas/chorangiomatosis was verified by CD31, CD34, FVIII expression. Myofibroblastic spindle cells expressing VEGF and NEP may be precursor cells in these peculiar angiomatous tumours. Although activating c-kit mutation Asp 816 to Val was not detected by PCR, the presence of KIT protein (CD117)-positive intratumoral myofibroblastic spindle cells in chorioangiomas and chorangiomatosis might suggest involvement of the stem cell factor (SCF)-receptor in pathologically enhanced angiogenesis. Placenta (2003), 24, 758–766  2003 Elsevier Ltd. All rights reserved.

INTRODUCTION Chorioangiomas are the most common placental tumours, occurring in approximately one per cent of all pregnancies (Fox, 1978). As early as 1798, chorioangioma was first described by Clarke (Clarke, 1798). These benign tumours are thought to be hamartomatous malformations. The typical chorioangioma is composed of fetal blood vessels and surrounded by only scant connective tissue (Benirschke and Kaufmann, 2000). Most of these tumours are small without clinical relevance. However, larger chorioangiomas may influd

To whom correspondence should be addressed. Tel.: +49-451-5002700; Fax: +49-451-500-4851; E-mail: [email protected] 0143-4004/03/$–see front matter

ence pregnancy. Complications include abruptio placentae, premature labour, fetal anaemia, hydrops, cardiomegaly and polyhydramnios (Fox, 1978; Tonkin, Setzer and Ermocilla, 1980; Rodan and Bean, 1983; Benirschke and Kaufmann, 2000). While the majority of chorioangiomas present as single nodules, they can also occur multiple, then referred to as chorangiomatosis. The exact pathogenesis of chorioangiomas has not yet been entirely established. Chorioangioma probably arise as malformations from primitive angioblastic tissue (Fox, 1978). Hypoxia and genetic factors are discussed to be predisposing factors for chorioangioma (Soma, Watanabe and Hata, 1995).  2003 Elsevier Ltd. All rights reserved.

Noack et al.: VEGF-, KIT-, and CD10-positive Myofibroblasts in Chorioangiomas

Prior to this study we investigated a rare case of chorangiomatosis by immunohistochemistry using a large scale of antibodies. Screening enabled us to detect disseminated spindle cells coexpressing VEGF, KIT protein and NEP. The 34–46 kDa glycoprotein VEGF has been characterized as potent angiogenic growth factor, which acts on both endothelial and trophoblastic cells (Ferrara et al., 1992). It is selectively mitogenic for endothelial cells and appears to play a key role in the mediation of vasculogenesis, i.e. de novo differentiation of endothelial cells to form new vessels, as well as angiogenesis, i.e. new capillary arising from pre-existing vessels (Ferrara et al., 1992; Ferrara, 2000). KIT protein (CD117) is a 145–160 kDa transmembrane type III tyrosine kinase and is expressed on haematopoietic stem cells, mast cells (MCs), germ cells, melanocytes, luminal epithelium of breast, and Cajal cells of the gastro-intestinal tract (Williams et al., 1990; Zsebo et al., 1990; Tsuura et al., 1994). The extracellular domain of the KIT protein binds stem cell factor (SCF), a pleiotropic glycoprotein with a molecular mass of 25–36 kDa also known as Kit ligand or mast cell growth factor. Among solid spindle-shaped mesenchymal tumours, KIT protein is consistently expressed in gastrointestinal stromal tumours (GIST). The 90–100 kDa membrane-bound metallopeptidase neutral endopeptidase 24.11 (NEP) regulates biological activities of peptide substrates. Human NEP has been cloned and is identical with the common acute lymphoblastic leukaemia antigen (CALLA or CD10) (Shipp, Vijayaraghavan and Schmidt, 1989), which serves as an important cell surface marker in the diagnosis of human acute lymphoblastic leukaemia and germinal centre lymphomas. NEP is widely distributed in nonhaematopoietic cells and certain solid tumour cell lines (Chu and Arber, 2000). VEGF is an important angiogenic growth factor and thought to be important for placental development (Jackson et al., 1994). NEP- and KIT protein expression have been demonstrated in normal placentae (Imai et al., 1992; Kauma et al., 1996; Ino et al., 2000). NEP regulates local concentrations of circulating biologically active peptides at the fetal– maternal interface (Imai et al., 1992). KIT protein is known to be involved in the control of proliferative and invasive processes in the placenta (Benirschke and Kaufmann, 2000). Since a possible involvement of VEGF, NEP and KIT protein in angiogenesis and tumorigenesis of chorioangiomas/ chorangiomatosis has not been studied so far, we investigated expression of these factors in chorioangioma and chorangiomatosis reviewing all cases of chorioangioma diagnosed in our institute between 1992 and 2002.

MATERIAL AND METHODS Generally only placentae with presumed maternal and/or fetal pathology are submitted for pathological examination to our institute. On average we receive about 100 placentae p.a. From our archive we identified eight cases of chorioangiomas and

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one case of chorangiomatosis diagnosed in between 1992 and 2002. However, from two cases no suitable material was available. Formalin-fixed and paraffin-embedded tissues from chorioangiomas (n=6) and chorangiomatosis (n=1) were retrieved from the archives of our institute. In all cases referring clinicians had not been conspicuous of chorioangioma or chorangiomatosis (Table 1). Furthermore, tissue from six placentae without pathological findings (12, 13, 16, 23, 40, and 41 weeks of gestation) and from four cutaneous haemangiomas (three juvenile haemangiomas and one cavernous haemangioma) were used as controls.

Immunohistochemistry Paraffin blocks were cut at 4 µm. Haematoxylin and eosin (H&E), Giemsa, and naphthol AS-D choroacetate esterase (CAE) stains were performed. Immunhistochemical staining was carried out either by the ABC or APAAP method. Antibodies used in this study are summarized in Table 2. Two observers independently evaluated the immunoreactivity according to a semi-quantitative score: no immunostaining (=0); weak, focal staining (=+); moderate and diffuse positivity (=++); and strong positivity (=+++).

Mutation analysis Whole microtome sections cut from paraffin-embedded tissue blocks of the chorioangiomas were analysed for c-kit mutation Asp 816 to Val. Online mutation detection during nested PCR amplification of c-kit DNA using mutation-specific probes and the LightCycler sequence detection system (ROCHE, Mannheim, Germany) was performed as recently described elsewhere (Sotlar et al., 2002). In brief, after digestion with proteinase-K at 55(C for 12 h, PCR mixtures were added to each PCR tube. To increase the sensitivity of subsequent amplification, nested PCR with outer primers c-kit S (5#-CAG CCA GAA ATA TCC TCC TTA CT-3#) and c-kit C (5#-CAT TAT GAA AGT CAC GGA AAC AAT-3#), and inner primers c-kit U (5#-CAC AAA GAT TTG TGA TTT TGG TCT A-3#) and c-kit B (5#-TTG CAG GAC TGT CAA GCA GAG-3#) was performed. The first round of amplifications (40 cycles) was performed in a Perkin–Elmer GeneAmp 9700 thermocycler (PE Applied Biosystems, Weiterstadt, Germany). Nested PCR amplification followed with 2 µl of the PCR product as template. LightCycler online mutation detection was performed using the DNA Master Hybridization Probes kit. Briefly, hybridization probes consisted of two different oligonucleotides hybridizing to an internal sequence of the c-kit gene. Hybridization probe c-kit sensor (5#-AGC CAG AGA CAT CAA GAA TGA TTC TA-3#) spanning the point mutation at nt 7176 was 3#-end labelled with fluorescein and hybridization probe c-kit anchor (5#-ATG TGG TTA AAG GAA ACG TGA GTA CCC A-3#) was 5#-labelled with the LightCycler-Red 640 (TIB

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Table 1. Clinical and pathological characteristics of placentae with chorioangiomas (case 1–6) and chorangiomatosis (case 7) Case no.

Age and pariety of mother

Weeks of gestation

Clinical diagnosis

Size and weight of placenta

Size and number of chorioangiomas

Other pathological findings/anomalies

1

30 years, 2G, 2P

32+2

2

35 years, 2G, 1P

33+1

3

27 years, 1P, 1P

39+3

Gemini, pre-eclampsia, postpartal HELLP syndrome Stillbirth, fetal diaphragmatic defect Gemini

Several chorioangiomas, diameter up to 0.5 cm Single chorioangioma, diameter 2.5 cm II: Single chorioangioma, diameter 0.5 cm

Diamniotic dichorionic placenta, increased perivillous fibrin Unevenly accelerated maturation, chronic infarctions Diamniotic dichorionic placenta

4

28 years, 1G, 1P

32+0

Pre-eclampsia, SGA newborn

5

31 years, 3G, 1P

36+2

Adherent placenta

6

25 years, 1G, 1P

31+0

Gemini, PROM

Single chorioangioma, diameter 1 cm Single chorioangioma, diameter 0.6 cm II: single chorioangioma, diameter 544 cm

Hypotrophy, unevenly accelerated maturation, chronic infarcts Acute chorioamnionitis, slightly delayed maturation Diamniotic dichorionic placenta

7

33 years, 3G, 2P

35+2

SGA fetus, oligohydramnios

21142 cm; 600 g 13112.5 cm; 260 g I: 22182 cm; 440 g II: 20171.8 cm; 380 g 14112.2 cm; 260 g 19.515.52 cm; 395 g I: 18182 cm; 350 g II: 19172.5 cm; 390 g 15154 cm; 450 g

Multiple chorioangiomas, measuring up to 1.5 cm

None

PROM=premature rupture of fetal membranes; SGA=small-for-gestational-age

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Table 2. List of antibodies with working dilution and source Antibody
-HCG CD34 CD68 Factor VIII-related antigen (F VIII) KIT protein (CD117) NEP (CD10) PECAM-1 (CD31) Tryptase Ulex europaeus agglutinin VEGF

Clone QBEnd10 104D2 56C6 JC/70A AA1

Isotype

Dilution

Source

Polyclonal IgG1 IgG3 Polyclonal IgG1 IgG1 IgG1 IgG1 Polyclonal Polyclonal

1 : 3000 1 : 100 1 : 50 1 : 1500 1 : 100 1 : 50 1 : 40 1 : 100 1 : 400 1 : 100

DAKO A/S, Denmark Biocarta Europe, Hamburg, Germany DAKO A/S, Denmark Novocastra, Newcastle-upon-Tyne, UK DAKO A/S, Denmark Novocastra, Newcastle-upon-Tyne, UK DAKO A/S, Denmark DAKO A/S, Denmark Vector, Burlingame, CA, USA Santa Cruz, CA, USA

MOLBIOL, Berlin, Germany). After hybridization of the two probes to the target PCR product in a head-to-tail fashion, the two fluorescent dyes come into close proximity. During melting curve analysis, slowly increasing the temperature from 40(C to 80(C, fluorescein is excited by the light source of the LightCycler instrument. The excitation energy is transferred to the receptor fluorophore, LightCycler-Red 640, and the emitted fluorescence is continuously measured by photohybrids of the instrument while the temperature is slowly increased. When one of the probes melts off the target, the two fluorophores are no longer in close proximity, resulting in a decrease in emitted fluorescence. The presence of a mutation within the target region of the sensor probe will result in its melt off at lower temperatures as compared to the perfectly matching wild type sequence.

RESULTS

Table 3. Results of immunohistochemical staining of chorioangiomas (n=7) with antibodies directed against VEGF, KIT protein, and NEP. (0)

(+)

(++)

(+++)

VEGF Endothelial cells Stroma Cytotrophoblast Syncytiotrophoblast

6 2 1 4

1 3* 0 1

0 2* 1 2

0 0 2 0

KIT protein Endothelial cells Stroma Cytotrophoblast Syncytiotrophoblast

7 3 4 7

0 2* 0 0

0 1* 0 0

0 1* 0 0

NEP Endothelial cells Stroma Cytotrophoblast Syncytiotrophoblast

7 0 3 0

0 3* 0 0

0 3* 1 3

0 1* 0 4

* =spindle shaped myofibroblasts

Endothelial cell markers Unanimously, vascular cells in chorioangiomas and chorangiomatosis showed expression of endothelial markers. Immunoreactivity with the following antibodies was observed in descending order: CD34 (+++), CD31 (+++), FVIII (++), and the lectin ulex europaeus (+). In the surrounding normal placental tissue as well as in all control placentae all villous capillaries were also stained with the endothelial cell markers in the above-mentioned decreasing order of intensity. The haemangiomas, especially the juvenile haemangiomas stained for endothelial markers in a similar fashion as the chorioangiomas.

cells in the stroma of chorioangiomas (Table 3). These spindle cells coexpressed vimentin and smooth muscle-actin (Figure 1), and were therefore identified as myofibroblasts. By contrast, all control placentae did not display such VEGF-positive myofibroblasts in the villous stroma. VEGF was also expressed by
-HCG-positive cytotrophoblastic and syncytiotrophoblastic cells. In two cases of early gestation, we found VEGFexpression in scattered CD68-positive Hofbauer cells. In haemangiomas, only sporadic weak VEGF staining of capillaries was seen (data not shown). VEGF-positive spindle cells in the stroma of the haemangiomas were not detected.

VEGF

KIT protein

No VEGF expression could be demonstrated in endothelial cells of all chorioangiomas. However, in five of seven cases positivity of VEGF could be demonstrated in spindle-shaped

No expression of KIT protein could be demonstrated in the trophoblastic layer of the chorioangiomas/chorangiomatosis, whereas weak KIT protein staining was observed in the

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Figure 1. H&E staining of chorioangiomas (A). Identification of the myofibroblastic origin of the spindle cells in the stroma of the chorioangiomas by strong positive staining for vimentin (B) and smooth muscle actin (C). A significant proportion of myofibroblastic spindle cells showed positivity of VEGF (D), KIT protein (E), and NEP (F). Scale bar in A=400 µm, in B–E=100 µm.

syncytiotrophoblast of normal control placentae throughout gestation (Table 4). In four of seven examined cases of chorioangioma, KIT protein-positive spindle-shaped myofibroblasts were observed (Figure 1). These spindle cells were definitively not MC, which was proven by negativity of these cells with anti-tryptase, Giemsa, and naphthol AS-D choroacetate esterase stains. Scattered KIT protein-positive myofibroblasts could also be observed in early gestational control placentae up to 23 weeks of gestation (Table 4). An increase in MCs, however, was noted in all juvenile haemangiomas. Almost all MCs in haemangiomas were KIT protein-positive (Figure 2). However, no KIT protein-positive myofibroblasts were found in the stroma of the haemangiomas.

Mutation analysis In none of the chorioangiomas, PCR analysis unveiled c-kit point mutation Asp to Val at codon 816. Likewise, no c-kit mutations were found in control placentae and haemangiomas. As positive controls, HMC-1 DNA was used, resulting in the detection of a wild type peak at 63.5(C and a mutation-specific peak at 59.5(C (Figure 3).

NEP In chorioangiomas, NEP was expressed by the cytotrophoblast (1/7) and syncytiothrophoblast (7/7). In all chorioangiomas,

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Table 4. Results of immunohistochemical staining of chorionic villi in normal placentae from 12 to 40 weeks of gestation. VEGF Endothelial cells Villous stroma Cytotrophoblast Syncytiotrophoblast

0 +1 ++ +

0 +1 + +

0 0 + +

0 0 + +

0 0 0 +

0 0 0 +

KIT protein Endothelial cells Villous stroma Cytotrophoblast Syncytiotrophoblast

0 +1,2 0 +

+ ++1,2 0 +

+ +2 0 0

0 +++1,2 0 +

0 0 0 +

0 0 0 0

0 ++2 + ++ 12 weeks’ gestation

0 ++2 + + 13 weeks’ gestation

0 +++2 + ++ 16 weeks’ gestation

0 +++2 0 ++ 23 weeks’ gestation

0 0 0 ++ 36 weeks’ gestation

0 0 0 +++ 40 weeks’ gestation

NEP Endothelial cells Villous stroma Cytotrophoblast Syncytiotrophoblast

1 2

=weak immunostaining in scattered Hofbauer cells =perivascular spindle shaped myofibroblasts

we found NEP-positive spindle-shaped myofibroblasts. Normal placentae exhibited NEP-expression in the cytotrophoblasts in early gestation, consistently at apical surfaces of the syncytiotrophoblast and also in chorionic mesenchymal cells. Up to 23 weeks of gestation, we also observed NEPpositive myofibroblasts in normal placentae, primarily in the perivascular stroma. Apart from weak basal membrane-like NEP staining of the capillaries, no NEP-expression could be observed in the haemangiomas, in particular no NEP-positive myofibroblasts were discovered. The immunolocalizations of VEGF, KIT protein, and NEP in chorioangiomas are summarized in Table 3. The quantity of spindle cell shaped myofibroblasts exhibiting all three antibodies were numerous in two cases and rare in two cases.

N-terminal amino acid residues 1–20 of human VEGF, whereas the other groups including ours used commercially obtained antibodies. Yet, VEGF expression of myofibroblasts in chorioangiomas has not been described so far. This study revealed intense VEGF expression in myofibroblasts in four of the seven chorioangiomas. This result suggests involvement of VEGF in the pathogenesis, i.e. angiogenesis of chorioangiomas. Sweiki et al. proposed hypoxia to be the initial stimulus of VEGF-mediated angiogenesis (Sweiki et al., 1992). Shore and colleagues showed that hypoxia induces VEGF-expression in term trophoblasts (Shore et al., 1997). Increased expression of VEGF in fibroblasts in first-trimester placentae as a response to hypoxia was demonstrated by Wheeler, Elcock and Anthony (1995). Hypoxia could also be an important stimulus of VEGF expression in myofibroblasts in chorioangiomas and consequent excessive angiogenesis.

DISCUSSION VEGF

KIT protein

In accordance with Jackson et al. and Asif et al., we found VEGF expressed in normal placentae in the cytotrophoblast and Hofbauer cells in early gestation, and in the syncytiotrophoblast throughout gestation. No immunostaining, however, was observed in endothelial cells (Jackson et al., 1994; Asif et al., 1995). Moreover Shiraishi and coworkers demonstrated VEGF-positive fibroblasts or fibroblast-like mesenchymal stromal cells in first trimester placentae (Shiraishi et al., 1996). Like other groups, we were unable to demonstrate VEGF-positive fibroblasts in the stroma of first trimester placentae (Jackson et al., 1994; Asif et al., 1995). Shirashi and coworkers discussed the use of different antibodies as possible explanation of the different findings: Shirashi’s group used rabbit anti-IgG against a synthetic peptide composed of

Sharkey and coworkers demonstrated KIT protein to be solely expressed by Hofbauer cells, and by decidual macrophages at all stages in pregnancy (Sharkey et al., 1994). In contrast to Sharkey’s study, Kauma et al. found KIT protein mainly immunolocalized in both syncytiotrophoblast and cytotrophoblast in early placenta and at term (Kauma et al., 1996) Our results revealed that KIT protein was expressed by Hofbauer cells in normal placentae as well as by syncytiotrophoblastic cells, but not by cytotrophoblastic cells. The use of different antibodies as well as antigen-unmasking procedures may be responsible for these differing results (Kauma et al., 1996). KIT protein expression of myofibroblasts in chorioangiomas has not been described previously. Not much is known about the possible role of c-kit and KIT protein in

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deletions in the receptor intracellular juxtamembrane region disrupting inhibitory control of the receptor kinase activity. The juxtamembrane c-kit mutations appear more commonly in human gastrointestinal stroma tumours (Hirota et al., 1998; Longley et al., 1999). We were unable to detect c-kit activating 816 Asp to Val in myofibroblasts of all chorioangiomas/ chorangiomatosis. However, we only surveyed c-kit 816 Asp to Val mutations in this study to endorse clonal or neoplastic orgin of chorioangioma/chorangiomatosis. Alternative c-kit mutations should also be analysed in order to prove clonality definitely.

NEP In early gestation, NEP has been demonstrated in chorionic mesenchymal cells of normal placentae (Toki et al., 2001). NEP expression was also shown in the cytotrophoblast and the apical surface of the syncytiotrophoblast in normal placentae (Imai et al., 1992; Ino et al., 2000; Toki et al., 2001; Uehara et al., 2001). However, no NEP immunoreactivity was yet observed in stromal cells or fetal vessels of the placental villi (Ino et al., 2000). In this study, myofibroblasts in chorioangiomas revealed NEP expression. NEP has many peptide substrates including enkephalins, bombesin, and endothelins (Erdo¨s and Skidgel, 1989). Salani et al. demonstrated that endothelin-1 (ET-1) is a potent mitogen for endothelial cells. Angiogenic response of cultured endothelial cells and stimulation of neovascularization in concert with VEGF was shown in vivo (Salani et al., 2000).

Different mechanism of angiogenesis in chorioangioma and haemangioma

Figure 2. H&E staining of a juvenile haemangioma (A). Increase in mast cells is demonstrated in (B) by anti-tryptase staining. A significant part of the MCs is positive for KIT protein (C). Scale bars=100 µm.

the maturing placenta. It remains a matter of speculation, whether KIT protein plays a role in placental angiogensis and also in the pathogenesis of chorioangiomas. Additionally to the immunohistochemical presence of KIT protein we investigated the tissues for activating c-kit mutation Asp 816 to Val by PCR. In humans, the proto-oncogene c-kit is located on chromosome 4 (4q11–12) and encodes the KIT protein. Activating c-kit mutations can be divided into two groups. Mutations in the tyrosine-kinase domain, most often involving codon 816, resulting in a substitution of valine to aspartartic acid are usually found in sporadic adult-onset mastocytosis and in a subset of paediatric cases with a more aggressive mastocytosis (Longley et al., 1999). The other group includes single residue substitutions and inframe insertions or

In comparison with chorioangiomas, we found accumulating MCs in juvenile haemangiomas. A significant part of these MCs exhibited KIT protein (shown in Figure 3), but no VEGF or NEP. Increase in MCs has been associated with tumour angiogenesis in haemangiomas (Glowacki and Milltan, 1982; Qu et al., 1995). However, the functional significance of MC infiltration in haemangiomas is not entirely clear (Ribatti, 2001). Nakanishi et al. designated increase in MCs as a host response to neoplasia (Nakanishi et al., 1994). MCs comprise preformed and newly synthesized cytokines and growth factors that initiate or monitor the angiogenic process. Infiltration of MCs was demonstrated in experimentally induced tumours before the beginning of tumour-induced angiogenesis (Kessler et al., 1976). Furthermore, MC-deficient mice demonstrated reduced vascularity and fewer metastases (Starkey, Crowle and Taubenberger, 1988). Altogether, these findings indicate a direct involvement of MCs in tumour angiogenesis (Ribatti, 2001). Absent VEGF expression of MCs in haemangiomas in this study may partially reflect a biological behaviour of the life cycle beyond the proliferating phase (Takahashi et al., 1994; Bielenberg et al., 1999). KIT protein could play a crucial role

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Figure 3. Melting point analysis of PCR products in the successfully amplified samples of chorioangiomas (n=6) respectively chorangiomatosis (n=1). In each of the seven samples a melting point of 59.5(C was observed, specific for the wild type sequence. An additional peak of 63.5(C was generated by melting point analysis of amplified HMC-1 DNA, specific for the mutation Asp 816 to Val.

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