Fetal and Pediatric Pathology, 33:216–225, 2014 C Informa Healthcare USA, Inc. Copyright ISSN: 1551-3815 print / 1551-3823 online DOI: 10.3109/15513815.2014.913748
ORIGINAL ARTICLE
Splenic Hamartomas in Alagille Syndrome: Case Report and Literature Review
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Aaron W James,1 Alan Nguyen,1 Jonathan Said,1 Scott Genshaft,2 Charles R Lassman,1 and Michael Teitell1 1
Department of Pathology and Laboratory Medicine; 2 Department of Radiology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
Alagille syndrome is a rare autosomal dominant disorder with characteristic findings of paucity of intrahepatic bile ducts, congenital heart disease, and vertebral, ocular, and renal abnormalities. We present a unique autopsy case of an 18-year-old female with Alagille syndrome and splenic hamartomas. Autopsy findings included growth restriction, Tetralogy of Fallot, paucity of intrahepatic bile ducts, end-stage renal disease with mesangiolipidosis, and splenomegaly with two well-circumscribed, splenic tumors. Histologic findings of the splenic tumors revealed disorganized vascular channels lined by cells without cytologic atypia. Immunohistochemical analysis demonstrated CD8+ CD31+ endothelial cells, consistent with splenic hamartomas. In summary, Alagille syndrome is a rare genetic disorder characterized by JAG1 mutations and disrupted Notch signaling. Review of the literature highlights the importance of Notch signaling in vascular development and disorders. However, to our knowledge this is the first description of splenic hamartomas in Alagille syndrome. Keywords: Alagille syndrome, JAG1, Notch signaling, paucity of intrahepatic bile ducts, splenic hamartoma, vasculogenesis
INTRODUCTION Alagille syndrome is an autosomal dominant disorder with characteristic findings of paucity of intrahepatic bile ducts, congenital heart disease, and vertebral, ocular, and renal abnormalities. It is characterized in the majority of cases by mutations in JAG1, an activating ligand of the Notch signaling pathway. Dysregulated Notch signaling has been implicated in benign and malignant vascular lesions, including hemangiomas [1], infantile hemangiomas [2], and even angiosarcoma [3]. We present a unique autopsy case of an 18-year-old female with Alagille syndrome and splenic hamartomas. To our knowledge, this is in the only case of splenic hamartomas in Alagille syndrome, and illustrates the importance of Notch signaling in vasculogenesis in health and disease.
Received 03 April 2014; Revised 00 0000; accepted 07 April 2014. Address correspondence to Dr Aaron W James MD, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, 10833 Le Conte Ave, Rm. A3-251 CHS, Los Angeles, CA, 90095 USA. E-mail: [email protected]
Splenic Hamartomas in Alagille Syndrome
MATERIALS AND METHODS Autopsy An unrestricted autopsy was performed per institutional protocols. Use of any medical records and tissues for research purposes was authorized by the family. Routine H&E sections were prepared of all vital organs with adjunctive immunohistochemical and special stains as needed.
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Special Stains and Immunohistochemistry Special stains were performed per routine protocol, including PAS with and without diastase, trichrome and reticulin staining. Indirect immunohistochemistry was performed against cytokeratin 7, CD8, and CD31 as previously described [4]. Electron microscopy was performed per institutional protocols. Case History The patient was an 18-year-old female with Alagille syndrome with known cardiac, hepatic, and renal manifestations of disease. The patient died suddenly, and her death was ascribed to cardiac causes. Her cardiac history included Tetralogy of Fallot with pulmonary atresia. She underwent a central shunt placement at 6 weeks of life, which subsequently thrombosed. A modified left Blalock-Taussig shunt was performed at age 9, but the patient subsequently suffered from persistent hypoxia, with an oxygen saturation of 60–80% on room air. Liver manifestations included persistent hyperbilirubinemia due to presumed paucity of intrahepatic bile ducts, treated with ursodiol. Renal manifestations included at least a 5-year history of end-stage renal disease of undetermined etiology resulting in hemodialysis dependence for the last 3 years. The patient complained intermittent diplopia, but had not undergone ophthalmologic examination. Although osteopenia and sclerosis characteristics of renal osteodystrophy were observed on multiple imaging studies, vertebral defects characteristics of Alagille syndrome were not seen. Her course was complicated by multiple catheter infections, as well as secondary hyperparathyroidism requiring subtotal parathyroidectomy. She had poor feeding requiring gastrostomy tube placement at age 14 and resulting in global growth and developmental delay. The patient was admitted for replacement of an indwelling, thrombosed hemodialysis catheter. The day after admission, the patient lost consciousness while sitting, but with a rapid return to neurologic baseline. Determination of cardiac enzyme activity, electrocardiography, chest X-rays, and computerized tomography (CT) of the head disclosed no abnormalities. The next day, while walking in her hospital room the patient again lost consciousness. She showed agonal breathing and was found to be pulseless. Cardiopulmonary resuscitation was performed for 45 minutes before the patient was pronounced dead. At the time of autopsy, gross and microscopic examination revealed a constellation of findings characteristic of Alagille syndrome. External findings included growth retardation (weight 45 kg) and facial features characteristic of Alagille syndrome (including broad forehead, deep set eyes, and long nose). Cardiac findings included Tetralogy of Fallot with pulmonary atresia [including overriding aorta, right ventricular hypertrophy (0.7 cm thickness), boot-shaped heart, rudimentary pulmonary valve, and stenosis of the right pulmonary artery], as well as postsurgical changes associated with two shunt procedures. No definitive cardiac cause of death could be identified. The liver was grossly unremarkable, and microscopically showed minimal portal fibrosis by Masson Trichrome stain (Figure 1a). High magnification confirmed an absence of bile ducts in small or medium-sized portal tracts (Figure 1b,c). Immunoperoxidase stain for cytokeratin 7 confirmed the complete absence of small bile C Informa Healthcare USA, Inc. Copyright
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ducts (not shown). In addition, mild to moderate zone 3 sinusoidal dilatation was present, and a reticulin stain demonstrated nodular regeneration characteristic of chronic abnormal blood flow (Figure 1d). The kidneys were atrophic (Right kidney: 40 gm, Left kidney: 30 gm), with a coarsely granular surface and multiple cysts less than 1 cm in diameter. Microscopically, the cysts were lined by partially denuded and flattened epithelium containing amorphous granular material. Extensive scarring involved up to 50% of the parenchyma (Figure 1e). Non-globally scarred glomeruli demonstrated a variety of changes including ischemic-type wrinkling, segmental sclerosis and mesangial expansion due to matrix material which focally had a foamy appearance (Figure 1f). Electron microscopy confirmed an increase in mesangial matrix which contained lipid droplets, consistent with mesangiolipidosis, the most common renal finding in Alagille syndrome (Figure 1g) [5]. There was minimal arterial and arteriolar sclerosis. Both ovaries demonstrated multiple cysts less than 6 mm in diameter. Microscopically, multiple cystic follicles were observed, covered by a dense fibrous capsule. These constituted polycystic ovary disease. The spleen was large (360 gm), and two well-circumscribed, thinly encapsulated, red, solid masses were observed (2.5 and 4.0 cm) (Figure 2a). Histologic examination showed both splenic masses to be composed of disorganized vascular channels lined by bland-appearing, slightly plump endothelial cells. No lymphoid aggregates were observed in the tumors, which both had the appearance of red pulp with compression of the normal splenic parenchyma (Figure 2b). There was no significant cytologic atypia, mitotic activity or necrosis (Figure 2c). Immunohistochemistry demonstrated that the luminal lining cells were positive for CD31 and CD8 (Figure 2d,e), and a diagnosis of splenic hamartomas was rendered. As these findings had not been appreciated antemortem, a retrospective review of prior imaging studies was performed. Although no dedicated abdominal imaging studies had been performed, both noncontrast CT and MR imaging for cardiac evaluation in the prior 2 years showed the presence of two vaguely discernable, well-circumscribed splenic masses which were stable in size and consistent with splenic hamartomas (not shown). DISCUSSION In summary, the patient demonstrated classic findings of Alagille syndrome, including paucity of intrahepatic bile ducts, as well as severe cardiac and renal disease. Although no definitive cause of death could be identified in this case, Tetralogy of Fallot in adults is associated with increased incidence of cardiac arrhythmias and sudden death [6, 7]. Therefore her death was ascribed to probable cardiac causes. Splenic hamartomas are strikingly unusual, with an estimated incidence of 0.024% to 0.13% (review of autopsies [8]). Even more uncommon, the incidence of Alagille syndrome is estimated at 1 in 70,000 newborns (or 0.0014%) [9], although this number is commonly presumed to be an underestimate [10]. With these numbers in mind, the incidence of random, coexistent splenic hamartomas in Alagille syndrome would be somewhere between an estimated 1 in 0.5 million to 1 in 3 million people. The finding of splenic hamartomas in Alagille syndrome does, however, highlight the role of Notch signaling in vascular differentiation, development, and disease. Overview of Notch Signaling Transduction Since its discovery, the Notch signaling pathway has been identified in regulating cell fate specification, growth, differentiation, as well as organ/organism patterning [11]. Multiple Notch family receptors, along with their respective Jagged and Delta ligands, are expressed during critical stages of embryonic and postnatal development in the vertebrate cardiovascular system [11]. In fact, early functional/knockout Fetal and Pediatric Pathology
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Splenic Hamartomas in Alagille Syndrome
Figure 1. Findings characteristic of Alagille syndrome. (a–d) Histologic appearance of the liver. (a) Low magnification of trichrome-stained sections, demonstrating minimal fibrosis and no cholestasis (Masson trichrome, 40×). (b,c) Small and medium-sized portal tracts, demonstrating complete absence of bile ducts (PAS stain with diastase, 200×). (d) Reticulin-stained section, demonstrating nodular regeneration (Reticulin stain, 100×). (e,f) Histologic appearance of the kidneys. (e) At low magnification, extensive parenchymal scarring with global glomerulosclerosis was observed (Masson trichrome stain, 100×). (f) At higher magnification, “foamy” mesangial expansion was present in nonscarred glomeruli (PAS stain, 1000×). (g) Electron microscopy of kidney sections, demonstrating lipid accumulation within the mesangial matrix, (mesangiolipidosis). C Informa Healthcare USA, Inc. Copyright
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Figure 2. Splenic hamartomas. (a) Gross appearance of splenic tumors (4 cm mass shown). Cross section of spleen after fixation demonstrates a well-circumscribed, solid lesion with similar color to surrounding splenic parenchyma. (Red arrows indicate edges of tumors. Black scale bar: 2 cm). (b) Histologic appearance of splenic lesions at low magnification, with partially circumscribed mass (lower right) compressing splenic parenchyma (upper left) (H&E 100×). (c) The tumor is composed of disorganized vascular channels lined by endothelial cells without significant cytological atypia (PAS with diastase stain, 200×). (d) Immunohistochemical staining for CD31, demonstrating widespread positivity within the lesion (200×). (e) Immunohistochemical staining for CD8, demonstrating positive staining of luminal lining cells (200×). Black scale bar: 2 cm.
studies in mice, zebrafish, cell culture, and tumor models demonstrate a critical function of Notch signaling in angiogenesis and arteriogenesis. Specifically, Notch signaling has been shown to control the angiogenic growth of blood vessel networks, proliferation of endothelial cells, as well as the differentiation of arteries versus veins [11]. More recently, Notch ligands such as Delta-like 4 have been identified as primary regulators of tumor angiogenesis [11]. The importance of Notch signaling in the vascular system is evidenced by human genetic diseases such as Alagille syndrome and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), which will be discussed in subsequent sections [11]. The Notch signaling pathway mechanism remains highly conserved across invertebrate and vertebrate animals [11]. In mammals, four Notch molecules (Notch1, Notch2, Notch3, and Notch4) have been identified along with five ligands (Delta-like 1, Delta-like 3, Delta-like 4, Jagged1, and Jagged2) [12]. The Notch receptors themselves are 300-kDa transmembrane proteins containing a large extracellular domain with epidermal growth factor (EGF)-like repeats [11, 13]. In the first step of Notch signaling, ligand-activation triggers proteolytic cleavage of the transmembrane Notch receptor via γ -secretase [12, 14] (Figure 3). Subsequently, the intracellular domain (NICD) translocates to the nucleus to bind with the CSL complex and interact with DNA-binding protein RBPJK [14]. Here, it functions as a transcriptional regulator over Notch target genes, the principal ones being the HES/HEY family of transcriptional Fetal and Pediatric Pathology
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Splenic Hamartomas in Alagille Syndrome
Figure 3. Illustration of Notch signaling transduction. Ligand activation triggers proteolytic cleavage of the transmembrane Notch receptor via γ -secretase. Intracellular domain (NICD) then translocates to the nucleus, where it binds with the CSL complex to interact with DNA-binding protein RBPJκ. This functions as a transcriptional regulator over Notch target genes, HES/HEY.
regulators [14]. (Please see the following reviews for a more in-depth description of Notch signal transduction [15–17]). Notch Signaling in Vasculogenesis Studies on zebrafish and mice have shown that the Notch signaling pathway is required for normal vascular remodeling and stabilization, arteriovenous specification, and vascular homeostasis [11–13, 18]. First, Notch signaling is required for embryonic vascular remodeling and for stabilization as evidenced by loss of function studies. Via the development of knockout animals, the necessity of several Notch receptors as well as ligands for normal vascular morphogenesis has been shown [12]. For example, mice lacking Jagged1, Notch1, or both Notch1 and Notch4 die at embryonic stage E9.5-E10 from disorganized vasculature [19–22]. Expectedly, loss of Notch downstream target genes Hey1 and Hey2 in combination also results in embryonic death at E9.5, with global lack of vascular remodeling and hemorrhage [23]. While initial vasculogenesis remained unaffected, the subsequent developing vessels in the embryo and yolk sac of these Notch1-deficient and Notch1/4-deficient mice show impaired remodeling of the primitive plexus into normal branching architecture of larger to smaller vessels; the absence of remodeling is also present in cerebral vascular plexus [20–22]. Further, remaining larger arteries in both Notch1 and Hey1/2 knockout mice express neither endothelial marker CD44 nor ephrin-B2, suggesting that Hey1/2 are transducers of Notch in vascular development [23]. Interestingly, a similar phenotype is induced through deletion for Notch2, Notch3, and Notch4 in combination, but not alone. Jagged1 and Delta-like 4 (Dll4) ligands have also been shown to interact with Notch1 during early stages of mouse development [13]. In addition to the Notch pathway’s role in vascular remodeling, various studies on zebrafish, mouse, and humans suggest Notch to work with other angiogenic pathways to pattern and stabilize vasculature. It is believed that the Notch signaling pathway has significant cross-talk with other angiogenic signaling pathways, including vascular endothelial growth factor (VEGF), ephrins, angiopoietin, and platelet-derived growth factor (PDGF). In endothelial cells, VEGF-A induced expression of Notch ligand Dll4 in tip cells, thereby inhibiting excess sprouts in adjacent endothelial cells [24]. Interestingly, in angiogenesis, Notch is believed to regulate the relative influence of VEGF receptor (VEGFR)-2 and VEGFR-3, an interaction found to be crucial for proper blood vessel development [25]. Furthermore, both Notch and VEGFR-2 independently C Informa Healthcare USA, Inc. Copyright
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regulate expression of VEGFR-3 in opposing directions. VEGFR-3, in turn, supports Notch signaling through VEGF-C activation in order to stabilize the tip of cells at the site of vessel fusion [25]. Crosstalk with Notch also occurs with ligand-receptor pair, Ephrin-B2 and EphB4, which play a role in artery-vein endothelium specification and function downstream to both Notch and VEGF pathways [26]. The inhibition of both Dll4 (via allelic deletion) and Ephrin-B2 (via sEphB4-Alb) showed greater inhibition of tumor growth than either alone, suggesting that while Dll4/Notch signaling induces Ephrin-B2, their effects do not overlap [26, 27]. For an in-depth review of Notch interaction with associated pathways, please see the following references [19, 25]. The Notch signaling pathway also serves to maintain control over arteriovenous specification. During the early developmental stage of mice (E11.5), Notch1, Notch4, and Dll4 can be detected in primitive vascular plexus in venous and arterial endothelial cells [28]. By E12.5, expression shifts to arteries and/or capillaries. A detailed analysis found that by E13.5, Notch1, Notch4, Jagged 1, Jagged2, and Dll4 were present in arterial endothelium, but not veins [29]. Capillaries show expression of Notch1 and Dll4, while arterial smooth muscle show low levels of Notch1 but high expression of Notch3 and Jagged1 [29]. These studies suggest a role for Notch signaling in arteriovenous specification. One of the first steps toward underscoring this relationship was via studies involving zebrafish, where Notch is only expressed in arteries. In zebrafish, a lack of Notch signaling resulted in a loss of ephrin-B2 expression in the arterial tree and consequently failure of remodeling from larger to smaller vessels [30]. Lawson et al. demonstrated that Notch signaling pathway lies upstream of Eph-B4/ephrin-B2 [31]. Disruption of ephrin-B2 in mice led to death at E11.5, also with resultant vascular abnormalities [13]. Finally, VEGF was shown to upregulate Notch1 expression in endothelial cells, along with Dll4, in order to specify arterial fate [21]. Thus, with arteries and veins differing in function, morphology, and gene expression profiles, arteryspecific differentiation may be controlled by the Notch signaling pathway [19]. Notch Signaling in Vascular Tumors Notch signaling has been studied in a number of benign and malignant vascular tumors. In adults, blood vessels normally maintain a nonangiogenic state but will preserve growth potential for wound healing [11]. Notch signaling is most well-studied in the benign vascular tumor, infantile hemangioma (IH). The expression patterns of Notch signaling ligands and receptors have been studied in IH [1]. Findings include high expression of Notch1, Notch4, and Jagged1 in hemangioma endothelial cells (HemECs) [1, 2]. In addition, Delta-like ligand-4 (Dll4) showed intermediate expression in HemECs, while Notch3 was expressed in hemangioma-associated perivascular cells (i.e. hemangioma stem cells) [1, 2]. Notch signaling activity has been verified by expression of target genes of the Notch pathway HES and HEY. These genes, part of the HESR (hairy enhancer split related) family of genes [32], are found in both HemECs and hemangioma stem cells (HemSCs) in a cell-type-specific manner. For example, in HemSCs there is high expression of HEY1, HEYL, and HES1. In HemECs, there is high expression of HEY2 [32]. This suggests a specificity in Notch receptor downstream signaling, or alternatively, that distinct HES/HEY genes are specific to different cell types within hemangiomas. Additional studies by Boscolo et al. found the Jagged1 gene to be critical in stem cell to pericyte differentiation in a murine model of hemangioma [33]. Thus, abundant data suggests a significant role for Notch signaling in the growth and even potential genesis of benign vascular neoplasms. Notch signaling aberrations have also been associated with malignant vascular tumors, or angiosarcoma [3]. Loss of function in Notch signaling has been generally been associated with unrestricted angiogenesis. For example, hepatic-specific deletion of Notch1 in mice (via Mx-Cre–specific deletion) in liver sinusoidal endothelial Fetal and Pediatric Pathology
Splenic Hamartomas in Alagille Syndrome
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cells (LSEC) led to de-differentiation, vascular remodeling of hepatic microvasculature, and intussusceptive angiogenesis [3]. In this study, Notch1 inhibition or deletion triggered an increase in LSEC proliferation rates, a deregulation of pro-angiogenic growth factor expression, an increase in vascular tube formation in vitro, and complete loss of endothelial fenestrae in vivo (a characteristic of highly-differentiated endothelium) [34]. Similarly, another group found inhibition of Dll4, via a Notch1 decoy, to also restrict new sprout development and result in a paradoxic hypersprouting of nonfunctional vessels [35]. Finally, hepatic-specific Notch1 knockout mice developed spontaneous hepatic angiosarcomas [3]. This occurred at 50 weeks after plpC injection at a frequency of 83%, and was characterized by blood-filled tumors with highly dysplastic endothelium. Thus, loss or repression of Notch signaling is associated with endothelial cell proliferation, disorganized growth, and potentially malignant transformation. Notch Signaling Resulting in Clinical Vascular Anomalies Vascular anomalies are a hallmark of Alagille syndrome, although vascular tumors are not reported at increased frequency. The most common cardiovascular anomalies include peripheral pulmonic artery stenosis, coarctation of aorta, Tetralogy of Fallot, and atrial and ventricular septal defects [36]. Of 200 subjects with Alagille syndrome and demonstrated JAG1 mutation, 94% had probable cardiovascular involvement, and 75% had imaging documented cardiovascular anomalies [36]. Overall, the most common anomaly was stenosis/hypoplasia in branches of the pulmonary artery. Tetralogy of Fallot was present in 11.5% of the subjects, associated with a pulmonary atresia in several cases, as in our patient [36]. Similar vascular anomalies have been reported in mice with Jagged1 and Notch2 double heterozygote [37–39]. Less commonly, Alagille syndrome manifests with cerebrovascular abnormalities, and in particular Moyamoya syndrome. In a case study by Emerick et al. evaluating the vascular anomalies of 26 Alagille syndrome patients, of which 10/26 patients were detected to have cerebrovascular abnormalities. Of those, three were confirmed and five were suspected to have Moyamoya syndrome [40]. Moyamoya syndrome is an uncommon chronic vasculopathy, with only a few reported in the literature, which involves stenosis of the terminal portion of the internal carotid artery and its branches, associated with development of compensatory collateral vessels at base of brain [41]. Those with Alagille syndrome complicated by Moyamoya syndrome exhibit vascular anomalies that can affect all main cerebral arteries [41]. Other cerebrovascular findings in Alagille have included stenoses of the internal carotid arteries unilaterally, basilar artery aneurysm, and middle cerebral artery aneurysm [41]. Cerebrovascular complications can also occur with Notch3 mutations, or CADASIL syndrome, a degenerative disorder resulting in atypical vascular smooth muscle cells [19, 20]. CONCLUSION In summary, this is the first description of splenic hamartomas in Alagille syndrome. Despite the panoply of vascular anomalies observed in Alagille syndrome, the association between Alagille syndrome and vascular tumors has not yet been made. However, as Notch signaling is intimately related to vasculogenesis and endothelial cell specification, splenic hamartoma may be more common in Alagille syndrome than currently understood. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. C Informa Healthcare USA, Inc. Copyright
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[30] Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 1998;93(5):741–753. [31] Lawson ND, Vogel AM, Weinstein BM. Sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell 2002;3(1):127–136. [32] Adepoju O, Wong A, Kitajewski A, et al. Expression of HES and HEY genes in infantile hemangiomas. Vasc Cell 2011;3:19. [33] Boscolo E, Stewart CL, Greengerger S, et al. JAGGED1 signaling regulates hemangioma stem cellto-pericyte/vascular smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 2011;31(10): 2181–2192. [34] Nedredal GI, Elvevold KH, Yterbo LM, et al. Liver sinusoidal endothelial cells represents an important blood clearance system in pigs. Comp Hepatol 2003;2(1):1. [35] Dufraine J, Funahashi Y, Kitajewski J. Notch signaling regulates tumor angiogenesis by diverse mechanisms. Oncogene 2008;27(38):5132–5137. [36] McElhinney DB, Krantz ID, Bason L, et al. Analysis of cardiovascular phenotype and genotypephenotype correlation in individuals with a JAG1 mutation and/or Alagille syndrome. Circulation 2002;106(20):2567–2574. [37] Xue Y, Gao X, Lindesell CE, et al. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet 1999;8(5):723–730. [38] McCright B, Lozier J, Gridley T. A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development 2002;129(4):1075–1082. [39] McDaniell R, Warthen DM, Sanchez-Lara PA, et al. NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway. Am J Hum Genet 2006;79(1):169–173. [40] Emerick KM, Krantz ID, Kamath BM, et al. Intracranial vascular abnormalities in patients with Alagille syndrome. J Pediatr Gastroenterol Nutr 2005;41(1):99–107. [41] Rocha R, Soro I, Leitao A, et al. Moyamoya vascular pattern in Alagille syndrome. Pediatr Neurol 2012;47(2):125–128.
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ORIGINAL ARTICLE
Splenic Hamartomas in Alagille Syndrome: Case Report and Literature Review
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Aaron W James,1 Alan Nguyen,1 Jonathan Said,1 Scott Genshaft,2 Charles R Lassman,1 and Michael Teitell1 1
Department of Pathology and Laboratory Medicine; 2 Department of Radiology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
Alagille syndrome is a rare autosomal dominant disorder with characteristic findings of paucity of intrahepatic bile ducts, congenital heart disease, and vertebral, ocular, and renal abnormalities. We present a unique autopsy case of an 18-year-old female with Alagille syndrome and splenic hamartomas. Autopsy findings included growth restriction, Tetralogy of Fallot, paucity of intrahepatic bile ducts, end-stage renal disease with mesangiolipidosis, and splenomegaly with two well-circumscribed, splenic tumors. Histologic findings of the splenic tumors revealed disorganized vascular channels lined by cells without cytologic atypia. Immunohistochemical analysis demonstrated CD8+ CD31+ endothelial cells, consistent with splenic hamartomas. In summary, Alagille syndrome is a rare genetic disorder characterized by JAG1 mutations and disrupted Notch signaling. Review of the literature highlights the importance of Notch signaling in vascular development and disorders. However, to our knowledge this is the first description of splenic hamartomas in Alagille syndrome. Keywords: Alagille syndrome, JAG1, Notch signaling, paucity of intrahepatic bile ducts, splenic hamartoma, vasculogenesis
INTRODUCTION Alagille syndrome is an autosomal dominant disorder with characteristic findings of paucity of intrahepatic bile ducts, congenital heart disease, and vertebral, ocular, and renal abnormalities. It is characterized in the majority of cases by mutations in JAG1, an activating ligand of the Notch signaling pathway. Dysregulated Notch signaling has been implicated in benign and malignant vascular lesions, including hemangiomas [1], infantile hemangiomas [2], and even angiosarcoma [3]. We present a unique autopsy case of an 18-year-old female with Alagille syndrome and splenic hamartomas. To our knowledge, this is in the only case of splenic hamartomas in Alagille syndrome, and illustrates the importance of Notch signaling in vasculogenesis in health and disease.
Received 03 April 2014; Revised 00 0000; accepted 07 April 2014. Address correspondence to Dr Aaron W James MD, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, 10833 Le Conte Ave, Rm. A3-251 CHS, Los Angeles, CA, 90095 USA. E-mail: [email protected]
Splenic Hamartomas in Alagille Syndrome
MATERIALS AND METHODS Autopsy An unrestricted autopsy was performed per institutional protocols. Use of any medical records and tissues for research purposes was authorized by the family. Routine H&E sections were prepared of all vital organs with adjunctive immunohistochemical and special stains as needed.
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Special Stains and Immunohistochemistry Special stains were performed per routine protocol, including PAS with and without diastase, trichrome and reticulin staining. Indirect immunohistochemistry was performed against cytokeratin 7, CD8, and CD31 as previously described [4]. Electron microscopy was performed per institutional protocols. Case History The patient was an 18-year-old female with Alagille syndrome with known cardiac, hepatic, and renal manifestations of disease. The patient died suddenly, and her death was ascribed to cardiac causes. Her cardiac history included Tetralogy of Fallot with pulmonary atresia. She underwent a central shunt placement at 6 weeks of life, which subsequently thrombosed. A modified left Blalock-Taussig shunt was performed at age 9, but the patient subsequently suffered from persistent hypoxia, with an oxygen saturation of 60–80% on room air. Liver manifestations included persistent hyperbilirubinemia due to presumed paucity of intrahepatic bile ducts, treated with ursodiol. Renal manifestations included at least a 5-year history of end-stage renal disease of undetermined etiology resulting in hemodialysis dependence for the last 3 years. The patient complained intermittent diplopia, but had not undergone ophthalmologic examination. Although osteopenia and sclerosis characteristics of renal osteodystrophy were observed on multiple imaging studies, vertebral defects characteristics of Alagille syndrome were not seen. Her course was complicated by multiple catheter infections, as well as secondary hyperparathyroidism requiring subtotal parathyroidectomy. She had poor feeding requiring gastrostomy tube placement at age 14 and resulting in global growth and developmental delay. The patient was admitted for replacement of an indwelling, thrombosed hemodialysis catheter. The day after admission, the patient lost consciousness while sitting, but with a rapid return to neurologic baseline. Determination of cardiac enzyme activity, electrocardiography, chest X-rays, and computerized tomography (CT) of the head disclosed no abnormalities. The next day, while walking in her hospital room the patient again lost consciousness. She showed agonal breathing and was found to be pulseless. Cardiopulmonary resuscitation was performed for 45 minutes before the patient was pronounced dead. At the time of autopsy, gross and microscopic examination revealed a constellation of findings characteristic of Alagille syndrome. External findings included growth retardation (weight 45 kg) and facial features characteristic of Alagille syndrome (including broad forehead, deep set eyes, and long nose). Cardiac findings included Tetralogy of Fallot with pulmonary atresia [including overriding aorta, right ventricular hypertrophy (0.7 cm thickness), boot-shaped heart, rudimentary pulmonary valve, and stenosis of the right pulmonary artery], as well as postsurgical changes associated with two shunt procedures. No definitive cardiac cause of death could be identified. The liver was grossly unremarkable, and microscopically showed minimal portal fibrosis by Masson Trichrome stain (Figure 1a). High magnification confirmed an absence of bile ducts in small or medium-sized portal tracts (Figure 1b,c). Immunoperoxidase stain for cytokeratin 7 confirmed the complete absence of small bile C Informa Healthcare USA, Inc. Copyright
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A. W. James et al.
ducts (not shown). In addition, mild to moderate zone 3 sinusoidal dilatation was present, and a reticulin stain demonstrated nodular regeneration characteristic of chronic abnormal blood flow (Figure 1d). The kidneys were atrophic (Right kidney: 40 gm, Left kidney: 30 gm), with a coarsely granular surface and multiple cysts less than 1 cm in diameter. Microscopically, the cysts were lined by partially denuded and flattened epithelium containing amorphous granular material. Extensive scarring involved up to 50% of the parenchyma (Figure 1e). Non-globally scarred glomeruli demonstrated a variety of changes including ischemic-type wrinkling, segmental sclerosis and mesangial expansion due to matrix material which focally had a foamy appearance (Figure 1f). Electron microscopy confirmed an increase in mesangial matrix which contained lipid droplets, consistent with mesangiolipidosis, the most common renal finding in Alagille syndrome (Figure 1g) [5]. There was minimal arterial and arteriolar sclerosis. Both ovaries demonstrated multiple cysts less than 6 mm in diameter. Microscopically, multiple cystic follicles were observed, covered by a dense fibrous capsule. These constituted polycystic ovary disease. The spleen was large (360 gm), and two well-circumscribed, thinly encapsulated, red, solid masses were observed (2.5 and 4.0 cm) (Figure 2a). Histologic examination showed both splenic masses to be composed of disorganized vascular channels lined by bland-appearing, slightly plump endothelial cells. No lymphoid aggregates were observed in the tumors, which both had the appearance of red pulp with compression of the normal splenic parenchyma (Figure 2b). There was no significant cytologic atypia, mitotic activity or necrosis (Figure 2c). Immunohistochemistry demonstrated that the luminal lining cells were positive for CD31 and CD8 (Figure 2d,e), and a diagnosis of splenic hamartomas was rendered. As these findings had not been appreciated antemortem, a retrospective review of prior imaging studies was performed. Although no dedicated abdominal imaging studies had been performed, both noncontrast CT and MR imaging for cardiac evaluation in the prior 2 years showed the presence of two vaguely discernable, well-circumscribed splenic masses which were stable in size and consistent with splenic hamartomas (not shown). DISCUSSION In summary, the patient demonstrated classic findings of Alagille syndrome, including paucity of intrahepatic bile ducts, as well as severe cardiac and renal disease. Although no definitive cause of death could be identified in this case, Tetralogy of Fallot in adults is associated with increased incidence of cardiac arrhythmias and sudden death [6, 7]. Therefore her death was ascribed to probable cardiac causes. Splenic hamartomas are strikingly unusual, with an estimated incidence of 0.024% to 0.13% (review of autopsies [8]). Even more uncommon, the incidence of Alagille syndrome is estimated at 1 in 70,000 newborns (or 0.0014%) [9], although this number is commonly presumed to be an underestimate [10]. With these numbers in mind, the incidence of random, coexistent splenic hamartomas in Alagille syndrome would be somewhere between an estimated 1 in 0.5 million to 1 in 3 million people. The finding of splenic hamartomas in Alagille syndrome does, however, highlight the role of Notch signaling in vascular differentiation, development, and disease. Overview of Notch Signaling Transduction Since its discovery, the Notch signaling pathway has been identified in regulating cell fate specification, growth, differentiation, as well as organ/organism patterning [11]. Multiple Notch family receptors, along with their respective Jagged and Delta ligands, are expressed during critical stages of embryonic and postnatal development in the vertebrate cardiovascular system [11]. In fact, early functional/knockout Fetal and Pediatric Pathology
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Splenic Hamartomas in Alagille Syndrome
Figure 1. Findings characteristic of Alagille syndrome. (a–d) Histologic appearance of the liver. (a) Low magnification of trichrome-stained sections, demonstrating minimal fibrosis and no cholestasis (Masson trichrome, 40×). (b,c) Small and medium-sized portal tracts, demonstrating complete absence of bile ducts (PAS stain with diastase, 200×). (d) Reticulin-stained section, demonstrating nodular regeneration (Reticulin stain, 100×). (e,f) Histologic appearance of the kidneys. (e) At low magnification, extensive parenchymal scarring with global glomerulosclerosis was observed (Masson trichrome stain, 100×). (f) At higher magnification, “foamy” mesangial expansion was present in nonscarred glomeruli (PAS stain, 1000×). (g) Electron microscopy of kidney sections, demonstrating lipid accumulation within the mesangial matrix, (mesangiolipidosis). C Informa Healthcare USA, Inc. Copyright
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Figure 2. Splenic hamartomas. (a) Gross appearance of splenic tumors (4 cm mass shown). Cross section of spleen after fixation demonstrates a well-circumscribed, solid lesion with similar color to surrounding splenic parenchyma. (Red arrows indicate edges of tumors. Black scale bar: 2 cm). (b) Histologic appearance of splenic lesions at low magnification, with partially circumscribed mass (lower right) compressing splenic parenchyma (upper left) (H&E 100×). (c) The tumor is composed of disorganized vascular channels lined by endothelial cells without significant cytological atypia (PAS with diastase stain, 200×). (d) Immunohistochemical staining for CD31, demonstrating widespread positivity within the lesion (200×). (e) Immunohistochemical staining for CD8, demonstrating positive staining of luminal lining cells (200×). Black scale bar: 2 cm.
studies in mice, zebrafish, cell culture, and tumor models demonstrate a critical function of Notch signaling in angiogenesis and arteriogenesis. Specifically, Notch signaling has been shown to control the angiogenic growth of blood vessel networks, proliferation of endothelial cells, as well as the differentiation of arteries versus veins [11]. More recently, Notch ligands such as Delta-like 4 have been identified as primary regulators of tumor angiogenesis [11]. The importance of Notch signaling in the vascular system is evidenced by human genetic diseases such as Alagille syndrome and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), which will be discussed in subsequent sections [11]. The Notch signaling pathway mechanism remains highly conserved across invertebrate and vertebrate animals [11]. In mammals, four Notch molecules (Notch1, Notch2, Notch3, and Notch4) have been identified along with five ligands (Delta-like 1, Delta-like 3, Delta-like 4, Jagged1, and Jagged2) [12]. The Notch receptors themselves are 300-kDa transmembrane proteins containing a large extracellular domain with epidermal growth factor (EGF)-like repeats [11, 13]. In the first step of Notch signaling, ligand-activation triggers proteolytic cleavage of the transmembrane Notch receptor via γ -secretase [12, 14] (Figure 3). Subsequently, the intracellular domain (NICD) translocates to the nucleus to bind with the CSL complex and interact with DNA-binding protein RBPJK [14]. Here, it functions as a transcriptional regulator over Notch target genes, the principal ones being the HES/HEY family of transcriptional Fetal and Pediatric Pathology
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Splenic Hamartomas in Alagille Syndrome
Figure 3. Illustration of Notch signaling transduction. Ligand activation triggers proteolytic cleavage of the transmembrane Notch receptor via γ -secretase. Intracellular domain (NICD) then translocates to the nucleus, where it binds with the CSL complex to interact with DNA-binding protein RBPJκ. This functions as a transcriptional regulator over Notch target genes, HES/HEY.
regulators [14]. (Please see the following reviews for a more in-depth description of Notch signal transduction [15–17]). Notch Signaling in Vasculogenesis Studies on zebrafish and mice have shown that the Notch signaling pathway is required for normal vascular remodeling and stabilization, arteriovenous specification, and vascular homeostasis [11–13, 18]. First, Notch signaling is required for embryonic vascular remodeling and for stabilization as evidenced by loss of function studies. Via the development of knockout animals, the necessity of several Notch receptors as well as ligands for normal vascular morphogenesis has been shown [12]. For example, mice lacking Jagged1, Notch1, or both Notch1 and Notch4 die at embryonic stage E9.5-E10 from disorganized vasculature [19–22]. Expectedly, loss of Notch downstream target genes Hey1 and Hey2 in combination also results in embryonic death at E9.5, with global lack of vascular remodeling and hemorrhage [23]. While initial vasculogenesis remained unaffected, the subsequent developing vessels in the embryo and yolk sac of these Notch1-deficient and Notch1/4-deficient mice show impaired remodeling of the primitive plexus into normal branching architecture of larger to smaller vessels; the absence of remodeling is also present in cerebral vascular plexus [20–22]. Further, remaining larger arteries in both Notch1 and Hey1/2 knockout mice express neither endothelial marker CD44 nor ephrin-B2, suggesting that Hey1/2 are transducers of Notch in vascular development [23]. Interestingly, a similar phenotype is induced through deletion for Notch2, Notch3, and Notch4 in combination, but not alone. Jagged1 and Delta-like 4 (Dll4) ligands have also been shown to interact with Notch1 during early stages of mouse development [13]. In addition to the Notch pathway’s role in vascular remodeling, various studies on zebrafish, mouse, and humans suggest Notch to work with other angiogenic pathways to pattern and stabilize vasculature. It is believed that the Notch signaling pathway has significant cross-talk with other angiogenic signaling pathways, including vascular endothelial growth factor (VEGF), ephrins, angiopoietin, and platelet-derived growth factor (PDGF). In endothelial cells, VEGF-A induced expression of Notch ligand Dll4 in tip cells, thereby inhibiting excess sprouts in adjacent endothelial cells [24]. Interestingly, in angiogenesis, Notch is believed to regulate the relative influence of VEGF receptor (VEGFR)-2 and VEGFR-3, an interaction found to be crucial for proper blood vessel development [25]. Furthermore, both Notch and VEGFR-2 independently C Informa Healthcare USA, Inc. Copyright
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regulate expression of VEGFR-3 in opposing directions. VEGFR-3, in turn, supports Notch signaling through VEGF-C activation in order to stabilize the tip of cells at the site of vessel fusion [25]. Crosstalk with Notch also occurs with ligand-receptor pair, Ephrin-B2 and EphB4, which play a role in artery-vein endothelium specification and function downstream to both Notch and VEGF pathways [26]. The inhibition of both Dll4 (via allelic deletion) and Ephrin-B2 (via sEphB4-Alb) showed greater inhibition of tumor growth than either alone, suggesting that while Dll4/Notch signaling induces Ephrin-B2, their effects do not overlap [26, 27]. For an in-depth review of Notch interaction with associated pathways, please see the following references [19, 25]. The Notch signaling pathway also serves to maintain control over arteriovenous specification. During the early developmental stage of mice (E11.5), Notch1, Notch4, and Dll4 can be detected in primitive vascular plexus in venous and arterial endothelial cells [28]. By E12.5, expression shifts to arteries and/or capillaries. A detailed analysis found that by E13.5, Notch1, Notch4, Jagged 1, Jagged2, and Dll4 were present in arterial endothelium, but not veins [29]. Capillaries show expression of Notch1 and Dll4, while arterial smooth muscle show low levels of Notch1 but high expression of Notch3 and Jagged1 [29]. These studies suggest a role for Notch signaling in arteriovenous specification. One of the first steps toward underscoring this relationship was via studies involving zebrafish, where Notch is only expressed in arteries. In zebrafish, a lack of Notch signaling resulted in a loss of ephrin-B2 expression in the arterial tree and consequently failure of remodeling from larger to smaller vessels [30]. Lawson et al. demonstrated that Notch signaling pathway lies upstream of Eph-B4/ephrin-B2 [31]. Disruption of ephrin-B2 in mice led to death at E11.5, also with resultant vascular abnormalities [13]. Finally, VEGF was shown to upregulate Notch1 expression in endothelial cells, along with Dll4, in order to specify arterial fate [21]. Thus, with arteries and veins differing in function, morphology, and gene expression profiles, arteryspecific differentiation may be controlled by the Notch signaling pathway [19]. Notch Signaling in Vascular Tumors Notch signaling has been studied in a number of benign and malignant vascular tumors. In adults, blood vessels normally maintain a nonangiogenic state but will preserve growth potential for wound healing [11]. Notch signaling is most well-studied in the benign vascular tumor, infantile hemangioma (IH). The expression patterns of Notch signaling ligands and receptors have been studied in IH [1]. Findings include high expression of Notch1, Notch4, and Jagged1 in hemangioma endothelial cells (HemECs) [1, 2]. In addition, Delta-like ligand-4 (Dll4) showed intermediate expression in HemECs, while Notch3 was expressed in hemangioma-associated perivascular cells (i.e. hemangioma stem cells) [1, 2]. Notch signaling activity has been verified by expression of target genes of the Notch pathway HES and HEY. These genes, part of the HESR (hairy enhancer split related) family of genes [32], are found in both HemECs and hemangioma stem cells (HemSCs) in a cell-type-specific manner. For example, in HemSCs there is high expression of HEY1, HEYL, and HES1. In HemECs, there is high expression of HEY2 [32]. This suggests a specificity in Notch receptor downstream signaling, or alternatively, that distinct HES/HEY genes are specific to different cell types within hemangiomas. Additional studies by Boscolo et al. found the Jagged1 gene to be critical in stem cell to pericyte differentiation in a murine model of hemangioma [33]. Thus, abundant data suggests a significant role for Notch signaling in the growth and even potential genesis of benign vascular neoplasms. Notch signaling aberrations have also been associated with malignant vascular tumors, or angiosarcoma [3]. Loss of function in Notch signaling has been generally been associated with unrestricted angiogenesis. For example, hepatic-specific deletion of Notch1 in mice (via Mx-Cre–specific deletion) in liver sinusoidal endothelial Fetal and Pediatric Pathology
Splenic Hamartomas in Alagille Syndrome
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cells (LSEC) led to de-differentiation, vascular remodeling of hepatic microvasculature, and intussusceptive angiogenesis [3]. In this study, Notch1 inhibition or deletion triggered an increase in LSEC proliferation rates, a deregulation of pro-angiogenic growth factor expression, an increase in vascular tube formation in vitro, and complete loss of endothelial fenestrae in vivo (a characteristic of highly-differentiated endothelium) [34]. Similarly, another group found inhibition of Dll4, via a Notch1 decoy, to also restrict new sprout development and result in a paradoxic hypersprouting of nonfunctional vessels [35]. Finally, hepatic-specific Notch1 knockout mice developed spontaneous hepatic angiosarcomas [3]. This occurred at 50 weeks after plpC injection at a frequency of 83%, and was characterized by blood-filled tumors with highly dysplastic endothelium. Thus, loss or repression of Notch signaling is associated with endothelial cell proliferation, disorganized growth, and potentially malignant transformation. Notch Signaling Resulting in Clinical Vascular Anomalies Vascular anomalies are a hallmark of Alagille syndrome, although vascular tumors are not reported at increased frequency. The most common cardiovascular anomalies include peripheral pulmonic artery stenosis, coarctation of aorta, Tetralogy of Fallot, and atrial and ventricular septal defects [36]. Of 200 subjects with Alagille syndrome and demonstrated JAG1 mutation, 94% had probable cardiovascular involvement, and 75% had imaging documented cardiovascular anomalies [36]. Overall, the most common anomaly was stenosis/hypoplasia in branches of the pulmonary artery. Tetralogy of Fallot was present in 11.5% of the subjects, associated with a pulmonary atresia in several cases, as in our patient [36]. Similar vascular anomalies have been reported in mice with Jagged1 and Notch2 double heterozygote [37–39]. Less commonly, Alagille syndrome manifests with cerebrovascular abnormalities, and in particular Moyamoya syndrome. In a case study by Emerick et al. evaluating the vascular anomalies of 26 Alagille syndrome patients, of which 10/26 patients were detected to have cerebrovascular abnormalities. Of those, three were confirmed and five were suspected to have Moyamoya syndrome [40]. Moyamoya syndrome is an uncommon chronic vasculopathy, with only a few reported in the literature, which involves stenosis of the terminal portion of the internal carotid artery and its branches, associated with development of compensatory collateral vessels at base of brain [41]. Those with Alagille syndrome complicated by Moyamoya syndrome exhibit vascular anomalies that can affect all main cerebral arteries [41]. Other cerebrovascular findings in Alagille have included stenoses of the internal carotid arteries unilaterally, basilar artery aneurysm, and middle cerebral artery aneurysm [41]. Cerebrovascular complications can also occur with Notch3 mutations, or CADASIL syndrome, a degenerative disorder resulting in atypical vascular smooth muscle cells [19, 20]. CONCLUSION In summary, this is the first description of splenic hamartomas in Alagille syndrome. Despite the panoply of vascular anomalies observed in Alagille syndrome, the association between Alagille syndrome and vascular tumors has not yet been made. However, as Notch signaling is intimately related to vasculogenesis and endothelial cell specification, splenic hamartoma may be more common in Alagille syndrome than currently understood. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. C Informa Healthcare USA, Inc. Copyright
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