Early regrowth of juvenile cerebral arteriovenous malformations: report of 3 cases and immunohistochemical analysis

1. Introduction 

Cerebral AVMs are complex abnormal blood vessels thought to result from a failure of embryogenesis in the otherwise-normal differentiation of primordial vascular channels into mature arteries, capillaries, and veins [16], [17]. Complete surgical resection documented by postoperative angiography is believed to eliminate the risk of subsequent hemorrhage. Because AVMs are developmental malformations and not neoplastic, recurrence is not expected after an angiogram has documented absence of residual nidus or early draining veins. Regrowth of a lesion with subsequent hemorrhage after angiographically documented obliteration has been observed in children [17], [4]. In addition, AVMs in adults are reported to be at risk of regrowth, despite an angiogram confirming complete removal [2], [4]. However, the mechanism by which regrowth occurs has not been clarified; neither is it clear exactly when regrowth occurs. After recurrence of AVMs was reported, we routinely performed serial cerebral angiography on patients showing dysplastic vessels or capillary blush without A-V shunting on postoperative angiograms. In the present study, we report cases showing regrowth of AVMs on postoperative angiogram performed 3 months after surgery. We analyzed the protein levels of various factors, which may influence AVM regrowth. These include CD31 (PECAM), CD34, and CD105 (endoglin), which are endothelial or endothelial progenitor markers [1], [11]; VEGF, a growth factor that may influence AVM regrowth [3], [12]; and PCNA, a marker of proliferating cells [13]. In addition, we show that pERK has a potential role in AVM regrowth.

2. Materials and methods 

2.1. Patients 

Between January 2001 and January 2007, 54 patients with cerebral AVM were operated on at Kyoto University Hospital (Kyoto, Japan). Among them, 11 patients younger than 20 years were operated on. One or 2 weeks after surgical treatment, we routinely performed cerebral angiography to reconfirm the removal of nidus. In 6 patients, perinidal dysplastic vessels or capillary blush was detected. Three patients showed an increase in the size of nidus and A-V shunting, which indicated regrowth of the AVM. All patients were younger than 20 years. All specimens for immunohistochemistry were obtained from patients during the surgical procedures. Clinical data on the patients are summarized in Table 1.

Table 1. Clinical summary of cases

	Case	Age/sex	Location	Onset	S-M grade	Neurologic deficit	Preoperative embolization	Convulsion	Deep drainer
	1	11/M	Insula	Hemorrhage	III	Hemiparesis	(−)	(−)	(+)
	2	6/F	Insula	Hemorrhage	III	Hemiparesis	(−)	(−)	(+)
	3	4/F	Parietal	Convulsion	II	(−)	(−)	(+)	(+)

3. Results 

3.1. Illustrative cases 

3.1.1. Case 1 

An 11-year–old boy patient presented with intracerebral hemorrhage. Angiography study demonstrated an S-M grade III AVM. The nidus was located in the insula and fed by the middle cerebral artery and the lateral lenticulostriate artery. The drainers were the sylvian vein and the internal cerebral vein (Fig. 1A, B). He showed right hemiparesis at the time of admission. The surgery was performed through a transsylvian approach. After the surgical resection, postoperative angiogram showed that the A-V shunting had vanished (Fig. 1C, D). Three months later, a follow-up angiogram showed that the AVM had regrown and that there was further A-V shunting (Fig. 1E, F). Stereotactic radiosurgery was performed, and the next follow-up angiogram showed disappearance of the nidus.

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Fig. 1. Case 1: 11-year–old boy. A and B: Preoperative cerebral angiogram indicates an S-M grade III cerebral AVM located in the insula and fed by the middle cerebral artery and the lateral lenticulostriate artery. C and D: Postoperative angiogram showed that the A-V shunting had disappeared. E and F: Three months later, a follow-up angiogram showed that the AVM had regrown and revealed A-V shunting. Arrows indicate the regrowth of AVM.

3.1.2. Case 2 

A 4-year–old girl presented with generalized convulsion. Cerebral angiogram disclosed a parietal S-M grade II AVM. The AVM was fed by the anterior parietal and parietooccipital arteries and drained into the superior sagittal sinus and the internal cerebral vein (Fig. 2A, B). Direct surgical treatment after parietal craniotomy was performed. After the surgery, cerebral angiogram indicated total nidus resection with capillary blush without A-V shunting (Fig. 2C, D). Three months later, a follow-up angiogram showed that the AVM had regrown and revealed the presence of A-V shunting (Fig. 3E, F). Reoperation was performed, and the next follow-up angiogram showed the disappearance of the A-V shunt.

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Fig. 2. Case 2: 4-year–old girl. A and B: Cerebral angiogram disclosed a parietal S-M grade II cerebral AVM fed by the anterior parietal and parietooccipital arteries. C and D: After surgery, cerebral angiogram indicated complete nidus resection with tiny vascular blush without A-V shunting. E and F: Three months later, a follow-up angiogram showed that the AVM had regrown and revealed the presence of A-V shunting. Arrows indicate the regrowth of the nidus.

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Fig. 3. Case 3: 6-year–old girl. A and B: Cerebral angiography indicated an S-M grade III AVM fed by the middle cerebral artery and the lateral lenticulostriate artery. C and D: After surgical resection, postoperative angiogram showed that the nidus was removed. E and F: A follow-up angiogram showed that the AVM had regrown. Arrows indicate the regrowth of the nidus.

3.1.3. Case 3 

A 6-year–old girl presented sudden onset of hemiparesis and consciousness disturbance due to intracerebral hemorrhage. Cerebral angiography indicated an S-M grade III AVM. The nidus was located in the insula and was fed by the middle cerebral artery and the lateral lenticulostriate artery. The drainers were the sylvian vein and the internal cerebral vein (Fig. 3A, B). The surgery was performed through a transsylvian approach. After the surgical resection, postoperative angiogram showed that the nidus had been removed (Fig. 3C, D). Perinidal dysplastic vessel was confirmed. Three months later, a follow-up angiogram showed that the AVM had regrown and revealed A-V shunting (Fig. 3E, F). Stereotactic radiosurgery was performed, and the next follow-up angiogram performed 1 year after the treatment showed the disappearance of the A-V shunt.

3.2. Immunohistochemical analysis 

The results of immunohistochemical analysis are summarized in Table 2. In our analysis, intense VEGF immunoreactivity was not detected in any of the cases (Fig. 4A, B). Proliferating cell nuclear antigen immunoreactivity was also weak in all cases (Fig. 4C). Perivascular intense pERK immunoreactivity was detected in 2 of the 3 cases. Next, we analyzed various endothelial markers used for the assessment of microvessels including CD31, CD34, and CD105. All sections showed positive staining with all of these markers. CD31 immunoreactivity was detected in the endothelial layer of arterial and venous components, but not in the perivascular region. CD34 immunoreactivity was also detected in the endothelial layer of arterial and venous components and in the perivascular region. CD105 immunoreactivity was also detected in the endothelial layer and perivascular area of the specimens. It was stronger in the arterial component than in the venous one.

Table 2. Results of immunohistochemical study

	Antibody	Site	Case 1	Case 2	Case 3
					1st	2nd
	VEGF	Arterial	1	1	1	1
		Venous	1	1	1	1
		Perivascular	2	1	2	2
	PCNA	Arterial	1	1	1	1
		Venous	1	1	1	1
		Perivascular	0	0	1	1
	pERK	Arterial	1	1	1	1
		Venous	1	1	1	1
		Perivascular	3	1	2	3
	CD105 (endoglin)	Arterial	2	2	2	1
		Venous	3	3	3	2
		Perivascular	3	1	1	2
	CD34	Arterial	2	3	3	2
		Venous	3	2	2	2
		Perivascular	1	1	2	1
	CD31(PECAM)	Arterial	2	2	3	3
		Venous	3	3	3	3
		Perivascular	0	0	0	0

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Fig. 4. Immunohistochemical study of VEGF and PCNA. A: Faint immunoreactivity for VEGF is shown in the perivascular region. B: Immunoreactive cells are mainly neuronal cells (arrow). C: Weak immunoreactivity for PCNA is shown.

In addition, we compared the immunoreactivity between specimens obtained at the first surgery and those obtained at the second surgery (case 3). In the second specimen, immunoreactivity for CD31 was the same as in the first specimen (Fig. 5A, B). Immunoreactivity for CD34 was weaker in the second specimen (Fig. 5C, D). In the second specimen, stronger perivascular immunoreactivity for CD105 and pERK was detected (Fig. 5E, F).

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Fig. 5. Immunohistochemical study of CD31, CD34, CD105, and pERK in specimens obtained at the first and second surgeries (case 2). A: Intense immunoreactivity for CD31 in endothelial layer is shown. B: A similar pattern of immunoreactivity is detected in the regrowth specimen. C: Intense immunoreactivity for CD34 in endothelial layer and perivascular region is shown. D: In the second specimen, perivascular immunoreactivity for CD34 is not obvious. E: As for CD105, immunoreactivity in the first specimen is mainly seen in the endothelial layer of arterial and venous components. F: In the second specimen, immunoreactivity in the perivascular region is increased. G: Moderate immunoreactivity for pERK in the perivascular region is shown. H: Increased immunoreactivity is detected in the perivascular region in the regrowth specimen.

4. Discussion 

In this study, we describe juvenile cases with regrowth cerebral AVMs. The phenomenon of AVM regrowth and rebleeding after negative postoperative angiography has been reported in children. Yasargil [17] has published the largest series of such AVM regrowths. He described 6 pediatric patients ranging in age from 4 to 17 years. However, only 1 patient had a documented negative angiogram obtained 10 days postoperatively. The recurrence was discovered after 7 years. In addition, Kader et al [4] described a series of 5 pediatric patients with recurrent AVMs who presented initially with hemorrhage at 6 to 11 years of age. All 5 patients had negative postoperative angiograms [4]. Kondziolka et al [5] found that, among 132 children with AVMs of the brain, 2 developed rehemorrhage due to regrowth of AVMs despite a negative postoperative angiogram.

There are no definite proven mechanisms to explain why congenital anomalies such as cerebral AVMs recur after total extirpation in patients with mature brain vasculature. Recently, plausible mechanisms have been proposed. One is angiogenesis dysregulated by various growth factors, especially VEGF [3], [13]; the other is that recurring AVMs represent a new anatomical entity, the so-called hidden compartments [10], [12]. The level of VEGF in pediatric and adult AVMs was investigated by Sonstein et al [12]. Although VEGF is one of the main angiogenic factors and its important role in fetal brain and pathologic neovascularization has been reported, the roles of other humoral factors cannot be excluded. The synthesis of VEGF might be insufficient to explain the recurrence of cerebral AVMs because Sonstein et al also found VEGF-positive staining in nonrecurrent patients. In this study, we could not show intense immunoreactivity for VEGF in recurrent cases.

In this study, we analyzed various endothelial markers. Recently, histomorphologic aspects of angiogenesis and neoangiogenesis, both quantitative and qualitative, and their applications in the prognostic evaluation of neoplastic diseases have been intensively analyzed [6], [7], [14]. Among them, CD31, CD34, and CD105 have been used as markers for assessing microvessels [7], [14], [8]. Microvessels are considered to be new networks that are morphologically and functionally primitive [7], [14], [8]. CD31 and CD34 are panendothelial antibodies and generally react better with larger vessels than microvessels. CD31 is not only an endothelial marker, but is also involved in the regulation of endothelial cell-cell interactions and angiogenesis [15]. CD34 is also considered to be a panendothelial marker [7]. CD34 is used to distinguish endothelial progenitor cells from blood cells. In addition, it stains more immature microvessels than von Willebrand factor. Thus, CD34 reacts with more immature endothelial cells than von Willebrand factor. In our study, all specimens were positive for CD31 and CD34. CD34 intensely stained the endothelial layer of arterial portions, but CD31 stained equally those of arterial and venous portions. On the contrary, CD105 is a marker of neoangiogenesis and only stains a smaller proportion of blood vessels. It is a proliferation-associated and hypoxia-inducible protein and is preferentially expressed in the activated endothelial cells participating in neoangiogenesis. It is a receptor for TGF-β1 and TGF-β3 and modulates TGF-β signaling [8]. Tanaka et al [14] have shown that CD105 expression is the best marker of angiogenesis and is a significant predictor of death-free survival, superior to CD34. CD105 is also known as endoglin. Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1, a disease associated with AVMs and characterized by haploinsufficiency [9]. In this study, CD105 expression was confirmed in AVM specimens, as reported previously [9]. CD105 was more intensely expressed in venous components than in arterial ones. In addition, stronger immunoreactivity for CD105 in venous and perivascular components was detected in regrowth AVM specimens obtained at the second operation. This observation may be related to a role for CD105 in regrowth. In a previous report [9], up-regulation of CD105 in the perivascular region was proposed to have a role in AVM. We also detected that pERK immunoreactivity was increased in the perivascular region of a specimen obtained during the second operation. We have already reported the potential role of pERK in AVM regrowth. This observation also indicates that pERK may have a role in AVM regrowth [15], [13], [17].

In summary, we report juvenile cases of AVM regrowth at 3 months after surgery. It is necessary to perform serial angiography to confirm the removal of the nidus several months after surgery, especially in juvenile cases.