Imaging and imagination: understanding the endo-lysosomal system

Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis. The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning. Here, we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system. We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network (TGN) to the endo-lysosomal system. These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN. In addition, we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo. Throughout our review, we highlight some examples in the literature showing how imaging, especially electron microscopy, has made major contributions to our understanding of the endo-lysosomal system today.
Introduction
Lysosomes are the terminal degradative compartments of the endocytic pathway. They receive extracellular components via endocytosis and intracellular material via autophagy, as well as via the biosynthetic pathway (Eskelinen 2005; Klionsky 2007; Luzio et al. 2007; Sachse et al. 2002a). By modulating the degradation of a multitude of proteins, lysosomes are key organelles in the regulation of cellular homeostasis. Moreover, these catabolic functions are complemented by a group of cell type-specific lysosome-related organelles, which store and/or secrete key factors involved in a diversity of functions, such as blood clotting, antigen presentation, pigmentation and alveolar surface tension regulation (dell’Angelica et al. 2000a; Raposo et al. 2007; Weaver et al. 2002). Because of their many functions, defects in lysosome functioning can have devastating consequences, as is illustrated by the rapidly growing number of human disorders displaying a primary or secondary defect in the lysosomal system. To date, lysosomal aberrations have been found in the ‘classical’ lysosomal storage disorders, as well as in muscular and neurological disorders, numerous types of cancer, immune-deficiency diseases and pigmentation-bleeding disorders (Futerman and van Meer 2004; Kroemer and Jäättelä 2005; Mohamed and Sloane 2006; Nixon 2005; Saftig et al. 2001).
Unraveling the intrinsic complexity of the endo-lysosomal system is a major challenge for cell biologists. Here, we will review some of the current insights in the mechanisms involved in lysosome biogenesis and functioning. Imaging of the endo-lysosomal system combined with imaginative visions has been instrumental for furthering the conceptual understanding of the endo-lysosomal system. Therefore, on the special occasion of the 50th anniversary of the Journal of Histochemistry and Cell Biology, we will also highlight some examples in the literature showing how imaging, especially electron microscopy (EM), has contributed to our understanding of the endo-lysosomal system today.
The discovery of the lysosome
The famous story of the discovery of the lysosome reads as a compelling detective novel (Bainton 1981; de Duve 2005). A major question that was faced by the cell biological society of the fifties was how cells could exert proteolytic activity without being digested by autolysis. The first indications on the existence of lysosomes came from studies designed to answer an entirely different question-i.e. to clarify the action of insulin on the liver-in which acid phosphatase activity (nowadays known as a lysosomal activity) measurements were used as a control. A combination of experiments from the labs of Christian de Duve and Albert Claude led to the intriguing observation that acid phosphatase in fresh fractions, prepared by a relatively gentle protocol, showed astonishingly low activity levels. By contrast, samples that were obtained by a crude fractionation protocol or stored for some days before assaying showed elevated levels of phosphatase activity. When this same hide-and-seek strategy was found for four additional hydrolytic enzymes, all with an acidic pH optimum and the same sedimentation characteristics as acid phosphatase, de Duve concluded that these enzymes must be contained together in particles surrounded by a membrane ‘to prevent the enzymes getting out and the substrate getting in’. The term lysosome, for lytic particle or digestive body, was introduced (de Duve et al. 1955).
Novikoff, together with de Duve, provided the first EM pictures of these liver fractions that in addition to numerous mitochondria also showed an enrichment of electron dense bodies (Fig. 1). These were tentatively recognized as the ‘pericanalicular dense bodies’ previously found in intact liver cells (Novikoff et al. 1956). In these same years, Gomori had developed a protocol for acid phosphatase cytochemistry (Gomori 1952), which was subsequently optimized for EM by Novikoff, Holt, Barka and Anderson (Barka and Anderson 1962; Holt 1959; Novikoff 1961). The circle was closed when the acid phosphatase staining was applied to the ultrastructural level, thereby providing the direct evidence that acid phosphatase activity localized to the dense bodies of the liver parenchyma cells (Essner and Novikoff 1961) (Fig. 2), as well as similar bodies in various other cell types.
Fig. 1Reproduction of the first electron micrograph ever that indicated the existence of lysosomes (arrows), as originally published in 1956 by Novikoff and colleagues. The sample represents a liver fraction that also contains mitochondria (MT) and endoplasmic reticulum (ER). Reproduced from Novikoff et al. (1956)Fig. 2Reproduction from Essner and Novikoff (1961). This picture shows for the first time at the ultrastructural level that acid phosphatase activity is localized in lysosomes (LY) of liver cells. BC bile canaliculus, ER endoplasmic reticulum, EX extraneous precipitate, MI microvilli, V vacuole
Another major clue was provided by Werner Straus. He found that ‘droplets’ of unknown function within the proximal tubules of the kidney contained reabsorbed (i.e. endocytosed) material as well as acid phosphatase activity and other lysosomal hydrolases (Straus 1954, 1956). This observation provided the first link between lysosomal digestion and endocytic uptake of extracellular material. Hence, the concept was born of the lysosome as a membrane-bound organelle that contains acid hydrolases involved in the digestion of substances that enter the cell via endocytosis (de Duve 1959). It explained that cells digest without autolysis by setting apart the degradative enzymes together with their endocytosed substrates in a membrane-bound compartment. The answer to the riddle was found.
The endocytic pathway in a nutshell
Lysosomes receive extracellular substances for degradation via endocytosis: the invagination and pinching-off of membrane-bound vesicles from the plasma membrane (Fig. 3a). There are at least five highly controlled different entries into the cell, of which the ‘classical’ clathrin-mediated pathway is best-documented. However, the importance of non-clathrin-mediated pathways is becoming increasingly evident (Conner and Schmid 2003; Kirkham and Parton 2005; Mayor and Pagano 2007; Nichols 2003). Clathrin-mediated endocytosis begins with the sorting of receptors into a clathrin-coated pit at the plasma membrane, which then buds off to form a transport vesicle. These vesicles fuse with the first intracellular sorting station, the early endosome (Fig. 3a). Early endosomes have a mildly acidic pH that triggers the dissociation of some ligands from their receptors. Emptied receptors enter membrane tubules that emerge from the endosomal vacuole, resulting in transport via the recycling endosome back to the plasma membrane or, alternatively, the trans-Golgi network (TGN) (Fig. 3a). Receptors that remain ligand-bound are sequestered into small 50–80 nm diameter intraluminal vesicles (ILVs) that bud from the endosomal-limiting membrane into the endosomal lumen. Through a series of such vesicle fusion and fission events involving protein sorting, early endosomes gradually transform into late endosomes, a process called endosomal maturation. The formation of ILVs can continue up to the late endosome, which is why during endosomal maturation the number of these internal vesicles increases and late endosomes are often referred to as multivesicular bodies (MVBs, Fig. 3a). Degradation of cargo starts in late endosomes/MVBs and continues in the lysosome. Late endosomes/MVBs develop into lysosomes by multiple fusion and fission cycles (Futter et al. 1996; Luzio et al. 2007). After degradation of the lysosomal content, transporter molecules translocate sugars, amino acids and lipids across the limiting lysosomal membranes into the cytosol where they function as building blocks in the synthesis of new products.
Fig. 3Sorting and transport within the biosynthetic and endo-lysosomal pathways. a Schematic representation of the interplay between the biosynthetic pathway and endo-lysosomal system depicting the main endosomal compartments that are discussed in the text. b Detailed overview of the various sorting steps at the early endosome. At the endosomal vacuole or sorting endosome, receptor–ligand complexes destined for degradation accumulate in the bilayered clathrin coat (1), upon which they are sorted into intraluminal vesicles (ILV) that form by inward budding of the vacuolar limiting membrane. Recycling cargo by default enters the membrane tubules of the tubular sorting endosome (2) from which it can recycle back to the plasma membrane via the recycling endosome, or is actively sorted towards late endosomes/lysosomes (AP-3 pathway for lysosomal membrane proteins) or TGN (AP-1 pathway). A third exit from the vacuolar endosome is provided by the endosome-to-TGN carriers (3) that mediate SNX1-dependent recycling of MPRs and sortilin to the TGN. c From the TGN, multiple pathways arise that mediate transport to the endo-lysosomal system and plasma membrane. It is still an open question to what extent these pathways use different or overlapping molecular machinery and transport carriers. AP adaptor protein complex, ER endoplasmic reticulum, M6P mannose 6-phosphate, MPR mannose 6-phosphate receptor, SAP sphingolipid activator protein, SNX1 sorting nexin 1, TGNtrans-Golgi network
Defining early and late endosomes
When studying the endo-lysosomal system, one is forced to continuously pose the question how to discriminate between the distinct endosomal intermediates. The distinction ‘early’ and ‘late’ endosomes often has too limited discriminating power to describe the dynamic continuum of the endo-lysosomal pathway. In a recent EM study (Mari et al. 2007), a novel method to distinguish early and late endosomes was introduced by combining morphological, kinetic and molecular criteria. Early endosomes are by definition accessible to internalized transferrin (Tf) that recycles to the plasma membrane. As a result, late endosomes contain only marginal levels of Tf. With this in mind, the number of ILVs within the endosomal vacuoles was correlated with the occurrence of internalized Tf. Tf-positive early endosomal vacuoles on average contained one to eight ILVs, whereas endosomal vacuoles with nine or more ILVs were mostly devoid of Tf and therefore designated as late endosomes. A similar analysis with various other established early endosomal marker proteins corroborated and extended this definition. For example, the early endosomal antigen 1 (EEA1) appeared to be connected with only the very early maturation stages of early endosomes (one to five ILVs), whereas Rab11 appeared only on the later maturation stages of early endosomes (four ILVs or more). The early endosomal markers Rab4 and Hrs were more evenly distributed. Although the interpretation of this study is currently limited to the cell line in which the analysis was performed, i.e. the human hepatoma cell line HepG2, these observations illustrate that distinct stages of endosomes can be better monitored with increasing means of analysis.
TGN-to-endosome transport pathways
To fulfill their degradative function, lysosomes must also receive lysosomal proteins. These are synthesized in the endoplasmic reticulum and Golgi complex, after which they enter the TGN (Fig. 3a). From the TGN, multiple pathways to the endosomal system arise, mediating delivery of essential components to various endosomal intermediates. Thus, lysosomes receive their components via both the endocytic pathway and biosynthetic pathway.
Two classes of proteins together are essential for lysosomal functioning: the soluble lysosomal hydrolases and lysosomal membrane proteins. Each lysosomal hydrolase targets specific substrates for degradation and presently more than 50 different types are known. The heavily glycosylated lysosomal membrane proteins encompass a group of proteins with various functions. The major lysosomal membrane proteins (Marsh et al. 1987) are the lysosome-associated membrane proteins (LAMP)-1 and -2, the lysosomal integral membrane protein (LIMP)-2 and the tetraspanin CD63 [originally identified as platelet-activating glycoprotein (Pltgp40) and also often referred to as melanoma-associated antigen ME491, LIMP-1 or LAMP-3]. Initially, lysosomal membrane proteins were thought to mainly play a role in lysosomal stability and integrity, but currently new functions emerge involving chaperone-mediated autophagy and macroautophagy (reviewed by Eskelinen et al. 2003). Thus, the proper targeting of lysosomal proteins from the TGN to the endo-lysosomal system is an essential process in the biogenesis and maintenance of lysosomes. The best-known pathway for TGN-to-endosome transport is the delivery of soluble lysosomal hydrolases by the 300 kD cation-independent (CI) and 46 kD cation-dependent (CD) mannose 6-phosphate receptors (MPR) (Kornfeld and Mellman 1989; von Figura 1991). However, as will be outlined in the next paragraphs, increasing evidence indicates the existence of multiple additional or alternative pathways from the TGN to endosomes.
MPR-dependent transport of soluble lysosomal hydrolases
Both the CI and CD-MPR are present in almost all mammalian cell types. The lumenal domain of the CI-MPR is composed of 15 repetitive units, with two mannose 6-phosphate (M6P)-binding sites, whereas the CD-MPR has a lumenal domain of 159 amino acids, resembling one such unit, and has a single M6P-binding site. The M6P-binding sites recognize M6P-moieties, which are specifically present on the precursor forms of newly synthesized lysosomal hydrolases. Both receptors predominantly function as non-covalently bound homodimers. A specific role for each MPR in the targeting of lysosomal hydrolases has not been established, but distinct lysosomal hydrolases can exhibit different affinities for CI or CD-MPR (Qian et al. 2008; Sleat and Lobel 1997). CI-MPR can endocytose extracellular lysosomal hydrolases, whereas under physiological conditions CD-MPR is not involved in endocytosis. CD-MPR shows enhanced ligand binding in the presence of divalent cations (Hoflack and Kornfeld 1985a, b). Interestingly, the CI-MPR also has several other functions; it regulates the levels of circulating insulin-like growth factor II by binding it at the plasma membrane for delivery to lysosomes. Furthermore, it facilitates the activation of the precursor of transforming growth factor-β1, which is a growth inhibitor, and also binds retinoic acid, thereby mediating retinoic acid-induced apoptosis and growth inhibition, further supporting the idea that CI-MPR could be a tumor suppressor (reviewed by Ghosh et al. 2003a).
Together, the two MPRs mediate the targeting of the huge majority of newly synthesized lysosomal hydrolases from the TGN to the endo-lysosomal system (Fig. 3c) (Hoflack and Kornfeld 1985a, b; Sahagian et al. 1981). In steady state, both CD and CI-MPR have been localized by immuno-EM to the TGN, plasma membrane, endosomes and associated vesicles (Bleekemolen et al. 1988; Geuze et al. 1984, 1985; Griffiths et al. 1988; Klumperman et al. 1993, 1998). Sorting of MPRs at the TGN was long thought to predominantly depend on binding to the heterotetrameric adaptor-protein complex (AP)-1, which also mediates the recruitment of clathrin (Fig. 4). The discovery of the Golgi-localized, γ-ear-containing, Arf-binding family of proteins (GGA) as clathrin adaptors changed this view (Boman et al. 2000; dell’Angelica et al. 2000b; Hirst et al. 2000; Poussu et al. 2000). GGAs are monomeric proteins and three different forms are known in mammals, GGA1, GGA2 and GGA3. GGAs and AP-1 might function in parallel to generate distinct MPR-containing vesicles at the TGN, thereby allowing delivery to different endosomal compartments. Alternatively or additionally, GGAs may facilitate entry of MPR into clathrin-coated vesicles by interacting with AP-1. A major clue in favor of the latter hypothesis was provided by immuno-EM showing that GGAs and AP-1 colocalize in the same clathrin-coated TGN buds (Doray et al. 2002; Puertollano et al. 2003). Furthermore, the three GGAs were found to interact with each other and also to bind directly with their hinge domain to the γ-ear domain of AP-1. Depletion of each GGA resulted in decreased levels of the other GGAs, their redistribution to the cytosol and impaired sorting of CI-MPR and the lysosomal hydrolase cathepsin D (Doray et al. 2002; Ghosh et al. 2003b). However, these findings do not rule out the possibility that different adaptor proteins can also act separately to a certain extent. Interestingly, it is not precisely known where the MPRs deliver their bound ligands. A small percentage of the MPRs travels to the cell surface, from where they are internalized by clathrin-mediated endocytosis and routed to the early endosomes. However, the majority takes a direct intracellular pathway to the endo-lysosomal system, and it is generally believed that both early and late endosomes can be recipients for TGN-derived MPR. The involvement of multiple adaptors could provide a means to establish these distinct pathways.
Fig. 4Immuno-EM of a human hepatoma cell (HepG2) showing the occurrence of CIMPR (represented by 10 nm gold particles) in AP-1 (represented by 15 nm gold particles) coated TGN membranes (arrowheads). CI-MPR and AP-1 positive membranes also show the characteristic cytoplasmic dense coat indicative for the presence of clathrin. The TGN area is enclosed by distinct golgi stacks (G). Bar, 200 nm
MPR-independent transport of soluble lysosomal hydrolases
In addition to the well-characterized MPR pathway, several additional transport pathways for lysosomal hydrolases exist (Fig. 3c). For example, the lysosomal hydrolases lysosomal acid phosphatase and β-glucocerebrosidase do not acquire M6P residues and their transport is not depending on MPRs (Aerts et al. 1988; Waheed et al. 1988).
Lysosomal acid phosphatase is a type I transmembrane protein that follows the constitutive secretory pathway to the plasma membrane and reaches the lysosome via endocytosis. In lysosomes, the transmembrane precursor is processed into a soluble form, but both proteins are enzymatically active (Waheed et al. 1988). Lysosomal acid phosphatase contains the tyrosine-based motif YRHV in its cytosolic domain, which is required for endocytosis, but does not mediate direct TGN-to-endosome targeting (Obermüller et al. 2002; Peters et al. 1990; Pohlmann et al. 1988).
β-Glucocerebrosidase has no membrane spanning domain (Erickson et al. 1985). After synthesis, it is glycosylated upon which it becomes membrane-associated (Rijnboutt et al. 1991). The machinery required for proper transport of β-glucocerebrosidase was long unknown. Recently, Paul Saftig and colleagues unexpectedly identified the lysosomal membrane protein LIMP-2 as a specific receptor for β-glucocerebrosidase. LIMP-2 binds β-glucocerebrosidase in a pH-dependent manner, enabling association in the ER and guiding it all the way to the lysosome, where the complex likely dissociates because of the acidic pH (Reczek et al. 2007). Whether LIMP-2 recycles between endosomes and TGN is not known. This study for the first time implies a lysosomal membrane protein in the transport of a lysosomal hydrolase. It will be very interesting to establish whether LIMP-2 acts as a more general binding partner for lysosomal hydrolases. In addition, characterization of the molecular machinery required for transport will provide further insight into this TGN-to-endosome transport pathway.
Lysosomal hydrolases that are normally modified with a M6P residue can also traffic in an MPR-independent way to the lysosomes (Fig. 3c). This became apparent by comparing different cell types from patients with I-cell disease, also known as Mucolipidosis type II. In this disease, soluble lysosomal hydrolases do not acquire M6P groups due to a deficient activity of the enzyme GlcNAc-phosphotransferase (Hasilik et al. 1981; Reitman et al. 1981). As a consequence, the lysosomal hydrolases follow the default constitutive pathway to the plasma membrane (Fig. 3c) and are thus secreted. However, in cells of non-mesenchymal origin, most lysosomal hydrolases are still targeted correctly to the lysosome (Fig. 5) (Little et al. 1987; Owada and Neufeld 1982; Waheed et al. 1982), suggesting the existence of alternative pathways. The same phenomenon was observed in a very recently established mouse model system for I-cell disease, achieved by GlcNAc-phosphotransferase knockout (Gelfman et al. 2007). Furthermore, in mice that are deficient of both MPRs, the intracellular activities of several lysosomal hydrolases are comparable to the control situation (Dittmer et al. 1999). These findings indicate that lysosomal hydrolases can follow a pathway to the lysosome that is not mediated by MPRs.
Fig. 5
Immuno-EM of a B lymphoblast derived from a patient with I-cell disease showing a lysosome (L) positively labeled for the lysosomal hydrolase cathepsin D (represented by 10 nm gold particles). This picture indicates that although the MPR pathway is impaired in these cells, lysosomal enzymes can still reach lysosomes. Bar 200 nm
The mechanisms and pathways of MPR-independent transport are still poorly defined. Recent studies, however, have implicated a role for the multiligand receptor sortilin as an alternative receptor for a subset of lysosomal proteins, called sphingolipid activator proteins (SAPs), which are non-enzymatic cofactors required for the degradation of glycosphingolipids. Two SAPs are known; the precursor protein prosaposin, which renders saposin A, B, C and D after proteolysis, and GM2 activator protein (AP), an essential cofactor for β-hexosaminidase A. Studies from Lefrancois and colleagues have shown that sortilin interacts with both prosaposin and GM2AP, which is independent of M6P tags, whereas depletion of sortilin induced their secretion (Lefrancois et al. 2003). It remains to be established, however, whether sortilin is the exclusive receptor for SAPs, or whether MPR has an additional role in their transport. Interestingly, sortilin, together with SorLA and SorCS1–3, forms a protein family sharing homology to the lumenal domain of the yeast vacuolar sorting protein Vps10p, which directs carboxypeptidase Y to the vacuole (Marcusson et al. 1994). Immuno-localization studies positioned sortilin predominantly in endosomes and the TGN (Mari et al. 2007), where it colocalized with CI-MPR in AP-1 and GGA3 positive clathrin-coated transport carriers, indicating that sortilin and MPRs travel via the same carriers to the endo-lysosomal system. Currently, the functions of the SorCS-subgroup are unknown, but since SorCS3 is predominantly localized at the plasma membrane it is unlikely to be involved in TGN-to-endosome trafficking (Westergaard et al. 2005). Whether sortilin or possibly SorLA could act as alternative receptors for lysosomal hydrolases is still unclear.
TGN-to-lysosome transport of lysosomal membrane proteins
Lysosomal membrane proteins that exit the TGN can be transported to lysosomes following either a direct or indirect pathway, involving passage over the plasma membrane. Virtually nothing is known about the carriers and molecular machinery that mediate the direct route of lysosomal membrane proteins to the lysosomes. Moreover, the contribution of this pathway remains debated (Carlsson and Fukuda 1992; Janvier and Bonifacino 2005). Important for the sorting of lysosomal membrane proteins to lysosomes are the tyrosine and dileucine-based consensus motifs within their cytosolic tails. LAMP-1, LAMP-2 and CD63 (LIMP-1) all bear a YXXØ motif, with an essential tyrosine residue, hydrophobic XX residues and the Ø residue being a bulky hydrophobic amino acid (reviewed by Bonifacino and Traub 2003). The YXXØ motif is required for endocytosis at the plasma membrane, but has also been implicated in the direct targeting from TGN to lysosomes. For TGN sorting, however, additional characteristics seem to be required; a glycine residue preceding the critical tyrosine, the XX residues being acidic and the entire motif positioned six to nine residues from the transmembrane domain.
LIMP-2, the recently identified receptor for β-glucocerebrosidase, has a dileucine-based sorting motif DXXLL, also referred to as acidic cluster-dileucine motif, which has also been implicated in both internalization and TGN-to-lysosome targeting. The YXXØ motifs are recognized by the μ subunits of AP-1, AP-2, AP-3 and AP-4 and the DXXLL motif by the VHS domain of the GGAs (reviewed by Bonifacino and Traub 2003). The role of adaptor proteins in TGN sorting of lysosomal membrane proteins remains to be established. Segregation of LAMP-1 into AP-1 positive vesicles was observed in a biochemical study and also confirmed by immuno-EM, indicating exit from the TGN in clathrin-coated vesicles (Höning et al. 1996; Hunziker and Geuze 1996). However, sorting of LAMP-1 is not strictly dependent on AP-1. In mice lacking the μ1A subunit, which impairs AP-1 binding and clathrin assembly at the TGN, there was no significant increase in plasma membrane levels of LAMP-1 (Meyer et al. 2000), whereas the protein was still found in lysosomes. Depletion of clathrin in HeLa cells did result in increased levels of LAMPs at the cell surface, but not a complete redistribution of LAMPs to the plasma membrane (Janvier and Bonifacino 2005), indicating the existence of a direct TGN-to-lysosome pathway independent of clathrin (Karlsson and Carlsson 1998). Interestingly, depletion of AP-1 in HeLa cells, like in mice, again had only modest effects on LAMP transport over the surface (Janvier and Bonifacino 2005). Together, these data indicate that in addition to AP-1 and clathrin, other factors might be involved in direct TGN-to-lysosome transport of LAMPs.
The presence of a clathrin-independent TGN-to-endosome pathway was also indicated by studies on cells that present antigens, like B lymphocytes, macrophages and dendritic cells, in the context of major histocompatibility complex (MHC) class II molecules (Fig. 3c). The newly synthesized MHC II molecules are transported from the TGN either via the plasma membrane or directly to so-called MHC II loading compartments (MIICs) that are lysosome-related organelles representing early and late endosomes in these cells (Kleijmeer et al. 1997). For proper transport of the integral membrane protein MHC II from TGN to MIICs, association with the type 2 transmembrane protein, invariant chain (Cresswell 1996), is important. Invariant chain contains [DE]XXX[LI]-type signals in its C-terminus, but also the cytoplasmic tail of class II was found to contain endosomal sorting information (Bakke and Nordeng 1999; Bonifacino and Traub 2003). The TGN-to-endosome pathway of MHC II/invariant chain was studied in detail in B lymphoblasts by a combined biochemical and immuno-EM approach (Glickman et al. 1996). This revealed that MHC II, together with cathepsin D, in both normal and I-cell disease B lymphoblasts entered TGN exit domains that did not contain AP-1 or clathrin and were devoid of CD-MPR. Further studies are required to establish the molecular machinery of this pathway.
Interestingly, a recent study linked the TGN exit of lysosomal membrane proteins to GGA3, the same component involved in the exit of MPRs. GGAs are monoubiquitinated at their GAT (GGA and TOM) domain, which supports the idea that ubiquitin is a universal sorting signal, acting at various intracellular sites. The lysosomal-associated protein transmembrane-5 (LAPTM5), which is specifically expressed in hematopoietic cells, associates in the TGN with both the ubiquitin ligase Nedd4 and GGA3 (Pak et al. 2006). This association is required for efficient targeting of LAPTM5 to lysosomes and indicates that ubiquitination of GGA3 is possibly accomplished by Nedd4. The finding that GGAs can be involved in TGN-to-lysosome transport of a lysosomal membrane protein might explain the mild effect of AP-1 knockdown. Clearly, further studies are required to clarify this direct TGN-to-endosome transport pathway of lysosomal membrane proteins.
The early endosome: from CURL to tubular sorting endosome
The combined data on TGN-to-endosome transport of lysosomal hydrolases and lysosomal membrane proteins suggest the existence of multiple TGN exits for direct delivery to the endosomal system (Fig. 3c). There may be several reasons for such different pathways. For example, they allow custom tailored regulation of the delivery of distinct types of cargoes and provide a back-up system in case a given pathway is no longer available (such as in I-cell disease). The existence of alternative TGN-to-endosome pathways would also allow different cargoes to enter the endo-lysosomal system at distinct stages, which may coincide with their function and mode of sorting. In addition to understanding TGN exits, it is therefore equally important to define the recipient endo-lysosomal intermediates in lysosome biogenesis. With the refining of EM techniques, our understanding of the endo-lysosomal system has equally evolved. The technique that we now know as ‘classical’ EM, i.e. thin sections of plastic-embedded samples of chemically fixed cells, allowed our first acquaintance with the fascinating complexity of the cell’s inner world. Immunocytochemistry added another dimension by allowing the localization of enzymatic activities to specific compartments (as illustrated for acid phosphatase). The next step was the development of immunocytochemical procedures, which greatly increased the repertoire of proteins that can be visualized in the cell.
One of the first published examples of immuno-EM also marked a major leap in our understanding of the endo-lysosomal system; i.e. the identification of the early endosome as sorting device. In 1983, a novel immuno-EM technique was introduced by Hans Geuze and Jan Slot, in which differentially sized gold particles were used to simultaneously localize two distinct proteins. By applying this technique to visualize a ligand-asialoglycoprotein (ASGP), which is endocytosed by liver cells for transport to lysosomes-as well as its receptor (ASGPR, which recycles after endocytosis), they for the first time visualized a protein-sorting event. The ASGPR was found in tubular membranes that lacked ASGP and were connected to vacuoles that were enriched for ASGP but depleted of ASGPR (Geuze et al. 1983) (Fig. 6, left panel). The ASGPR-containing tubules were implicated in the recycling of the ASGPR to the plasma membrane, and the ASGP-containing vacuoles were defined as ‘precursor compartments en route to the lysosome’. The entire compartment was called ‘CURL’: ‘compartment of uncoupling receptor and ligand’.
Fig. 6From CURL to tubular sorting endosome. Left panel reproduction of the first electron micrograph using double-immunogold labeling, showing the uncoupling of a ligand (asialoglycoprotein (ASGP), indicated by 5 nm gold particles) from its receptor (indicated by 15 nm gold particles) in early endosomes, at that time called CURL (compartment for uncoupling receptor and ligand). The ASGP receptor is found in tubules implicated in its recycling, whereas ASGP en route to lysosomes remains in the vacuole. Originally published by Geuze et al. (1983). Right panel, reproduction from Peden et al. (2004), showing by triple labeling that the adaptor protein AP-3 (15 nm gold particles), the lysosomal membrane protein LAMP-1 (10 nm gold particles) and the recycling ASGP receptor (5 nm gold particles) co-localize in the tubules emanating from endosomal vacuoles. This work gave rise to the concept of the tubular sorting endosome
Over the years, it became clear that CURL was involved in the sorting of multiple types of proteins and the modern structural equivalent of CURL is the early endosome. Indeed, the early endosome is the major cellular site, where cargo destined for recycling is sorted from cargo en route to late endosomes and lysosomes. To indicate this, early endosomes are often subdivided into the globular ‘vacuolar or sorting’ endosome (Fig. 3b), whereas the associated tubules were indicated as ‘recycling tubules’ or ‘recycling endosomes’. However, recent studies provided accumulating evidence that early endosome-associated tubules are more than just transport intermediates for recycling cargo; they are also actively involved in sorting (Fig. 3b).
With the increasing number of antibodies against distinct types of cargoes and by optimizing immuno-EM techniques (Slot and Geuze 2007), it became possible to localize less abundant cargoes. This revealed that at steady state conditions minor amounts of LAMP-1 and LAMP-2 are found in the early endosome-associated tubules (Fig. 6, right panel). The importance of this localization became apparent from studies on the pigmentation-bleeding disorder Hermansky-Pudlak-syndrome-2 (HPS-2). Cells from HPS-2 patients lack a functional AP-3 adaptor complex and show an increased transport of lysosomal membrane proteins over the plasma membrane (dell’Angelica et al. 1999). A combination of biochemical and immuno-EM approaches revealed that AP-3 mediates the exit of lysosomal membrane proteins from the early endosome-associated tubules, most likely for transport to late endosomes or lysosomes (dell’Angelica et al. 1998; Peden et al. 2004) (Fig. 3b). Impairment of AP-3 function results in an increased cycling of lysosomal membrane proteins between early endosomes and plasma membrane (Peden et al. 2004), which explains the increased levels of lysosomal membrane proteins at the plasma membrane of HPS-2 cells. Importantly, early endosomes can be reached by lysosomal membrane proteins via the plasma membrane, but possibly also via a direct pathway from the TGN. This may explain why in the literature AP-3 has been implicated in the direct as well as indirect pathway to lysosomes (Ihrke et al. 2004; Rous et al. 2002). In melanocytes, AP-3 also mediates a pathway from early endosomes to melanosomes, which are lysosome-related organelles (Theos et al. 2005).
Importantly, by multiple double- and triple immunogold labellings, it was shown that a particular early endosome-associated tubule could contain the recycling proteins Tf and ASGPR, the CD and CI-MPR, and LAMP-1 and LAMP-2. In addition, one single tubule could display multiple budding profiles that were positive for either the adaptor complex AP-1 or AP-3 (Peden et al. 2004). AP-1 on endosomal tubules has been implicated in the retrograde transport of the CD-MPR and of the Shiga toxin B subunit to the TGN (Meyer et al. 2000), as well as recycling of internalized Tf (Mallard et al. 1998; van Dam and Stoorvogel 2002). The presence of distinct types of recycling proteins and the association of multiple adaptor complexes indicated that the early endosome-associated tubules mediate sorting of cargo proteins to distinct destinations in the cell, i.e. plasma membrane, TGN and late endosomes or lysosomes. Therefore, these AP-3 positive compartments were introduced as tubular sorting endosomes (Figs. 3b and 6, right panel) (Peden et al. 2004). A recent review of Bonifacino and Rojas extends this concept (indicated here as ‘tubular endosomal network’), by ascribing all protein machinery currently known to be involved in endosome-to-TGN transport to distinct exits of the tubular sorting endosome (Bonifacino and Rojas 2006).
Together, the available data launch the concept of a two-step rocket. In the first step, at the vacuolar domain of early endosomes, proteins destined for late endosomes-lysosomes are sorted from recycling proteins that accumulate in the attached tubules. Then, in the second step, taking place in the tubular sorting endosome, recycling proteins are sorted out to distinct destinations in the cell. Within this model, the recycling endosomes are one type of transport intermediate that evolve from the tubular sorting endosomes, mediating the recycling of cargo to the plasma membrane (Fig. 3b).
Multiple sorting events at the early endosomal limiting membrane
Despite the relatively simple and attractive concept of the two-step rocket, sorting at the early endosomes displays another level of complexity; recycling carriers not only evolve from tubular sorting endosomes, but also from endosomal vacuoles. A recent study showed the formation of so-called endosome-to-TGN carriers (ETCs) from the limiting membrane of early endosomal vacuoles (Fig. 3b) (Mari et al. 2007). These ETCs are so far specifically involved in the endosome-to-TGN recycling of lysosomal protein receptors, i.e. the CD- and CI-MPRs and sortilin. Importantly, ETCs constitute an entirely different population of recycling carriers than those emanating from the tubular sorting endosome. With the exception of the MPRs, ETCs are devoid of any of the cargo or machinery proteins found in the tubular sorting endosome. The associated machinery proteins of ETCs are SNX1, SNX2 (Carlton et al. 2004, 2005; Mari et al. 2007) and hVps26p (Arighi et al. 2004; Mari, personal communication). ETCs also differ from tubular sorting endosomes by morphology. By 3D-tomographic analyses, ETCs were identified as non-branched, short tubules and vesicles, not organized in a reticulum.
Apart from segregating proteins into tubular sorting endosomes and ETCs, the limiting membrane of early endosomal vacuoles is involved in a third protein sorting event; i.e. segregation of proteins into a characteristic bilayered coated area that is involved in protein sorting into ILVs (Fig. 3b, see also below) (Bonifacino and Rojas 2006; Peden et al. 2004; Raiborg et al. 2002; Raposo et al. 2001; Sachse et al. 2002b). Thus, proteins present in early endosomal vacuoles may be sorted to at least three types of specialized sorting domains: tubular sorting endosomes, ETC or the bilayered coat. The formation of two types of exits from the non-coated regions of the early endosomal vacuoles predicts the existence of complementary molecular machineries that specifically recruit cargo to either of these recycling compartments (i.e. ETC or tubular sorting endosome). Future investigations are needed to further unveil the components of these machineries.
Sorting to late endosomes and lysosomes
The default pathway for molecules entering early endosomes is recycling back to the plasma membrane, a pathway that starts with the segregation of cargo into the tubular extensions of the early endosomes (Draye et al. 1988; Yamashiro et al. 1984). Endocytosed cargo from the plasma membrane that is not destined for recycling requires active sorting to late endosomes/lysosomes, a process mostly mediated by inclusion into ILVs (Gruenberg and Stenmark 2004; Katzmann et al. 2002; Sachse et al. 2002b).
Protein sorting into ILVs is critical for growth factors and their receptors, such as growth hormone (GH) (Sachse et al. 2002b) and epidermal growth factor (EGF) (Raiborg et al. 2003; Urbe et al. 2003). Sorting into ILVs segregates these ligand–receptor complexes from the cytoplasm, thereby terminating signaling. ILV sorting requires ubiquitination and involves among others the cytoplasmic proteins Hrs and STAM (Bache et al. 2003b; Raiborg et al. 2001, 2002; Urbe et al. 2003). By immuno-EM, it was shown that at the limiting membrane of the early endosomal vacuole the GHR and EGFR are concentrated in flat, bilayered coated areas with a fuzzy outer layer of clathrin and a dense inner layer of unknown protein composition. These bilayered coated domains were enriched in Hrs (Sachse et al. 2002b). These findings led to the proposition of the ‘sorting-by-retention’ model for endosomal sorting (Sachse et al. 2002b). This model describes that receptor–ligand complexes prior to their incorporation into ILVs are retained and concentrated in the bilayered coated areas of endosomal vacuoles, whereas recycling proteins pass through the vacuolar-limiting membrane and follow the bulk flow route towards recycling endosomes. A recent study showed that the recruitment of clathrin is responsible for concentrating Hrs in these endosomal microdomains, reinforcing an active role for clathrin in protein retention (Raiborg et al. 2006).
Downstream of Hrs, the targeting of ubiquitinated receptors into ILVs requires the ‘endosomal sorting complexes required for transport’ (ESCRT)-1, -2 and -3 in concert with Alix/AIP-4 and the AAA-ATPase (hVps4). hVps4 is required for disassembly and recycling of ESCRT complexes (Babst 2005; Bache et al. 2003a; Katzmann et al. 2003) and interestingly, also for the disassembly of the bilayered coat (Sachse et al. 2004). Impairment of hVps4p function gives rise to an elongated coat and decreased formation of ILVs, indicating that coat disassembly is required for the inward budding process to proceed (Sachse et al. 2004). Indeed, 3D-electron tomography of early endosomes showed that inward budding of ILVs is mostly seen at the edges of the coats (Murk et al. 2003). Notably, the sorting-by-retention model proposes a novel role for clathrin in addition to its role in the formation of transport vesicles, i.e. defining endosomal subdomains involved in protein retention.
Future developments
With the increasing number of molecular markers of endosomes and lysosomes, there is a growing awareness that distinct populations of endosomes and perhaps even lysosomes, with different functions, can coexist within one cell (Bökel et al. 2006; Lakadamyali et al. 2006; Möbius et al. 2003; White et al. 2006). For example, ILVs enriched in the lipid lysobisphosphatidic acid (LBPA) are found in distinct MVBs from those enriched in cholesterol (Möbius et al. 2003). Another example is illustrated in Fig. 7 showing three seemingly identical late endosomes, of which only two are reached by endocytosed BSA-gold after 3 h of uptake. One explanation is that cargo drives the formation of specific endocytic compartments. For example, addition of EGF to cells results in the specific increase of the type of MVBs that do not contain LBPA (White et al. 2006). Formation of cargo-dependent carriers can start at the plasma membrane. The recent identification of clathrin-independent endocytosis, as well as the discovery of cargo specific adaptors that lead to clathrin-coated pit formation-either in conjunction with or independent of AP-2-illustrates that alternative endocytic pathways can arise from the plasma membrane. If and how these pathways converge at the endosomal level is presently unclear (Benmerah and Lamaze 2007). EM is an indispensable tool to search for cargo-specific endosomal compartments, since by fluorescence microscopy only the labelled compartment is visible and a nearby compartment negative for the cargo under study will remain unseen.
Fig. 7Electron micrograph illustrating the heterogeneity of late endosomes (LE). HepG2 cells were allowed to internalize bovine serum albumin (BSA) conjugated to 5 nm gold particles (abundant labeling) for 3 h and processed for immuno-EM of cathepsin D (10 nm gold particles, scarce labeling). Three seemingly identical late endosomes containing multiple ILVs and partially degraded material are shown, two of which are heavily loaded with BSA-gold, whereas the upper one is devoid of the endocytic tracer. G Golgi stacks; Bar 200 nm
Another point of interest is that not all cargo might be recruited to ILVs by the same mechanism. Both in yeast (Bilodeau et al. 2002; Epple et al. 2003) and mammalian cells (Hislop et al. 2004), examples of cargoes are described that do not require direct ubiquitination. In case of the melanosomal protein Pmel17, which is also targeted to ILVs independently of ubiquitin and Hrs, two N-terminal lumenal subdomains seem necessary and sufficient for efficient transport (Theos et al. 2006). Possibly, these non-ubiquitinated cargoes associate with others for their association with the ESCRT machinery, but perhaps they use an alternative mechanism. Interesting in this respect is that LBPA can induce ILV formation in liposomes, in the absence of protein, suggesting that lipid-driven and ESCRT-dependent pathways may coexist (Matsuo et al. 2004). Also the lysosomal targeting of LAMP-1 and LAMP-2 remains an interesting open question, since these proteins are predominantly located on the limiting membranes of late endosomes and lysosomes, and incorporation into ILVs seems therefore not involved. By a more detailed characterization of the distinct endosomal maturation stages (Mari et al. 2007), it will be feasible to obtain a more accurate definition of the stage in which a specific cargo enters or exits the endo-lysosomal system. One of the questions that is currently addressed in our lab is whether lysosomal membrane proteins can travel directly to late endosomes or lysosomes, thereby bypassing the need of ESCRT sorting machinery.
Finally, with the development of live cell imaging methods and correlative live cell imaging EM, a unique tool is provided to integrate dynamic parameters with ultrastructural protein localization studies, which opens a whole new field of analysis.

imaging
lysosomes
protein sorting
trans-golgi network
mannose 6-phosphate receptor
endosomes
electron microscopy
lysosomal membrane proteins


Imaging findings in noncraniofacial childhood rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. This paper is focuses on imaging for diagnosis, staging, and follow-up of noncraniofacial RMS.
Introduction
Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma and constitutes 3–5% of all malignancies in childhood [1]. In children, the age-standardized annual incidence rate for RMS is between 4 and 7 per million [2]. This review focuses on the imaging of all RMS occurring outside the head and neck region, which comprise 40% of RMS; around 15% are genitourinary (GU) nonbladder prostate tumours (GU-NBP, i.e. paratesticular, vaginal and uterine tumours), 10% are bladder prostate tumours (BP), 15% occur in the limbs, and 20% occur in other sites (i.e. thoracic or abdominal tumours).
Clinical symptoms vary widely, based on the location of the primary tumour, but in general presenting symptoms often are indolent, with nonspecific or minimal symptoms, at the start mimicking innocent general paediatric diseases. Often the duration or progression of symptoms alerts the physician to the presence of a malignant tumour. Site-specific symptoms in GU-NBP tumours are a paratesticular mass in boys that may be painful or not, while girls may present with a grape-like (botryoid) vaginal extrusion of mucosanguineous tissue or micturition problems. BP tumours may present with urinary retention, haematuria, constipation or an abdominal mass. Limb tumours often present with a painless swelling and/or with enlarged regional lymph nodes, while presentation of tumours in other locations may vary from biliary obstruction in tumours of the biliary tract, to painless masses, all depending on their primary site.
Patients with metastatic disease present with more general symptoms of fatigue, weight loss, and low blood counts. As soon as a RMS is suspected an extensive work-up must be performed to get a histological diagnosis and correct staging of the tumour. Imaging of the primary site with US, MRI or CT is required in all patients at the start of the diagnostic work-up [3]. Once the diagnosis has been confirmed histologically, the most frequently involved metastatic sites will be investigated; CT of the lungs, imaging of regional lymph nodes, and a technetium bone scan are recommended for every patient. Furthermore, bone marrow aspirates and trephines should be performed in all patients, while examination of the cerebral spinal fluid is only required in patients with paraspinal and parameningeal locations. As soon as the work-up has been completed, the patient can be stratified to receive treatment according to the child’s risk group, based on the six significant prognostic factors for localized RMS that came out of a retrospective European analysis (European Paediatric Soft-tissue Sarcoma Study Group, EpSSG); histology, postsurgical status according to the Intergroup Rhabdomyosarcoma Study Group (IRSG), tumour site, node involvement, tumour size, and patient age (Table 1).
Table 1EpSSG prognostic factors [28] FavourableUnfavourableHistologyEmbryonalAlveolarIRSG statusHigher grades more unfavourableTumour siteHead and neck non-parameningealAll other sitesOrbitalGenitourinary – nonbladder/prostateNode involvementN0N1Tumour size (cm)≤5>5Age (years)<10≥10
Pathology
RMS is a fast-growing, primitive, high-grade, malignant mesenchymal tumour. Depending on their degree of differentiation, the tumour cells manifest features that more or less can be found in the cells of skeletal muscle. These features, essential for the diagnosis, are the presence of myofibrils and cross striations (on light and electron microscopy) and/or positive immunohistochemical staining for markers of muscle differentiation such as desmin and myoD1. For detailed descriptions of the histopathological aspects of RMS, reference should be made to Weiss and Goldblum [4] and Fletcher et al. [5].
Based on morphology, RMS is traditionally subdivided into embryonal, alveolar and pleomorphic. Pleomorphic RMS, in contrast to embryonal and alveolar RMS, almost exclusively occurs in adults (median age sixth decade), and is therefore not discussed further. Embryonal RMS is the most common type (60–70% of all RMS). The cells show a close resemblance to various stages in the embryogenesis of normal skeletal muscle (Fig. 1). Subtypes are botryoid RMS and spindle-cell RMS (Fig. 2). When arising in the submucosa, embryonal RMS may present as a fast-growing exophytic, polypoid mass. This macroscopic variant is called botryoid RMS (grape-like) and, due to its growth pattern (primary exophytic and not invasive), has a better prognosis.
Fig. 1Embryonal RMS: small cells and primitive spindle-shaped cells resembling the first stages of developing normal skeletal muscle (H&E, original magnification 10×20)Fig. 2Embryonal RMS, spindle-cell type: closely packed spindle cells arranged in perpendicular crossing fascicles giving a leiomyosarcoma-like appearance (H&E, original magnification 10×20)
Spindle-cell RMS comprising about 4% of all RMS, seems to behave less aggressively and is most often encountered in a paratesticular location (about 30% of all paratesticular RMS) and the head and neck [6, 7]. Histologically spindle cell RMS is characterized by parallel orientation of spindle cells with an eosinophilic, fibrillary cytoplasm and an elongated, hyperchromatic nucleus. The cells are arranged in broad fascicles (fibro-/leiomyosarcoma-like) or in short, interdigitating fascicles (storiform) and whirls with, in contrast to the broad fasciculated variant (leiomyomatous type), an abundant, partly hyalinized collagenous matrix.
Alveolar RMS is composed of ill-defined, dense aggregates of poorly differentiated round or oval tumour cells that frequently show loss of cohesion (Fig. 3). This loss of cohesion and the presence of thin fibrous septa result in an alveolar pattern. In cases where the tumour cells do not show loss of cohesion, the term ‘solid alveolar RMS’ is used. Alveolar RMS represents about 20% of all RMS and has two specific translocations with specific fusion transcripts that can be detected by RT-PCR. The majority (about 55%) show a t(2;13)(q35;q14) translocation with the corresponding fusion transcript PAX3-FKHR [8]. In about 22% a t(1;13)(p36;q14) translocation is found with fusion transcript PAX7-FKHR. In rare cases, RMS with a more or less alveolar pattern lacks these translocations.
Fig. 3Alveolar RMS: cluster of primitive cells with loss of cellular cohesion and bordered by dense fibrous septa, resulting in an alveolar pattern (H&E, original magnification 10×20)
Both embryonal and alveolar RMS may show rhabdoid tumour-like features and anaplasia (focal or diffuse) [9, 10]. More recently, under the heading sclerosing RMS, a variant with hyalin sclerosis has been described (Fig. 4) [11]. It is unclear whether this is a distinct subtype.
Fig. 4Sclerosing RMS: small cells, primitive spindle-shaped cells and eosinophilic rhabdomyoblasts in a background of hyaline sclerosis (H&E, original magnification 10×20)
In 1995, the IRSG and the International Society of Paediatric Oncology (SIOP) suggested a prognostically more significant classification (Table 2) [12].
Table 2RMS: IRSG and SIOP classification, extended with new variants [12]GroupPrognosisSubtypeIBetter prognosisEmbryonal RMS, botryoid typeEmbryonal RMS, spindle-cell typeIIIntermediate prognosisEmbryonal RMS (remaining)IIIWorse prognosisAlveolar RMSIVUnclear prognosisRMS with rhabdoid featuresEmbryonal RMS with diffuse anaplasiaSclerosing RMS
Imaging findings
Radiography
As RMS is a soft-tissue tumour, conventional radiology plays an insignificant role in its diagnosis. Localized bony erosion adjacent to the primary site is a recognized complication; this area may be hot on a 99mTc-MDP bone scan in the absence of metastatic disease in the skeleton. In contrast to the initial diagnostic work-up, where for the assessment of pulmonary metastases CT is mandatory, AP and lateral chest radiographs are used in the follow-up period.
Ultrasonography
US is often the first imaging modality used in children with soft-tissue masses because it is readily available, has high resolution, and can easily assess extent and vascularity of a mass. One should not forget that most soft-tissue lesions are benign, can readily be diagnosed with US, and do not need further diagnostic work-up or even treatment.
On US, RMS in general shows as a well-defined, slightly hypoechoic inhomogeneous mass that can show significantly increased flow (Fig. 5). In the particular case of paratesticular RMS, US is the imaging modality of choice, although CT of the abdomen is also necessary to evaluate for retroperitoneal lymphadenopathy. In all other RMS locations additional imaging using CT or MRI is mandatory.
Fig. 5A 6-year-old boy with a mass in the left scrotum. US image shows an ill-defined heterogeneous mass surrounding the testis (open arrow). The mass shows increased flow (solid arrow). Histopathology: embryonal RMS
US is also of use in image-guided biopsies. Recently Sebire and Roebuck [13] systematically reviewed the pathological diagnosis of paediatric tumours from image-guided needle-core biopsies. They concluded that image-guided biopsy material was sufficient to come to a diagnosis in 94% (95%; CI 92–96%) of patients. Complications needing treatment, mostly haemorrhage requiring transfusion, were reported in only 1% of patients. For image-guided biopsies the material obtained should be transported fresh to the pathology department. Fixation should not be performed as this precludes further cytogenetic studies.
CT
In order to assess pulmonary metastases from RMS, CT of the chest is a mandatory examination. However, assessment of pulmonary involvement can sometimes be difficult. Although criteria such as number and size of lesions, morphology (noncalcified, round and well-defined) and location (inferior lobes, subpleural spaces, branching vessels) have been applied in adult patients, none has shown 100% specificity. According to the EpSSG guidelines for evaluating chest CT the following criteria with respect to the diagnosis of pulmonary metastases should be applied: one pulmonary or one pleural nodule over 10 mm in diameter, two or more well-defined nodules of 5–10 mm or five or more lesions >5 mm [3]. This comes with the assumption that there is no other medical explanation for these lesions. When there is a high suspicion that a small lesion is metastatic, and appears to be the only site of metastatic disease, biopsy may be performed. In the EpSSG protocol, lung biopsy is not recommended.
With the introduction of multidetector CT (MDCT) the advantage of MRI of being a multiplanar imaging modality has been overtaken, and by virtue of its underlying physics, CT is superior to MRI in detecting osseous changes. The drawback of CT in children is the use of ionizing radiation. Since the seminal papers of Paterson et al. [14] and Brenner et al. [15], we should be aware of the theoretical risk of CT-induced cancer fatalities and take this into consideration especially in the work-up of children with cancer, as they have already proven their tendency to present with a malignancy early in life [14, 15].
MRI
With its superior ability to depict soft-tissue changes, MRI is the primary imaging modality in RMS [16]. Although imaging protocols should be tailored for each individual patient, they should at least consist of axial T1-W and T2-W images (for anatomic detail and assessment of neurovascular structures), T1-W images perpendicular to the axial plane, and imaging after gadolinium administration. It is important that at least two series should be identical, one before and one after contrast agent administration, to be able to discern enhancement. Contrast-enhanced series are mandatory and ideally be performed with fat saturation.
The imaging characteristics of RMS are relatively nonspecific. Like most soft-tissue tumours they have intermediate signal intensity on T1-W images (Fig. 6). On T2-W images they tend to be of intermediate-to-high signal intensity. If the tumour contains a high number of septa it may have a lobular shape. RMS in general show strong enhancement on postcontrast imaging (Fig. 6). In very rare instances the tumour may show a predominantly cystic appearance (Fig. 7). Dynamic series are useful in order to assess tumour vascularity, and to differentiate between postchemotherapy/surgery residual disease and fibrosis.
Fig. 6A 13-year-old girl who noticed a small lump near the anus. a T1-W MR image shows a well-defined pararectal lesion (arrow). b After intravenous gadolinium administration the lesion shows homogeneous enhancement (arrow) Histopathology: alveolar RMSFig. 7An 8-year-old girl with haemolysis, fever and a mass underneath the scapula. a Coronal STIR image shows a lesion with mixed signal intensity (open arrow) and multiple enlarged lymph nodes in the neck (solid arrow). b Axial T2-W image shows multiple cystic lesions with fluid-fluid levels (open arrow). Histopathology: stage IV embryonal RMS
For the surgeon, in order to plan surgery, it is of importance to describe the compartment in which the RMS is located [17]. Vascular involvement is considered to be absent if there is a normal tissue plane visible between the tumour and the vessel, or if the tumour has a less then a 180° circumferential relationship. If the tumour surrounds the vessel for more than 180°, it is considered to be encased.
Two studies have addressed the use of whole-body MRI (WB-MRI) in paediatric oncology [18, 19]. In the first study with various malignant tumours WB-MRI had a superior positive predictive value for skeletal metastases compared to bone scintigraphy (94 vs. 76%, respectively), and also a significantly higher sensitivity (99 vs. 26%, respectively) [18]. In the second study, WB-MRI had a higher sensitivity (82%) than skeletal scintigraphy (71%) for the detection of bone marrow metastases, but a lower sensitivity than FDG-PET (90%) [19]. The authors of both studies concluded that WB-MRI can replace bone scintigraphy. One advantage of this approach would be the implementation of a one-stop-shop approach to childhood RMS. A drawback of MRI in young children is, however, the need for general anaesthesia.
Bone scintigraphy
In the current EpSSG protocols, bone scintigraphy is mandatory as part of the work-up in patients with RMS. The finding of an isolated hot spot on the bone scan should be evaluated with conventional radiography or MRI.
Cogswell et al. [20] reported a retrospective series of 40, primarily adult, patients with RMS and found bone metastases in 18%. Bone scintigraphy in their study had a sensitivity of 70% and specificity 95% in the detection of metastatic disease. In contrast to this, in a Dutch study of 109 patients with soft-tissue sarcoma, bone metastases were found in only 8 patients (7%) [21]. However, of these eight patients, six reported bone pain and all had other sites of metastatic disease. The authors conclude that the yield of routine bone scintigraphy is low and that it should be reserved for symptomatic or high-risk patients only.
Positron emission tomography-CT
In PET-CT studies fluorine-18 fluorodeoxyglucose (18F-FDG), a radiolabelled glucose analogue, is used [22]. As 18F-FDG is a glucose analogue, it shows uptake in metabolically active cells, which most malignant tumour cells are. The combination of PET with CT, without moving the relative position of the patient, yields a higher diagnostic accuracy than PET alone (Fig. 8). In general, the CT scan will be low-dose CT scan only meant to identify anatomical structures. However, as the CT scanners in modern PET-CT systems are of high diagnostic quality, it is also possible to combine a diagnostic CT scan, e.g. for the depiction of pulmonary metastases, with a PET scan.
Fig. 8A 19-year-old boy with a history of treated metastatic RMS presented with low back pain. The PET-CT image shows intense 18F-FDG uptake in the spinal canal (open arrow). Physiological excretion of the radiopharmaceutical via the kidneys is visible (solid arrows). Histopathology: embryonal RMS
The literature on the use of PET-CT in children with RMS is limited to several case reports or small studies [23, 24]. Although in some cases PET-CT has been shown to be of benefit in individual patients, larger prospective studies are needed.
Staging and follow-up
Staging of RMS is of importance for the individual patient as it gives an indication of prognosis, and thus treatment stratification. From a broader perspective staging makes compiling data on larger patient groups for research purposes possible, enabling evaluation of the outcome of different treatment regimens.
The main staging system is the postsurgical staging system developed by the IRSG. This is currently used by study groups both in the USA and now in Europe also (Table 3). The IRSG was formed in 1972 and consisted of surgeons, pathologists, oncologists, and radiation oncologists. The absence of radiologists is striking, and paediatric radiologists are still infrequently involved in development of paediatric oncology study protocols, although in the EpSSG RMS 2005, paediatric radiologists were involved in the development of the protocol. For staging regional nodes it is important to be familiar with the regional node stations. Lymph node involvement has a negative impact on prognosis, as has been shown in the SIOP Malignant Mesenchymal Tumor 89 trial [25]. Overall 5-year survival was 60% for N1 patients versus 73% in N0 patients (P = 0.03). Distant lymph node involvement upgrades a patient to stage IV disease (Fig. 7), having an adverse impact on prognosis: overall 5-year survival becomes 24% [26].
Table 3IRSG classificationStageCharacteristicsILocalized disease completely resected (regional nodes not involved)A: Tumour confined to muscle or organ of originB: Tumour infiltrating outside organ of (muscle of) originIILocalized or regional disease with total resection of gross tumourA: Primary tumour grossly resected, with microscopic residual disease (negative findings in local nodes)B: Primary tumour and positive nodes completely resectedC: Primary tumour and positive nodes resected, with evidence of microscopic residual diseaseIIIIncomplete resection of tumour or biopsy, with gross residual diseaseIVDistant metastatic disease present at diagnosis
Table 4 lists the regional node stations by primary tumour site. Oval-shape lymph nodes and a short axis <1 cm are considered to be normal [3]. If the node shows peripheral enhancement or is round with a short axis of 1.5–2 cm then the node should be considered probably invaded by tumour. Besides surgical resection or needle-core biopsies, lymph node involvement can also be assessed using fine-needle aspiration (FNA). Klijanienko et al. [27] reported a review of the use of FNA in 180 tumours; 176 (97.8%) were either diagnosed accurately or as round-cell sarcoma.
Table 4Regional node stations by primary tumour site. Disease with involvement of other lymph nodes than those specified in the table should be classified as stage IVAnatomical siteNode stationExtremityLower extremityInguinal, femoral, popliteal nodes (rare)Upper extremityAxillary, brachial, epitrochlear, and infraclavicular nodesGenitourinaryBladder, prostate, cervix, uterus, paratesticularPelvic, retroperitoneal nodes at renal artery level or belowVaginaRetroperitoneal, pelvic nodes at or below common iliac inguinal nodes VulvaInguinal nodesThoracicIntrathoracicInternal mammary, mediastinal nodesRetroperitoneum/pelvisPelvic, retroperitoneal nodesTrunkAbdominal wallInguinal, femoral nodesChest wallAxillary, internal mammary, and infraclavicular nodesOtherBiliaryLiver hilar nodes Perianal/perinealInguinal, pelvic nodes (may be bilateral)
Only in patients with intraspinal or suspected meningeal extension (on imaging or clinical assessment) does the EpSSG RMS protocol state that craniospinal MR should also be performed. In the current EpSSG RMS 2005 protocol, risk stratification is based on six criteria that have emerged from the analyses of previous European studies: histology (embryonal vs. alveolar), postsurgical stage (IRSG), tumour site, node stage, tumour size and patient age (Table 1) [28].
During follow-up, tumour size is an important parameter in assessing tumour response. In the current EpSSG protocol complete remission is defined as disappearance of tumour both clinically and on imaging. Minor response is defined as >33% reduction in volume after three courses of chemotherapy; if not reached the patient is eligible for second-line chemotherapy. In recording tumour response the EpSSG uses volumetric evaluation; additionally the presence or absence of a posttherapeutic residue should be mentioned in the radiology report [28].
In studies in adults the use of Response Evaluation Criteria In Solid Tumours (RECIST) has been advocated by the European Organization for Research and Treatment of Cancer (EORTC), National Cancer Institute of Canada Clinical Trials Group and the National Cancer Institute (NCI) of the United States [29]. With respect to implementation in children, RECIST have been a matter of debate [30]. Recently RECIST were retrospectively applied to ten consecutive children with cancer [31]. The authors concluded that tumour size was underestimated and that in disseminated disease many lesions were either calcified or too small to measure and, therefore, that RECIST are not readily applicable in paediatric oncology. Currently the EpSSG has incorporated RECIST to be used alongside the volumetric measurements in their latest protocol in order to prospectively assess the validity of RECIST in a large patient population with a single tumour type.
Tumour relapse in patients most commonly presents with locoregional disease (51%) compared to distant relapse (41%) [32] (Fig. 9). In a retrospective case-based study the use of PET-CT was advocated; this, however, needs to be evaluated in larger prospective studies [33].
Fig. 9Two years after initial diagnosis the patient shown in Fig. 6 presented at the outpatient clinic complaining of back pain. a Coronal STIR image of the pelvis shows discrete increased signal intensity in the left ischium (open arrow). b Subsequently acquired PET-CT image confirms the presence of recurrent disease in the same location (open arrow). Note excretion of tracer into the urinary bladder (solid arrow). c PET-CT image also shows a second lesion in the thoracic spine (open arrow). Additional rib and pleural metastases were also visible (not visible on this image)
Tumour locations
Genitourinary
Approximately 25% of all RMS are GU RMS [34, 35]. As mentioned above, GU RMS can simply be subdivided into two subgroups based on different prognosis and subsequent treatment strategy, GU bladder/prostate (GU-BP) being an unfavourable location (Fig. 10). Tumours at other GU non-bladder/prostate (GU-NBP) sites, such as a paratesticular location (testes, epididymis and spermatic cord; Fig. 5), vagina or uterus (Fig. 11), have a favourable prognosis, and thus require less-intensive treatment [36–48]. There is a caveat to MRI of the bladder in RMS: after intravenous contrast medium administration, layering of contrast medium can occur making it difficult to appreciate bladder wall enhancement [49]. T2-W sequences can be particularly useful in this setting to assess bladder wall thickening. Additional cystoscopy is often warranted [50]. At the end of treatment, some residual soft-tissue thickening may persist, and on MRI it is impossible to decide whether this is residual scarring or tumour; in these cases endoscopic biopsy is mandatory.
Fig. 10A 3-year-old boy with RMS of the prostate. The sagittal T1-W contrast-enhanced MR image shows the lesion invading the bladder wall. A transurethral catheter has been inserted (open arrow). Histopathology: botryoid RMSFig. 11A 2-year-old girl presented with a mass in the vagina. a Axial T1-W contrast-enhanced image shows the mass with heterogeneous enhancement. The tumour has both solid (asterisk) and fluid (open arrow) compartments. b Sagittal T2-W MR image shows the mass with mixed signal intensity. The bladder is displaced anteriorly and the uterus cannot be visualized. Histopathology: embryonal RMS
Extremities
RMS of the extremities (Figs. 12 and 13) is almost always of alveolar histology, tends to occur in older children and young adults, is often present with positive regional lymph nodes, and has a propensity to metastasize to unusual sites; these negative prognostic factors contribute to the relatively poor prognosis of RMS in this location [51, 52]. In approximately 12% of patients, nodal involvement is seen on imaging; however, when nodal dissection is performed the rate of nodal involvement increases to almost 50% [53]. This discrepancy between imaging findings and nodal dissection might be reduced by using high-quality state-of-the-art US. In current treatment protocols systematic biopsy of regional nodes is advocated, even if the nodes are not palpable or enlarged on imaging; sentinel node procedures are recommended whenever feasible, although the value of upgrading a patient to a higher risk group based merely on a positive sentinel node has not been studied yet. As in all tumours of the extremities, preoperative imaging plays a vital role in the depiction of the relationship between neurovascular bundles and the tumour.
Fig. 12A 4-year-old girl presenting with a mass in the left lower leg. a Axial T1-W contrast-enhanced MR image shows an ill-defined mass circumferential to the fibula. Note the cortical thinning (open arrow) of the fibula. b Sagittal PD-weighted image shows diffuse bone marrow metastases. Histopathology: embryonal RMSFig. 13A 2-month-old boy with a mass in the third ray of the left foot. T1-W MR image shows a discrete lesion (open arrow) of intermediate signal intensity. Histopathology: embryonal RMS
Other
Chest wall
Chest wall RMS (Fig. 14) is a relatively rare finding with a reported incidence of 3.7% in the IRSG II and IRSG III studies [54]. Most of the reports of chest wall involvement are either case reports or small series [55–57]. In a retrospective analysis of 15 patients, Saenz et al. [57] found a 5-year survival of 67%.
Fig. 14A 4-year-old boy presenting with a mass on the right chest wall. a US image shows a heterogeneous mass in the pectoralis major muscle (the pectoralis minor is not involved; asterisk). b T1-W MR image of the chest shows a mass of intermediate signal intensity. c After intravenous contrast medium administration the lesion shows homogeneous enhancement. Histopathology: alveolar RMS
Pulmonary
There is an ongoing debate and controversy whether congenital cystic anomalies predispose children to intralesional development of RMS [58–62]. It has been reported that pleuropulmonary blastoma (PPB) has been mistaken for or classified as RMS arising in congenital cystic adenomatoid malformation (CCAM) on a number of occasions [63]. Despite the fact that the exact incidence in CCAM is unknown, it has prompted paediatric surgeons to resect even small pulmonary cystic lesions (Fig. 15) [64, 65].
Fig. 15A 3-year-old boy with dyspnoea. Chest CT image shows displacement of the trachea (open arrow) and oesophagus (solid arrow) to the right due to a large mass (asterisk) with accompanying pleural effusion. Histopathology: embryonal RMS
Biliary tree
RMS is the most common tumour of the biliary tree in childhood, although it only accounts for approximately 0.04% of all childhood tumours [66]. The initial diagnosis will, in most patients, be made on US, which may show a solid or cystic mass situated in the liver hilum, and intrahepatic bile duct dilatation (Fig. 16). MRI is mandatory for presurgical evaluation, where magnetic resonance cholangiopancreatography (MRCP) can depict the biliary tree (Fig. 16). In many patients, however, endoscopic retrograde cholangiopancreatography (ERCP) will have to be performed in order to depict intraductal irregularities (Fig. 16). Biliary tree RMS is a tumour that does not necessarily need to be fully resected in order to achieve long-term survival, as long as adequate radiotherapy is added [67]. Intraperitoneal metastases, which can also be found on follow-up, should also be born in mind.
Fig. 16An 8-year-old boy presented with abdominal pain and jaundice. a US image shows a central process in the liver hilum (open arrow) and dilatation of the intrahepatic bile ducts (solid arrow). b T2-W MR image shows a circumscribed lesion with increased signal intensity (open arrow). c MRCP image shows intrahepatic bile duct dilatation. Note that the right and left duct systems do not communicate (open arrow). d ERCP image (ERCP performed in order to insert a stent in the common bile duct). Histopathology: embryonal RMS
Other locations
In extremely rare instances RMS can be found in other organs such as the heart, the diaphragm (Fig. 17), the omentum, the urachus and the digestive tract [68–76].
Fig. 17A 4-year-old boy was shown to have a right-sided pleural effusion on a chest radiograph. Balanced FFE sagittal MR image shows a mass (open arrow) arising from the diaphragm (courtesy of S.G.F. Robben, Academic Hospital Maastricht, The Netherlands)
Congenital
RMS has been reported to occur as a congenital tumour (Fig. 18) [77–81]. In congenital alveolar RMS the prognosis is reported to be extremely poor, despite otherwise adequate treatment [79]. Orbach et al. [82] reported the SIOP data on soft-tissue sarcoma in the first year of life. In their study population of 16 newborns, with a follow-up of 1.8–10.0 years, 3 out of 5 newborns with RMS survived. It has been noted that in congenital RMS the disease may be metastatic at the time of birth, with metastases described in a number of organs and in the placenta [81].
Fig. 18A 4-day-old girl born with a lump on the left foot. Antenatal ultrasonography at 20 weeks showed no abnormalities. a T1-W MR image shows a large inhomogeneous mass arising from the left foot. b Abdominal US image shows popliteal and inguinal nodal invasion, and hepatic and pancreatic metastases (open arrow). Due to the poor prognosis, no therapy was given, and the child died several weeks later. Histopathology: poorly differentiated soft-tissue sarcoma without distinct translocations
Adult patients
Every once in a while paediatric radiologists and paediatric oncologists receive a request for help in the management of an adult patient (Fig. 19). RMS, although seen as a soft-tissue tumour of childhood, can also occur later in life [83–86]. Compared to childhood RMS, adult RMS has a poor outcome. In a large retrospective study of 171 patients 5-year overall survival was only 40% [85]. However, the patients in this series treated according to the guidelines for treatment of childhood RMS showed survival figures comparable to those seen in children. This suggests that treatment of adult RMS should be based on paediatric protocols tailored to adults, to increase survival in this age group. In the Academic Medical Centre Amsterdam we have a working group on childhood tumours in (often young) adults that specifically deals with this challenging population. This working group consists of medical oncologists, paediatric oncologists, radiation oncologists, (orthopaedic) surgeons and a paediatric radiologist. Imaging features will in general not be of help, as the pretest likelihood of RMS in an adolescent or adult is extremely low.
Fig. 19A 45-year-old man with a mass in the thigh. T1-W contrast-enhanced MR image shows a heterogeneous circumscribed mass in the vastus lateralis muscle of the right leg. Histopathology: alveolar RMS
Differential diagnosis
Given the wide variety of locations in which RMS can be found it is difficult to give a concise list of differential diagnoses. The site of the primary lesion determines the differential diagnosis. Keeping location out of the equation there are, however, certain tumours, such as haemangiomas/vascular malformations (Fig. 20), adult-type soft-tissue sarcomas, peripheral neuroectodermal tumours (PNET), infantile fibrosarcoma, aggressive fibromatosis, desmoplastic small round-cell tumours and rhabdoid tumours, and other more even rarer soft-tissue tumours such as nonosseous Ewing sarcoma (Fig. 21), that should be kept in mind when performing US or reading CT or MRI studies of soft-tissue tumours in childhood.
Fig. 20A 1-year-old girl with a mass on the left buttock. a Duplex US image shows a highly vascularized, well-delineated heterogeneous mass that was initially thought be a haemangioma. b Coronal STIR image shows a circumscribed solid lesion that invades the pelvis via the greater sciatic foramen (open arrow). c After initial resection, with incomplete margins, tumour recurrence was seen. MR image 2.6 years after initial diagnosis shows progression of disease extending to the abdominal wall (open arrow). Histopathology: alveolar RMSFig. 21A 22-month-old boy with a mass in the right groin. MRI shows a heterogeneous lesion adjacent to the gracilis muscle (open arrow). Histopathology: extraosseous Ewing sarcoma
Treatment and prognosis
Treatment of RMS requires a multidisciplinary approach, where chemotherapy, surgery and radiotherapy (RT) each has its own specific role.
Chemotherapy
Before the introduction of chemotherapy only 25% of patients with RMS survived, despite adequate local therapy. This indirectly indicates that the vast majority of patients had at least minimal disseminated disease at diagnosis, whereas, with state-of-the-art imaging techniques and bone marrow investigations, only 15% of patients with RMS present with overt metastatic disease. Besides treating minimal disseminated disease, chemotherapy enables local therapy to be more conservative. In most patients, surgery at diagnosis consists of biopsy only. Chemotherapy is given to all patients; it reduces tumour size and extension and often allows a delay in surgery. The tumour is more likely to be completely resected and mutilating surgery avoided. Most international protocols use vincristine and dactinomycin as standard drugs. The choice of alkylating agent differs between Europe (ifosfamide) and North America (cyclophosphamide). These drugs have proved to be equally effective, but differences in toxicity exist: ifosfamide is more nephrotoxic, whereas cyclophosphamide more gonadotoxic [87]. The addition of other antineoplastic agents to vincristine, dactinomycin and alkylators in the treatment of RMS has been investigated, but no significant improvement in outcome has been found [88].
Surgery
In children surgery often starts during the diagnostic phase with biopsy for histological studies, although here interventional radiology plays an increasing role. Excisional biopsy is not advocated except for paratesticular tumours. Most patients end up with postsurgical stage IRS group III. Surgery is generally delayed until after tumour reduction by chemotherapy. The surgical treatment of RMS is site-specific, but the current paradigm is complete wide excision of the primary tumour with a margin of uninvolved tissue whenever possible. Debulking and mutilating procedures should be avoided.
Radiotherapy
The philosophies underlying the treatment strategies in North America and Western Europe have differed in the past. North American protocols involved aggressive surgery and routine RT, except for those tumours that were microscopically radically resected at diagnosis, followed by prolonged chemotherapy regimens for up to 2 years. The SIOP-MMT (International Society for Paediatric Oncology–Malignant Mesenchymal Tumour) group advocated the use of chemotherapy and surgery in order to achieve complete remission in as many patients as possible in order to avoid RT in these often very young patients. RT often has devastating effects in growing children, potentially leading to significant cosmetic and functional problems. Radiation tolerance of growing bone is ≤20 Gy, but the radiation dose from the treatment of RMS ranges from 36 to 50.4 Gy. For certain sites this policy worked out well (e.g. orbital RMS), and in many patients RT could be avoided [89]. At most other sites, relapse rates were high, and salvage rates after relapse were low. The treatment protocols in Germany (Co-operative Weichteilsarcom Studie, CWS) and Italy (Italian Cooperative Soft Tissue sarcoma Group, ICG) were between that in North America and that of the SIOP-MMT group. In 2005, the former SIOP-MMT, CWS and ICG joined to form the EpSSG. As a result of extensive cooperation between the European and the North American groups, the similarities in treatment strategies now outnumber the differences [90]. Although RT is still deemed essential in many patients to achieve cure, the possibility of radiation-induced second malignant neoplasms must be kept in mind [91, 92].
European approach
Today more than 70% of nonmetastatic RMS are cured, but survivors may suffer from sequelae [93–98]. Therefore, the SIOP-MMT group has tried in their RMS75, MMT84, MMT89 and MMT95 studies to avoid mutilating surgery and RT [93, 99, 100]. Only children ≥3 years of age with parameningeal RMS at high risk of meningeal extension and children not achieving complete remission after chemotherapy and surgery were irradiated after intensive chemotherapy. Of all survivors, 49% were treated without significant local therapy [93]. Locoregional relapse occurred in 34%. This has lead to an event-free survival (EFS) of 57% and an overall survival (OS) of 71% [93]. Alveolar histology was associated with a significantly higher risk of relapse and a much higher risk of metastases.
North American approach
In the studies performed by the IRSG the need for RT is based on surgical radicality, localization, and size of the primary tumour. Only completely resected tumours (IRS group I) with a favourable location (stage I; Table 5), and small tumours (≤5 cm) at unfavourable locations (stage II) did not receive RT in the IRS-IV study [87]. Grossly resected tumours with microscopic residual disease (IRS group II), and incompletely resected tumours or tumours with gross residual disease after biopsy (IRS group III) were irradiated [87, 88, 101, 102]. With this approach in the IRS-IV study an EFS of 78% and an OS of 84% were obtained. Between the IRSG and SIOP-MMT group a significant difference in OS in alveolar RMS was seen (71% vs. 38%) [90]. The salvage rate after relapse of an alveolar RMS was low, and therefore in the current European trial, RT is mandatory for all patients with alveolar histology.
Table 5Pediatric Oncology Group and Children’s Cancer Group for the Study of Rhabdomyosarcoma classification [28]StageSitePrimary tumouraSizebRegional nodescDistant metastasisdIOrbitT1 or T2a or bN0, N1 or NXM0Head and neck (excluding parameningeal)Genitourinary, nonbladder nonprostateIIBladder and prostateT1 or T2aN0 or NXM0ExtremityCranial parameningealOther (including trunk, retroperitoneum, etc)IIIBladder and prostateT1 or T2aN1M0ExtremityCranial parameningealT1 or T2bN0, N1 or NXM0Other (including trunk, retroperitoneum, etc)IVAll sitesT1 or T2a or bN0 or N1M1aT1 tumour limited to original muscle or organ, T2 tumour has extension or fixation to the surrounding tissue.ba tumour equal to or less then 5 cm in greatest dimension, b tumour larger than 5 cm in greatest dimension.cN0 no clinical involvement of regional nodes, N1 clinical involvement of regional nodes, NX status of regional nodes unknown.dM0 no distant metastasis, M1 distant metastasis.
As described above, European and American approaches, although historically different, have converged based on the results of successive international trials and extensive cooperation between the European and North American groups [90]. As approaches now are very similar, in this review the current EpSSG approach for the different clinical groups is discussed.
Current EpSSG approach
IRS group I In the IRS-I study, a randomized trial in clinical IRS group I patients, no difference was seen in survival between patients treated with chemotherapy and those treated with chemotherapy and RT [101]. A subsequent retrospective study based on the subsequent IRS-I to IRS-III trials confirmed that for embryonal RMS although there was a small difference in failure-free survival (FFS) with and without RT, OS was not significantly different [103]. On the other hand, for alveolar RMS and undifferentiated RMS, 10-year FFS was 73% vs. 44%, and OS 82% vs. 52% for patients treated with and without RT, respectively [103]. Therefore the current EpSSG study recommends RT in non-embryonal RMS only.
IRS group II Evaluation of the role of RT in IRS group II patients in subsequent Cooperative Soft Tissue Sarcoma studies (CWS 81, 86, 91 and 96) showed that for embryonal RMS, EFS was significantly different for patients treated with and without RT [104]. However, OS at 5 years was not significantly different (84% vs. 77%). For patients with tumours of unfavourable histology (independent of site and size), EFS and OS were significantly better when RT was part of the treatment [104]. Therefore, in IRS group II patients, RT is recommended. It is compulsory in patients with high-risk features, but may be omitted in patients with favourable histology in whom RT may be considered too toxic when considering patient age and site of the tumour.
IRS group III In patients with IRS group III tumours, RT is the only available therapy in patients who cannot receive a secondary complete resection. Patients who do receive a delayed complete resection benefit from additional RT. In the CWS trials 81–96 5-year EFS was 77% and 58% for those treated with and without additional RT, respectively [28]. OS, however, was not significantly different between the two groups: 84% and 79%, respectively. RT is, therefore, usually indicated except in patients with a favourable site and histology.
Prognosis
As malignant tumours in childhood are rare diseases, most children with cancer have been included in international treatment protocols. This way survival for localized disease (85% of patients) has improved from 25% in the early 1970s to 75% in the most recent international trials [101, 105–107]. Although results for localized RMS have improved dramatically in the past decades, patients with disseminated disease still have a dismal prognosis, with a 5-year OS of 24% [26].
A way to reduce long-term sequelae of RT may be by using brachytherapy, especially in girls with genital tract RMS and patients with bladder-prostate, extremity, and head and neck RMS [100, 108–111]. Other options are the use of modulated RT (IMRT) and proton therapy [112, 113].
The postoperative patient
Image interpretation and management of the patient after surgery and often RT are challenges (Fig. 22). Most important is proper knowledge of the surgical procedure and/or the radiation field. The following postoperative changes can be encountered in children treated for RMS:
HaematomaOedemaSoft-tissue infection/abscessCalcificationForeign bodiesMuscle flaps/fat padsDistorted anatomyRadiation effect Figure 23 shows a possible decision tree to manage postoperative findings on follow-up imaging.
Fig. 22A 6-year-old boy with a history of treated bladder RMS. At surgical resection the urethra was damaged leading to a persistent urinoma in, after RT, nonvital tissue. MRI image after treatment shows a mass (asterisk) between the urethra (open arrow) and the rectum (solid arrow). This mass is a vascularized gracilis muscle flap used to repair the defect. Without proper knowledge of the surgical history of the patient, this might have been interpreted as tumour recurrence. Histopathology: embryonal RMSFig. 23Flow chart for posttreatment lesions found on MRI. SI signal intensity, CE contrast-enhanced [114]
Conclusion
In this review we have discussed the findings of RMS outside the craniofacial region. The treatment of RMS requires a multidisciplinary approach, in which paediatric oncologists, radiologists, paediatric surgeons, pathologists and radiation oncologists all play a vital role. Although they are the most common soft-tissue tumour of childhood, these still rare tumours should be evaluated and treated in specialized centres.


Delayed Intrathoracic Gastric Perforation After Obesity Surgery: A Severe Complication

We describe a case of a patient with an intrathoracic gastric perforation, 6 months after she underwent a gastric banding procedure for the treatment of morbid obesity. After an urgent laparotomy during which the stomach was replaced and oversewn, she recovered uneventfully. The possible mechanism of this severe complication is discussed.
Introduction
Gastric banding is a frequently performed operation for patients with morbid obesity. Complications have been described extensively. We describe a rare and severe complication of an intrathoracic stomach in a patient with gastric banding, 6 months after the initial procedure, and its treatment.
Case Report
A 37-year-old woman was admitted to our emergency department with acute onset of abdominal pain, vomiting, and dyspnea. Six months earlier, she had undergone laparoscopic adjustable gastric banding (Lap-Band®)for severe obesity. Physical examination now showed an anxious, dyspneic woman with a respiratory rate of 45 per minute. There was a tachycardia (140/minute) and blood pressure difference between the left (60/40 mmHg) and the right arm (110/70 mmHg). Auscultation of the chest revealed diminished breathing sounds on the left side. Chest x-ray showed an intrathoracic position of the stomach through a paraesophageal hernia with a shift of the mediastinum to the right (Fig. 1). A small pneumothorax was seen.
Fig. 1Intrathoracic position of the stomach with a shift of the mediastinum to the right
After a nasogastric tube was inserted, the patient was intubated because of respiratory insufficiency. A computed tomography (CT) scan of the chest showed a complete atelectasis of the left lung caused by an intrathoracic stomach and a left-sided pneumothorax (Fig. 2). The condition of the patient worsened and she was taken to the operating room for urgent laparotomy.
Fig. 2Complete atelectasis of the left lung caused by an intrathoracic stomach and a left-sided pneumothorax
During laparotomy an ischemic gastric perforation was identified at the orifice of the paraesophageal hernia, with spill of gastric content into the abdomen and thoracic cavity. The intrathoracic stomach was reduced into the abdomen. The gastric perforation was oversewn. The    Lap-band was removed, and a Nissen fundoplication was performed. At last, a thoracic drain was placed to treat the pneumothorax.
The patient was treated with intravenous antibiotics (metronidazole and cefuroxime) for 5 days. After 2 days, she was able to leave the Intensive Care Unit. Her postoperative course was uneventful. Thirteen days after surgery, she left the hospital.
Discussion
Gastric perforation after gastric banding has been described earlier [1]. In all reported cases, the perforation was caused by erosion of the band through the stomach with an incidence of 0.3–3% of the patients. Furthermore, herniation of the stomach into the thoracic cavity is a well-known entity. It has been described as a late complication after Nissen fundoplication, even in combination with intrathoracic perforation [2–4].
We describe the first case in which both perforation and herniation have occurred simultaneously after obesity surgery and even not as an immediate consequence of the operation. In our case, the gastric band was not the primary cause of the perforation. Most probably, the stomach had moved into the thoracic cavity through a preexistent paraesophageal hernia. Because of air-trapping distal to the band accompanied by having a copious meal, the intrathoracic stomach distended, rotated, and perforated at the orifice of the hernia. The acute onset of pain can be explained by the perforation and the dyspnea appears to be caused by mediastinal shift as well as the pressure atelectasis of the left lung.
Retrospectively, our patient was known to have a paraesophageal hernia before she underwent gastric banding. The possible severe complications of this combination should be considered. A Nissen fundoplication before or in the same operation could be an option for this subgroup of obese patients.

gastric perforation
complication
gastric banding
morbid obesity
bariatric surgery