Free-living flatworms under the knife: past and present

Traditionally, regeneration research has been closely tied to flatworm research, as flatworms (Plathelminthes) were among the first animals where the phenomenon of regeneration was discovered. Since then, the main focus of flatworm regeneration research was on triclads, for which various phenomena were observed and a number of theories developed. However, free-living flatworms encompass a number of other taxa where regeneration was found to be possible. This review aims to display and to compare regeneration in all major free-living flatworm taxa, with special focus on a new player in the field of regeneration, Macrostomum lignano (Macrostomorpha). Findings on the regeneration capacity of this organism provide clues for links between regeneration and (post-)embryonic development, starvation, and asexual reproduction. The role of the nervous system and especially the brain for regeneration is discussed, and similarities as well as particularities in regeneration among free-living flatworms are pointed out.
Introduction
Regeneration research in flatworms has a long standing history, but was focussed on triclads since its beginning in 1774, when Pallas (Pallas 1774) described the regeneration of the triclads Dendrocoelum lacteum and Bdellocephala punctata (from Brøndsted 1969). Since then, a vast amount of studies about regeneration in flatworms has appeared (for recent reviews, see Agata and Watanabe 1999; Sánchez Alvarado 2000, 2003, 2004, 2006; Saló and Baguñà 2002; Newmark and Sánchez Alvarado 2002; Agata 2003; Agata et al. 2003; Reddien and Sánchez Alvarado 2004; Reuter and Kreshchenko 2004; Sánchez Alvarado and Kang 2005; Saló 2006), but many of the old questions and many of the most intriguing phenomena that have been discovered cannot be explained today. Why can some animals regenerate, while others cannot? How is the duplication of heads or tails (heteromorphoses) possible in adult organisms? What conditions are sufficient and what are necessary for successful regeneration? Is regeneration a side-product of asexual reproduction, or the other way round? Is regeneration recapitulating pathways used in embryonic and postembryonic development? How are stem cells controlled and directed in regeneration? Are stem cells in adult flatworms totipotent, and can they be likened to embryonic blastomeres? This review provides a brief synopsis on what is known about the regeneration capacity in free-living flatworms, and addresses open questions about regeneration, with special emphasis on an emerging model organism, Macrostomum lignano (Egger et al. 2006a,b).
A comparison of the regeneration capacity of the macrostomorph flatworm M. lignano with other free-living flatworms necessarily involves a look at their phylogenetic relationship. The Macrostomorpha belong to the largest taxon Rhabditophora, which also encompasses the Polycladida, Lecithoepitheliata, Prolecithophora, Proseriata, Bothrioplanida, Tricladida, Rhabdocoela, and the parasitic Neodermata (Ehlers 1985; Rieger 1996; Tyler et al. 2006). Macrostomorpha are considered to be the most basal taxon within the Rhabditophora due to a simple pharynx, entolecithal eggs, and the lack of vitellaria (Ehlers 1985). Referring to their small size (millimeter range), macrostomorphans are members of the “microturbellarians”, a nonsystematic denomination. Polyclads and triclads are the only taxa belonging to the “macroturbellaria”, with forms generally in the centimeter range. Triclads, as more derived rhabditophorans, have vitellaria, a rather complicated embryonic development (e.g., Cardona et al. 2005) and an often very pronounced regeneration capacity (see Brøndsted 1969).
The status of the acoels
While many molecular (e.g., Baguñà and Riutort 2004a,b) and also some morphological studies (e.g., Reuter et al. 2001, summed up in Egger and Ishida 2005) have placed the acoels outside the Plathelminthes, a number of characteristics (apomorphies) speak in favor of acoels being flatworms (Ehlers 1985), most notably the stem cell system and the special mode of epidermal replacement (Smith et al. 1986; Tyler and Hooge 2004). In this review, acoels and nemertodermatids are considered members of the Plathelminthes.
What is a planarian?
In flatworm regeneration research, other groups than triclads are often neglected and can easily be overlooked. This may in part be due to an ambiguous use of the term “planarian”, which is most often referring merely to triclads (more specifically, to paludicolans, e.g., Agata 2003; Reddien and Sánchez Alvarado 2004), but is also commonly used as a synonym for all free-living flatworms (e.g., Baguñà 1998; Saló and Baguñà 2002) or even used interchangeably for free-living flatworms and triclads alike (e.g., Newmark and Sánchez Alvarado 2001). The term “freshwater planarian”, while somewhat more specific, is still ambiguous, as a number of nontriclad flatworms, e.g., Stenostomum and Microstomum, are freshwater dwellers as well. In publications dealing with several flatworm taxa, it is often unclear what the term planarian signifies.
It seems therefore desirable to agree on using less ambiguous denominations in scientific contexts, such as unequivocal scientific names, and to get rid of the term planarian.
Overview of regeneration capacity in free-living flatworm groups
All major free-living flatworm taxa with their tentative general regeneration capacities are given in Fig. 1. Asexually reproducing forms are only known for acoels, catenulids, macrostomorphans, and triclads. Within free-living flatworms, either catenulids (Ehlers 1985) or acoelomorphs (acoels and nemertodermatids, Rieger 1996) are considered as the basal-most taxon. All other flatworms are encompassed by the rhabditophorans, with macrostomorphans and polyclads at the base (Ehlers 1985; Rieger 1996).
Fig. 1The regeneration capacity of the major free-living flatworm taxa. In all groups with asexual reproduction, species with a pronounced regeneration capacity can be found (green shades). Not all species in the listed taxa are necessarily showing the same regeneration capacity; only a broad classification of regeneration capacities is given. The major taxa were compiled after Ehlers (1985), Rieger (1996), and Tyler et al. (2006)
Catenulida
The regeneration capacity of two catenulid genera, namely, Stenostomum and Catenula, has been extensively investigated. Probably linked with asexual reproduction (paratomy), excellent regeneration capacity has been reported after artificial amputation (Ritter and Congdon 1900; Child 1903a,b; Ruhl 1927a; van Cleave 1929; and Palmberg 1990 for Stenostomum; Moraczewski 1977 for Catenula). Regeneration from the so-called organ primordia was found to be possible for all organ systems.
Acoela
Based on experiments mainly with Polychoerus caudatus (Stevens and Boring 1905; Child 1907; Keil 1929) and Paramecynostomum diversicolor (Peebles 1913), Hyman notes that acoels “have limited powers of regeneration” (Hyman 1951, p 182). The studied species were not able to regenerate ganglia (brain) or the statocyst. Later studies with Amphiscolops langerhansi (Steinböck 1954, 1955, 1963a,b) and Hofstenia giselae (Steinböck 1966, 1967) illustrated a substantial regeneration capacity in these acoels, where H. giselae was found to even regenerate the statocyst after amputation (Steinböck 1966). Several species can regenerate their eyes (Hanson 1967 for A. langerhansi and Pseudohaplogonaria sutcliffei, Yamasu 1991 for Praesagittifera naikaiensis, and Åkesson et al. 2001 for Convolutriloba longifissura).
Acoels are now known to show all main modes of asexual reproduction, that is architomy (e.g., C. longifissura, Åkesson et al. 2001), paratomy (e.g., Paratomella unichaeta, Ax and Schulz 1959), and budding (e.g., C. retrogemma, Hendelberg and Åkesson 1988). Architomy is characterized by fission before formation of new organs, paratomy by formation of new organs before fission. Budding refers to a special case of paratomy, where the axis of the new piece is not corresponding to the axis of the old piece—in C. retrogemma, the axis of the buds is tilted by 180° in relation to the main animal (Hendelberg and Åkesson 1988, 1991).
Nemertodermatida
No account of regeneration potential or asexual reproduction has been given for animals from this taxon.
Macrostomorpha
As already observed in catenulids and acoels, the regeneration capacity of macrostomorphans is closely associated with the mode of reproduction. Thus, in the asexually reproducing taxa Microstomum lineare and Alaurina (paratomy), the regeneration capacity is most pronounced and all organs can be regenerated (von Graff 1908; Ruhl 1927a; Palmberg 1986, 1990, 1991). Regeneration research with the exclusive sexually reproducing genus Macrostomum started with Ruhl (1927a), who found that Macrostomum tuba cannot regenerate the head (brain, eyes, and pharynx) and requires at least a quarter of the gut in the anterior piece to be able to regenerate posterior parts, including copulatory organs. More recently, Ladurner et al. (1997) noted that in Macrostomum hystricinum marinum and Macrostomum pusillum the anterior part can fully regenerate the posterior part, while the posterior part cannot regenerate the anterior part. Similar findings were reported for M. lignano: cut in midbody, only the anterior part was found to regenerate the missing part (Rieger et al. 1999). Salvenmoser et al. (2001) described the regeneration of the musculature in M. lignano after the removal of the posterior end. Using the same species, Egger et al. (2006a,b) characterized the regeneration capacity at various cutting levels. Anterior regeneration is possible if the animals are cut in front of the brain; posterior regeneration can be successful from cutting levels starting with the posterior end of the pharynx. A distinct regeneration blastema occurs in M. lignano, but not in M. lineare, where organ primordia are formed without conspicuous accumulation of neoblasts at the wound site (Palmberg 1986).
Polycladida
The best-studied polyclad genus regarding regeneration is Leptoplana. Leptoplana alcynoe (now Letoplana alcinoi) and Leptoplana velutinus, as well as Thysanozoon brocchi and Cryptocelis alba, were shown to regenerate posterior parts by Monti (1900a). Using Leptoplana atomata, Schultz (1901, 1902) observed the regeneration of the posterior part, including gonads and copulatory organs, whereas head and pharynx were not regenerated. Similar results were obtained with Leptoplana tremellaris and L. alcinoi, although the pharynx was found to regenerate here (Child 1904a–c, 1905a; von Levetzow 1939). L. tremellaris can regenerate both anterior and posterior parts if only about half the brain is removed by transversal amputation. At amputation levels posterior of the brain, no complete anterior regeneration is possible (Child 1904a–c). Notoplana humilis shows a comparable regeneration capacity (Ishida 1998). Besides more posterior parts, T. brocchi is also able to regenerate the main tentacles, gut, and pharynx (Monti 1900a; von Levetzow 1939). Studying regeneration in Leptoplana saxicola, Pucelis litoricola, and Planocera californica, Olmsted (1922a) reports that these species follow the “polyclad rule for regeneration”, that is, regeneration of all amputated parts (including eyes) is possible if the brain remains intact. Several studies were dedicated to find out whether polyclads can regenerate the brain (Morgan 1905 for Leptoplana littoralis and von Levetzow 1939 for T. brocchi), and how the behavior changes after excision of the brain (Morgan 1905; Olmsted 1922b; von Levetzow 1939; Koopowitz et al. 1975, 1976) or after severance of the main lateral nerve cords (Olmsted 1922b; Koopowitz et al. 1975, 1976). Severed nerve cords and brain halves can fuse and resume their functions (Olmsted 1922b; Faisst et al. 1980), but the brain is not regenerated. Full anterior regeneration, including the brain, after complete removal of the brain was only observed in Cestoplana, provided the amputation level is a short distance behind the brain (Child 1905b). All mentioned species, except T. brocchi, are members of the taxon Acotylea (Tyler et al. 2006). From what is known, both Cotylea and Acotylea seem to share a comparable regeneration capacity.
Lecithoepitheliata
Only scarce reports about regeneration are available for this neoophoran taxon. For Prorhynchus stagnalis, Ruhl (1927a) remarks that no regeneration capacity whatsoever could be detected at anterior, posterior, and midbody cutting levels. The posterior pieces survived for 30 days. When cutting Geocentrophora baltica a short distance behind the pharynx, no regeneration could be observed (Steinböck 1927). All pieces died at the latest after 17 days. A closer examination of the regeneration capacity of Geocentrophora sphyrocephala revealed that these animals can approximately rebuild the mouth opening anteriorly in 3–5 weeks but cannot regenerate the brain. Both parts die after 40 days if the animals are cut just behind the pharynx. Cut in midbody or more posteriorly, the anterior part can regenerate the tail with adhesive organs in 2–3 weeks, although the animals were never observed to reach the original body length. The posterior part dies without regeneration after 1 day (Hagleithner 1946).
Prolecithophora
According to Pechlaner (1957), Plagiostomum lemani is reported to have a low regeneration capacity (Keller 1894), while Plagiostomum girardi can regenerate all amputated parts (Monti 1900b).
Proseriata
von Graff (1882) describes amputation experiments with Monocelis fusca, where posterior pieces resumed movement after 3–5 days. In M. fusca, Monocelis lineata and other monocelidids (Coelogynopora, Archilopsis), and also in otoplanids (Itaspiella, Bothriomolus), Giesa (1966) observed posterior regeneration of parts containing the brain in 3–4 days. Even pieces amputated just behind the brain can regenerate. The brain does not regenerate, though. In Otomesostoma auditivum, Pechlaner (1957) found promising regeneration potential, severely limited by a short life span of 9–10 months and low living temperatures (about 4°C). Gut, part of the gonads, and sensory organs can regenerate. First signs of pharynx, eye, and brain regeneration were observed after 9–15 weeks, but the animals died before regeneration was complete. The statocyst was not regenerated.
Bothrioplanida
While no extensive regeneration studies have been undertaken, Sekera (1911) found that Bothrioplana semperi is able to regenerate the pharynx posteriorly.
Tricladida
Dalyell (1814) noted that Planaria nigra (today: Polycelis nigra) could “almost be called immortal under the edge of the knife” (in Brøndsted 1969). However, not all triclad species are equally powerful regarding regeneration. Ŝivickis (1930) distinguished five groups of triclads according to their ability to regenerate a head. This classification was subsequently refined into eight types by Teshirogi et al. (1977). For the Phagocata velata type, the Dugesia dorotocephala type, the Polycelis auriculata type, and the Dendrocoelopsis lactea type 2 (Sapporo population), regeneration of a head is possible from any part of the body, but with varying probability of success. Animals of the Dendrocoelopsis lactea type 1 (Kuroishi population) fail to regenerate a head from tail pieces up to the male gonopore. The Dendrocoelopsis ezensis type and the Dendrocoelum lacteum type 1 are very similar in that they are generally only able to regenerate a head if amputated anterior to the pharynx, whereas the Bdelloura type (e.g., Bdelloura candida) completely lacks the ability to regenerate a head. While a vast amount of literature on regeneration in triclads is available, recent publications are not so much concerned with studying the regeneration capacities of different triclads, but more with unraveling cellular and molecular mechanisms of regeneration in only a few model triclads, such as Dugesia japonica and Schmidtea mediterranea.
Rhabdocoela
The typhloplanoid flatworm Mesostoma ehrenbergi cannot regenerate any organs, only approximately restore the original shape posteriorly (Steinmann and Bresslau 1913). Regeneration experiments with Mesostoma productum, Mesostoma lingua, and Mesostoma punctatum revealed that anterior regeneration of parts in front of the brain is possible, although often done imperfectly. The tail can regenerate; the more posterior the cut is made, the better is the regeneration. Brain and pharynx cannot be regenerated (Fulinski 1922). In Phaenocora unipunctata and Phaenocora megalops (also typhloplanoids), no regeneration capacity was found (Fulinski 1922). For the the dalyelliid Dalyellia millportiana and the kalyptorhynch Gyratrix hermaphroditus, Ruhl (1927a) remarks curtly that all animals died within 1 day after amputation and no regeneration being observable. Dalyellia viridis and Typhloplana viridata survive the amputation of the caudal-most part of the tail, but show no signs of regeneration, not even of the adhesive organs. In Rhynchomesostoma rostratum, another typhloplanoid, the amputated tail tip is regenerated, or at least a tail-like shape is restored (Hein 1928).
Comparison of the regeneration capacity between free-living flatworm taxa
While the experimental evidence for some groups is still patchy, we find three main types of regeneration capacity in free-living flatworms. Some taxa cannot regenerate at all, such as most rhabdocoels and some lecithoepitheliates. On the other end of the scope are species that can regenerate all organs, such as the acoel Convolutriloba, the catenulid Stenostomum, the macrostomorphan Microstomum, the polyclad Cestoplana, and many triclads. Most taxa however lie in between these extremes, lacking the ability to regenerate brain, eyes, pharynx, or statocyst (many acoels), but are able to regenerate gonads, copulatory organs, sucker, duo-gland adhesive systems, gut, and the anterior-most part in front of the brain.
The regeneration capacity of M. lignano can be likened most closely to triclads of the Bdelloura group, which cannot regenerate a head (brain or eyes). Most polyclads, although often able to regenerate a pharynx, are also roughly comparable to Macrostomum regarding their regeneration capacity. Members of other diverse taxa like lecithoepitheliates and rhabdocoels are bearing resemblance with regeneration in Macrostomum as well. Such similarities can be found crossing the boundaries of the major flatworm taxa, making the regeneration capacity a feature of no immediate phylogenetic value. Close relatives like Microstomum and Alaurina (both macrostomids) surpass Macrostomum’s regeneration capacity by far.
Asexual reproduction and regeneration
Not surprisingly, the ability to regenerate is often linked to asexual reproduction (Fig. 1, Brøndsted 1969; Sánchez Alvarado 2000). In taxa lacking asexual reproduction, the regeneration capacity is generally less pronounced than in taxa with asexual reproduction, where regeneration is necessarily involved (Brøndsted 1969; Reuter and Kreshchenko 2004). As described in the section “Acoela”, in architomy, regeneration events follow fission, while in paratomy and budding, the term “pregeneration” seems to be more accurate, as organs are duplicated in the presence of the old organs in the new animal (zooid) before fission occurs. The notable difference between regeneration and pregeneration is the presence of a brain in the latter, which is regarded as a decisive organ for regeneration in many species (Henley 1974; Kreshchenko et al. 2001). It is interesting to note that the catenulid Stenostomum leucops, reproducing asexually by paratomy, often fails to regenerate a head if amputated artificially (van Cleave 1929). This finding hints at an actual difference between paratomy and architomy regarding the influence on regeneration capacity.
Asexual reproduction in flatworms occurs in more basal taxa (Acoela, Catenulida, and Macrostomorpha) as well as in more derived taxa (Tricladida and Neodermata). Triclads, like acoels, show paratomy (Dugesia fissipara and Dugesia paramensis, Hyman 1951) as well as architomy (e.g., Dugesia tahitiensis, Peter et al. 2001). Just like regeneration capacity, asexual reproduction is too dispersed among diverse taxa to provide insight into phylogenetic relationships.
Why can some flatworm species regenerate while others cannot?
If a profound regeneration capacity is a plesiomorphic feature, there might have been a selection pressure against regeneration during evolution (Goss 1991; in Sánchez Alvarado 2000). Why would such a useful feature be reduced or abolished? Because regeneration is dangerous, as a plethora of evidence of double-headed or double-tailed flatworms testify. Such aberrant forms are easily produced in species that can regenerate (see section “Heteromorphoses”), but are not known from flatworms lacking regeneration capabilities. Other than as a side effect of asexual reproduction, the ability to regenerate is seemingly not useful enough to outweigh the inherent dangers. What are the chances that a predator spares a tail fragment that can regenerate to a complete animal in a couple of days if left alone? What are the chances that a disease only affects a part of the animal, but spares the rest to readily regenerate the damaged organs? A totipotent stem cell system out of control might be too high a risk for comparably little reward. This could be the reason why regeneration capabilities decreased in species that no longer propagate through asexual reproduction.
In this light, it seems more likely that in evolutionary terms, asexual reproduction is a primitive and not a derived feature (Rieger 1986; Gremigni 1992; Reuter and Kreshchenko 2004), and that regeneration capability results from asexual reproduction as an added bonus. Regeneration can be seen as a form of asexual reproduction, only triggered by different stimuli.
What is the decisive factor that determines the regeneration capacity of a species? Citing regeneration experiments with Dendrocoelum and Bdellocephala, Brøndsted (1969) comes to the conclusion “that the posterior part of the body in these two species simply lacks the potential to respond to head inducing substances”. This may also hold true for M. lignano, considering that in transversal amputations a head was never regenerated, but in longitudinal incisions a second head—in the presence of the original head—could be formed (Fig. 2c,d, Egger et al. 2006a,b). This experiment suggests that the ability to induce head formation, although present in adult M. lignano, is restricted to the head region only. Serotonin and possibly other substances related to the brain or nervous system might be key factors deciding on the regeneration capacity of flatworms.
Fig. 2Heteromorphoses resulting from embryogenesis and regeneration. a Twin embryo of M. lignano, 8-cell stage. The two embryos are not separated by a dividing wall. In some cases, such embryos may grow to siamese twins. Diameter of individual embryos is about 150 μm. b Different animal than a found in culture dishes. The juvenile animals are connected at the level of the pharynx, possibly the result of two embryos growing together. c Adult M. lignano, 1 week after longitudinal incision (arrowhead). Only a small indentation is visible. d Same specimen as in c 29 days after longitudinal incision. A second head with brain, a single eye, and pharynx has emerged. Scale bars are 100 μm. c, d Same scale bar
It is interesting to note that double-headed specimens of M. lignano can sometimes be observed in untreated culture dishes. These aberrant forms might rather be the result of twin embryos (Fig. 2a) that have grown together during embryonic development, than adults that were accidentally incised longitudinally (Sekera 1911; Steinmann and Bresslau 1913; Seilern-Aspang 1958).
Ovaries restricting regeneration?
The possible evolutionary origin and function of regeneration aside, what genetical, physiological, developmental, or environmental factors are responsible to determine to what extent an organism can regenerate? Even close relatives may differ vastly in regeneration capacity (e.g., Microstomum–Macrostomum), and also the same species in different developmental stages. Stenostomum alternates seasonally between asexual and sexual reproduction (protandric hermaphroditism). Asexual and male specimens of Stenostomum grande can regenerate all organs, but lose the ability to regenerate a head after the appearance of ovaries (van Cleave 1929). While the presence of ovaries and the loss of regeneration capacity might be coincidental, it appears possible that in S. grande ovaries are producing substances that inhibit regeneration. Could sexualization be directly affecting a general decrease of regeneration potential? At present, the molecular nature of such an inhibitory substance remains unclear. Vasa-related genes are expressed strongly both in gonads and in the early blastema in D. japonica and in M. lignano (Shibata et al. 1999; Pfister and Ladurner 2005), making an inhibitory function of vasa during regeneration improbable, at least in these two species. Also, contrary to the findings in Stenostomum, several triclads are known to regenerate all organs in the presence of gonads (e.g., S. mediterranea, Sánchez Alvarado 2003). Two possibilities may explain differences in the regeneration capacity in the presence of ovaries: either the ovaries in Stenostomum are producing a unique inhibitory substance that is not synthesized in Schmidtea, or Schmidtea is able to counteract the effects of such an inhibitory agent.
Regeneration in juveniles
If gonads, specifically ovaries, are indeed hindering regeneration in some species, then sexually immature juveniles could possibly be more potent regenerates than adults. In the rhabdocoel Mesostoma, young animals (the author does not specify their age) are reported to regenerate better than adults (Fulinski 1922). Contrastingly, in the acoel species H. giselae, no regeneration even of large juveniles (2.5 mm long) takes place (Steinböck 1967).
In most cases, however, juveniles regenerate as well (or bad) as adults. An explanation is that gene expression for the building of gonads is already occurring in immature animals, thus diminishing possible differences in the regeneration capacity between juveniles and adults. Regeneration in juvenile triclads has been studied in B. punctata by Brøndsted, who found that animals recently hatched from cocoons were able to regenerate the head at the same rate as mature adults (Brøndsted 1942, 1969). Pechlaner (1957) studied the regeneration of freshly hatched animals, juveniles and mature adults in the proseriate O. auditivum. He did not observe significant differences in the regeneration capacity between these age groups. Neither juvenile nor adult dalyellids were found to regenerate (Hein 1928). In the lecithoepitheliate G. sphyrocephala, the regeneration capacity of juveniles corresponds to that of adults (Hagleithner 1946). The results for M. lignano support these findings and show that even very young animals (1 day after hatching) are capable of regeneration, indicating that the neoblast stem cell system is already present and functional as found in adults. Accordingly, BrdU labeling of 1-day-old hatchlings revealed a similar pattern of S-phase cell distribution as in adults, with the majority of labeled cells along the sides of the animal, and no labeled cells in the rostrum. Just like adults, juveniles were not able to regenerate a head (Egger et al. 2006a,b).
Similarities between postembryonic development and regeneration
However similar, regeneration has some characteristics not shared with postembryonic development. Unlike postembryonic development, regeneration in flatworms is triggered by a disruptive event like fission or amputation (but not by chemical destruction of tissue, burning, or high voltage; see Brøndsted 1969). As in other flatworms, in M. lignano, the wound is soon closed after amputation by contraction of the ring musculature minimizing the wound surface, and by flattening of the surrounding epidermal cells (Salvenmoser et al. 2001). In the next days, undifferentiated cells (neoblasts) accumulate at the wound site, forming a distinct blastema, covered by new and old epidermal cells. Subsequently, differentiation of microorgans and organs becomes prevalent within the blastema, superseding the blastema proper (Egger et al. 2005, 2006a,b). Epidermal cells are not undergoing mitoses, as new epidermal cells and blastema cells are derived from neoblasts located in the mesodermal space. Different to triclads, neoblasts are proliferating also within the blastema, not only at the blastema border (Egger et al. 2005). While wound closure and blastema formation are regeneration-specific events, similarities between regeneration and postembryonic development become prevalent starting with differentiation in the (former) blastema. In extreme cases, where only a small piece remains for regeneration (e.g., in pharynx-level regenerates, Egger et al. 2006a,b), resemblance with postembryonic development is particularly striking (Fig. 3). The remaining organs in these regenerates undergo extensive morphallactic changes to accommodate to the new body proportions (Fig. 3n–o in Egger et al. 2006a).
Fig. 3Comparison of M. lignano hatchlings with posterior regenerates amputated at the pharynx level. b and d are depicting the same individual. a Hatchling up to 24 h old. b Pharynx level regenerate 7 days after amputation. c 7-day-old hatchling. d Pharynx level regenerate 14 days after amputation. Scale bar for all subpanels is 100 μm
One week after amputation, pharynx-level regenerates of M. lignano are confronted with similar challenges as freshly hatched juveniles: both have to build gonads and the male and female genital organs, grow a full set of duo-gland adhesive glands, and increase their size by five or six times (Figs. 3a,b, and 4). It seems plausible, that after wound healing and restoration of the basic functionality of the gut, pharynx-level regenerates are making use of similar or same developmental pathways that are used in hatchlings (Fig. 4) (cooption, Sánchez Alvarado 2000). This view is supported by the ability of hatchlings and juveniles to regenerate just like adults, and by the similarity of the stem cell system in hatchlings, juveniles, and adults (Ladurner et al. 2000; Egger et al. 2006a,b). Vasa gene expression is similar in hatchlings and in gonad-recovering regenerates as well (Pfister and Ladurner 2005). Also, the time needed for developing to a full-grown adult in M. lignano is about the same for hatchlings (Morris et al. 2004) and 1-week-old pharynx-level regenerates, that is about 2 weeks (Fig. 3 and Egger et al. 2006a,b). In the proseriate M. fusca, the regeneration of small anterior pieces also goes through stages similar to postembryonic development (Giesa 1966), which is observed in triclads as well (Baguñà 1998). Starved adults, reducing their size and breaking down the gonads (Nimeth et al. 2004), are also likely recapitulating their postembryonic development while growing back to full size (Fig. 4). Not only is regeneration akin to postembryonic development, amputated animals can even be rejuvenated by regeneration (Fig. 4 and Haranghy and Balázs 1964 for triclads, Egger et al. 2006a,b for M. lignano). While untreated animals were found to live for about 10 months in culture conditions, they were outlived by animals amputated 45 times over a period of 20 months, effectively doubling their life span so far (Egger et al. 2006a,b; Egger, unpublished observations). At the time of writing, many of these repeatedly amputated animals are still alive.
Fig. 4Similarities between (post-)embryonic development, regeneration, and starvation considering as example M. lignano. Successive developmental stages from left to right, from embryonic to postembryonic development (formation of gonads and genital organs) until adulthood. Regeneration If an adult animal is amputated just posterior of the pharynx, the anterior piece will pass stages similar to early juveniles and subadults before regeneration to a normal adult is completed. The same animal can be amputated repeatedly, each time seemingly repeating parts of its postembryonic development. Repeated regeneration was also shown to have a rejuvenation effect. A connection between the formation of organ primordia during embryonic development and the organ differentiation in regenerating animals is possible, but has not been shown yet for M. lignano. Some developmental pathways are likely shared between late embryos and regenerates as well, e.g., the initial building of microorgans like the duo-gland adhesive systems. Both in embryos and posterior regenerates, these organs are built from scratch. Starvation Animals starved for 30 days or longer break down their gonads and genital organs shrink, but can recover and regain sexual prowess after feeding. Starvation leading to an extended life span was shown for triclads and is also likely to occur in M. lignano
A fountain-of-youth effect through starvation was shown to occur in triclads (Haranghy and Balázs 1964), but was not yet proven for M. lignano (Fig. 4).
Embryonic development and regeneration
Early embryogenesis is vastly different between diverse free-living flatworm taxa (for recent publications, see Jondelius 2004 for nemertodermatids; Ramachandra et al. 2002 for acoels; Morris et al. 2004 for macrostomorphans, Younossi-Hartenstein and Hartenstein 2000 for polyclads, Cardona et al. 2005 for triclads, and Younossi-Hartenstein and Hartenstein 2001 for rhabdocoels). Even comparably close relatives show significant differences in their early embryonic development. Lacking vitellaria and therefore producing entolecithal eggs, Macrostomorpha and Polycladida are the only archoophoran Rhabditophora, placing them at the basal-most positions in this largest flatworm taxon (Ehlers 1985; Rieger 1996). Nonetheless, their early embryonic development starts to deviate after about the third cleavage (Fig. 5). If developmental pathways from the embryonic development are reused in regeneration, it is most likely from the formation of organ primordia and later stages (Fig. 4). Cardona et al. (2005) point out the similarities between embryogenesis and early regeneration in triclads concerning the building of a provisional epidermis and the development of muscles and nerves, suggesting that not only postembryonic, but also embryonic developmental pathways are utilized during regeneration, a viewpoint shared with Vannini (1966). A comparison of gene expression patterns between regenerates (see Reddien et al. 2005 for triclads), late embryos and hatchlings seem to be a worthwhile task.
Fig. 5Early embryonic development of two basal rhabditophorans. a The 12-cell stage of the polyclad Pseudostylochus intermedius. Egg without eggshell. b 12-cell stage of the macrostomorphan M. lignano. Different to the clear spiral cleavage of polyclads, M. lignano forms the so-called hull cells after the third cleavage to enclose the rest of the embryo. The diameter of the embryos is about 150 μm
Early blastomere ablation in polyclads has revealed a determinative development as in other spiralians (Boyer 1986), the fate of blastomeres depending on micromere–macromere interaction (Boyer 1989). Polyclads do not have an invariant cell lineage, however (Boyer 1992). Polyclad embryos can be obtained devoid of an eggshell (Fig. 5a) and still develop normally in a culture dish treated with antibiotics. Regeneration experiments with polyclad embryos may thus be relatively easy to carry out and could provide additional clues about the requirements for regeneration in flatworm embryos. Also, the regeneration capacity of polyclad larvae, as compared to hatchlings of directly developing polyclads, would be interesting to know. A connection between regulation in early embryonic development and regeneration capacity does not seem to exist. This notion is supported by regeneration experiments with embryos of the triclad P. nigra. Regeneration does not take place before organ systems, such as the nervous system, reach a certain degree of differentiation in the embryo, so that stage five embryos are unable to regenerate, while stage six embryos with a more differentiated nervous system readily regenerate lost parts (Le Moigne 1966). This author held that the nervous system plays a critical role in the formation of a blastema during regeneration.
Nervous system and role of the brain for regeneration
The nervous system, especially the brain, has long been considered to play a key role in regeneration (Kreshchenko et al. 2001). In many free-living flatworms, posterior parts can be regenerated, while the brain cannot. In anterior regenerates of M. lignano missing the brain, a blastema-like structure was observed, which was unable to give rise to all missing organs. The more posterior the animals were amputated, the less pronounced was this abortive anterior blastema (Egger et al. 2006a,b). In Macrostomum, are the brain and the nerve cell clusters associated with the pharynx (Fig. 6) required to release factors needed for the regeneration of brain, eyes, and pharynx? The study of gene expression in head-amputated regenerates of the triclads D. japonica and S. mediterranea revealed necessary genes for regeneration of the brain (Cebrià et al. 2002a; Koinuma et al. 2003; Cebrià and Newmark 2005). Also, with nou-darake a factor was identified that restricts brain tissue to the head region of D. japonica. If nou-darake is interrupted by RNA interference, brains appear in all body regions (Cebrià et al. 2002b). In species that are not able to regenerate a brain, nou-darake or a similar factor might be expressed not only in the brain region but also throughout the body, thus preventing the formation of a new brain even if the old brain was lost.
Fig. 6Immunocytochemical stainings of the nervous system of M. lignano. Confocal images. a GYIRFamidergic immunoreactivity. Clusters of immunoreactive cells are found lateral to the pharynx and in front of the eyes. b, c Serotonergic immunoreactivity in the head region. b Full stack of confocal images. Note the stained cell clusters lateral to the pharynx and in front of the eyes. c Subset of image stack in b. In this focal plane, stained cells anterior to the eyes become apparent. Arrowheads denote the level of the eyes
In M. hystricinum marinum and M. pusillum, large clusters of serotonergic cells have been found besides the posterior part of the pharynx (Ladurner et al. 1997). These results were corroborated for M. lignano by staining of the serotonergic and the GYIRFamidergic nervous system (Fig. 6). Amputees cut in the middle of the pharynx lose this cluster of nerve cells, as well as a large part of the pharynx, which is known to be a critical structure for regeneration in some triclads (Brøndsted 1969) and polyclads (Ishida 1998). Ladurner et al. (1997) held that the distribution of serotonergic nerve cells is correlated with the regeneration capacity. In species where serotonergic neurons are restricted to the head area, the posterior part of the animals cannot regenerate the head. In the asexually reproducing species M. lineare and S. leucops, serotonergic nerve cells are found over the whole length of the main nerve cords.
The inability of M. lignano to regenerate eyes after transversal amputation or oblique amputation between the eyes, is possibly linked with a severe brain damage caused by these amputations, and the loss of strongly immunoreactive cells in front of the eyes (Fig. 6c). In two cases of longitudinal incisions, lost eyes were regenerated or supernumerary eyes were built—but in these cases, the brain was only slightly injured. In a series of experiments, Lender (1950, 1951a–c) and Wolff and Lender (1950a,b) stress the importance of the head ganglia for the regeneration of eyes in P. nigra. If the brain was repeatedly excised or X-irradiated, no regeneration of eyes took place: an intact brain was obviously required to induce eye regeneration. These results were corroborated for Dugesia (now Schmidtea) lugubris by Török (1958). However, L. littoralis, L. saxicola, and P. litoricola can regenerate eyes even if the brain was excised (Morgan 1905; Olmsted 1922a).
Vannini (1965) observed in S. lugubris and P. nigra that the testes do not regenerate in the absence of a brain. Ghirardelli (1965) found that if a two-headed Dugesia was decapitated on one side, the testes on the same side degenerated. Contrastingly, the gonads in M. lignano are viable for an extended period of time after decapitation or oblique amputation between the eyes, and are only reduced if the pharynx is lost as well, causing malnourishment and starvation (Egger et al. 2006a,b). This may be an indication that the building of the gonads is not under the primary control of the brain in M. lignano, just like the tail plate can be built without the presence of a head. The regeneration of a tail in headless regenerates is possible in M. lignano as well as in triclads of the Dendrocoelum group (Brøndsted 1939, 1969), in the polyclads L. littoralis, L. saxicola, and P. litoricola (Morgan 1905; Olmsted 1922a), and also in the rhabdocoel Mesostoma (Fulinski 1922). Therefore, the head in all of these organisms is not required to control the regeneration of the tail. In Cestoplana and T. brocchi, the pharynx can regenerate in the absence of the brain (Child 1905c; von Levetzow 1939); in Cestoplana often a secondary pharynx is built in the presence of the old pharynx (Child 1905c). Also, the regulation of tissue breakdown (e.g., gonads) and body size reduction during starvation is possible in decapitated pieces, so the brain is not responsible for this regulation as well. Headless M. lignano with intact pharynx are able to feed, in accordance with the polyclads Notoplana acticola and Planocera gilchristi (Koopowitz et al. 1976). These animals are able to identify and devour food in the absence of a brain, while Enchiridium punctatum, a polyclad possessing a tubular instead of a plicate pharynx, cannot feed without the brain (Koopowitz et al. 1976). Similarly, some triclads, also equipped with a tubular pharynx, are not able to feed if the brain was amputated (Hyman 1951). Brainless M. ehrenbergi cannot feed but can move normally (Steinmann and Bresslau 1913), whereas brainless Phaenocera is reported to behave similarly to normal animals (Fulinski 1922). G. sphyrocephala, different to M. lignano, cannot adhere to the substrate after decerebration (Hagleithner 1946).
The main function of the brain in M. lignano is probably to be found in directed motion (e.g., light avoidance, predator avoidance; see also Koopowitz et al. 1976 and literature therein), satiation reflex (Koopowitz et al. 1976), mating behavior (Scharer et al. 2004), and chemical analyzation of the medium.
The totipotency of neoblasts
Pharynx-level regenerates of M. lignano have shown that the regeneration of gonads is possible after removal of all gonadal tissue. The viability of the regenerated gonads has been demonstrated by breeding experiments with fully regenerated animals. Besides the gonads, also the gut and the copulatory organs have been rebuilt de novo in pharynx-level regenerates (Egger et al. 2006a,b). In similar experiments, Morgan (1902) removed all gonads in Schmidtea lugubris, and the animals regenerated to fully functional sexual organisms. It is still possible that the remaining neoblasts in the amputee are only pluripotent and not totipotent, different types of neoblasts being competent for different (groups of) tissues. A convincing experiment to prove the totipotency of neoblasts would be the injection of a single neoblast into a lethally X-irradiated animal that is to be amputated some weeks after the injection. In case the animal survives, and is able to restore all removed organs, then either redifferentiation of cells would have taken place, or (at least some) adult neoblasts would be totipotent. A similar experiment, with the injection of 20,000–24,000 cells enriched in neoblasts (30–88% neoblasts) into X-irradiated S. mediterranea, was performed by Baguñà et al. (1989). This experiment was not replicated in the literature, but the results made highly probable that neoblasts as a whole are totipotent in routine cell turnover, leaving open the questions if this holds also true for regeneration and if this totipotency applies to one single cell type or to several different types of stem cells.
Are neoblasts in species with limited regeneration potential not totipotent, or are they just not receiving the right stimulus or guidance for regeneration? Supporting the latter notion is the ability of Macrostomum to duplicate heads after longitudinal incision (Fig. 2d and Egger et al. 2006a,b), while decapitated animals were never observed to regenerate a head (Egger et al. 2006a,b). In this species, it seems that stimuli from the brain are required to duplicate or regenerate the brain. Is there a specific subset of predetermined “brain” neoblasts in species that cannot regenerate a brain? These neoblasts could be located in the vicinity of the brain and therefore lost by amputation of the brain. Brain excision experiments in polyclads, where only the encapsuled brain was removed (von Levetzow 1939; Koopowitz et al. 1976), do not support the existence of such brain-specific neoblasts, as these animals were not able to regenerate the brain. Also, amputation experiments in M. lignano, where only brain and the neoblast-free rostrum were amputated, showed no brain regeneration (Egger et al. 2006a,b). While neoblasts are required for regeneration in general, they do not determine whether a species is able to regenerate a specific organ such as the head, as was found with grafting experiments in triclads (Brøndsted 1969).
Can a single neoblast from an adult develop to a whole organism in an adequate environment, just like a zygote? For the parasitic flatworm Taenia crassiceps, it was shown that single cells of trypsinated cysticerci are able to produce complete cysticerci in the mouse host (Toledo et al. 1997). So in these cestodes, a single totipotent cell, other than a zygote, is able to recover a whole animal. No such experiment is known for free-living flatworms. Besides the difficulty in providing a suitable culture medium, distinct differences between a single neoblast and a zygote are yolk content or surrounding yolk cells and maternal determinants that are present in eggs, but not in neoblasts. Do (all or some) neoblasts have embryonic characteristics, as some authors held (e.g., Keller 1894)? When and where do neoblasts origin in the embryo? Polyclads with their clear and traceable embryonic development appear to be suitable flatworms for investigating these questions.
Heteromorphoses
Hetermorphoses, i.e., the occurrence of supernumerary heads or tails, in species that cannot regenerate an amputated head, have shown that the regeneration potential of neoblasts is not to be equated with the regeneration capacity of the species. Heteromorphoses are also a placative warning sign for the dangers coming with the ability to regenerate (all) organs. Last but not the least, hetermorphoses in free-living flatworms show that the duplication of axes is not only possible during embryonic development (Fig. 2b, siamese twins), but also in adults, even after comparably minor surgery. The M. lignano specimen depicted in Fig. 2c,d shows that a longitudinal incision in the anterior part of the animal can establish a second anteroposterior body axis parallel to the original axis. The new anteroposterior axis reaches from the rostrum tip to the caudal tip and duplicates all organs including head, pharynx, female genital opening, and male copulatory organ. Even the breadth of the animal approximates that of two single specimens. Over time, however, the animal degenerated in both halves losing all eyes, and was not able to recover to a viable state (Egger et al. 2006a,b). A decreased viability in animals with heteromorphoses was also observed in Macrostomumappendiculatum and Bothrioplana bohemica (Sekera 1911), as well as in S. leucops (Ruhl 1927b).
In triclads, duplicated or even multiple heads (e.g., 10-headed D. lacteum, Lus 1924) and tails have been generated and studied by a great number of researchers (see Brøndsted 1969). Randolph (1897) made longitudinal and lateral incisions in Planaria maculata [today: Dugesia (Girardia) tigrina] leading to additional eyes and pharynges. It was found that when a duplicated head or tail was amputated close enough to the second main axis, regeneration was inhibited by the presence of the remaining head or tail (Rand and Mildred 1926). In the polyclad Leptoplana, double-heads and double-tails were experimentally produced by longitudinal incision (Child 1905a). With the same method, tails, but not heads, could be duplicated in the rhabdocoel Mesostoma (Fulinski 1922) and in the acoel P. caudatus (Keil 1929). Constrastingly, heads, but not tails could be duplicated by incision in the catenulid S. leucops (Ruhl 1927b).
Another type of heteromorphoses is created by provoking a body axis reversed to the original body axis. This was found in M. lignano after longitudinal incision in the head, that induced the creation of a tail plate appendage in the anterior body region (Egger et al. 2006a,b). Agata et al. (2003) suggested that one of the main functions of a blastema in triclads is producing positional signals and inducing intercalary regeneration between stump and blastema. An incision may cause the building of a very small blastema at the wound site, which provides anterior or posterior positional signals. Depending on the signal the blastema produces, either a head or a tail plate is subsequently formed.
Incisions may also have a long-range effect, as was observed by Randolph (1897) in triclads, where outgrowths appeared at body regions not affected by incision. A similar phenomenon as in M. lignano was observed by Steinböck (1967) in H. giselae, where an animal was divided into 10 pieces, one of which developed a tail-like structure at the anterior end besides the head. Just as was observed in M. lignano (Egger et al. 2006a,b), the anterior tail in H. giselae was gradually moving along the side of the animal towards the posterior end, when after 36 days the additional tail was located at the posterior tip of the animal.
Conclusion
A pronounced regeneration capacity and the existence of possibly totipotent stem cells in adults are two key features of many flatworms not found in this combination in any other bilaterian taxa. It could be shown that regeneration follows similar principles in many different taxa of free-living flatworms, even if the regeneration capacity can vastly differ. Such comparisons make it possible to recognize unique patterns, and also to form general ideas how, why, and when regeneration works. For this reason, it appears to be valuable to establish several flatworm model organisms from various taxa, to address old and upcoming questions regarding regeneration and the neoblast stem cell system. One of these comparably new model flatworms is M. lignano, a small but versatile worm that facilitates regeneration studies on the cellular and molecular level.

rhabditophora planarian catenulida acoela platyhelminthes


Treatment of patients with gastro-entero-pancreatic (GEP) tumours with the novel radiolabelled somatostatin analogue [177Lu-DOTA0,Tyr3]octreotate

Medical treatment and chemotherapy are seldom successful in achieving objective tumour reduction in patients with metastatic neuroendocrine tumours. Treatment with the radiolabelled somatostatin analogue [90Y-DOTA0,Tyr3]octreotide may result in partial remissions in 10–25% of patients. The newer analogue [DOTA0,Tyr3]octreotate (octreotate) has a ninefold higher affinity for the somatostatin receptor subtype 2 as compared with [DOTA0,Tyr3]octreotide. Also, labelled with the beta- and gamma-emitting radionuclide 177Lu, it has proved very successful in achieving tumour regression in animal models. The effects of 177Lu-octreotate therapy were studied in 35 patients with neuroendocrine gastro-entero-pancreatic (GEP) tumours who underwent follow-up for 3–6 months after receiving their final dose. Patients were treated with doses of 100, 150 or 200 mCi 177Lu-octreotate, to a final cumulative dose of 600–800 mCi, with treatment intervals of 6–9 weeks. Nausea and vomiting within the first 24 h after administration were present in 30% and 14% of the administrations, respectively. WHO toxicity grade 3 anaemia, leucocytopenia and thrombocytopenia occurred after 0%, 1% and 1% of the administrations, respectively. Serum creatinine and creatinine clearance did not change significantly. The effects of the therapy on tumour size were evaluable in 34 patients. Three months after the final administration, complete remission was found in one patient (3%), partial remission in 12 (35%), stable disease in 14 (41%) and progressive disease in seven (21%), including three patients who died during the treatment period. Tumour response was positively correlated with a high uptake on the octreoscan, limited hepatic tumour mass and a high Karnofsky Performance Score. Because of the limited efficacy of alternative therapies, many physicians currently adopt an expectant attitude when dealing with patients with metastatic GEP tumours. However, in view of the high success rate of therapy with 177Lu-octreotate and the absence of serious side-effects, we advocate its use in patients with GEP tumours without waiting for tumour progression.
Introduction
Neuroendocrine gastro-entero-pancreatic (GEP) tumours, which comprise pancreatic islet cell tumours, non-functioning neuroendocrine pancreatic tumours and carcinoids, are usually slow growing. If the tumour is localized, the therapy of choice is surgery. When a metastatic tumour causes a syndrome by hormonal overproduction (i.e. carcinoid syndrome, hypergastrinaemia), treatment with somatostatin analogues results in symptomatic relief in most cases. In terms of objective tumour reduction (complete and partial remission), however, treatment with somatostatin analogues is seldom successful, whether or not it is given in combination with interferon-α [1, 2, 3].
A new development in cytoreductive therapy for GEP tumours is the use of radiolabelled somatostatin analogues. Initial studies with high doses of [111In-DTPA0]octreotide (111In-octreotide; Octreoscan) in patients with metastatic neuroendocrine tumours were encouraging, although partial remissions were exceptional [4, 5]. However, 111In-coupled peptides are not ideal for peptide receptor radionuclide radiotherapy (PRRT) because of the small particle range and therefore short tissue penetration. Therefore, another radiolabelled somatostatin analogue, [90Y-DOTA0,Tyr3]octreotide, was developed. Using this compound, partial remissions have been reported in 10–25% of patients with neuroendocrine tumours [6, 7, 8].
Recently, it was reported that the somatostatin analogue [DOTA0,Tyr3]octreotate has a ninefold higher affinity for the somatostatin receptor subtype 2 as compared with [DOTA0,Tyr3]octreotide [9]. Also, labelled with the beta- and gamma-emitting radionuclide 177Lu, this compound was shown to be very successful in achieving tumour regression and animal survival in a rat model [10]. In a comparison in patients, we found that the uptake of radioactivity, expressed as a percentage of the injected dose of [177Lu-DOTA0,Tyr3]octreotate (177Lu-octreotate), was comparable to that after 111In-octreotide for kidneys, spleen and liver, but was three- to fourfold higher for four of five tumours [11].We concluded that 177Lu-octreotate potentially represents an important improvement because of (a) the higher absorbed doses that can be delivered to most tumours with about equal doses to potentially dose-limiting organs and (b) the lower tissue penetration range of 177Lu as compared with 90Y, which may be especially important for small tumours.
In this study we present the first data on the side-effects as well as the antitumoral effects of 177Lu-octreotate therapy in 35 patients with GEP tumours, who had a follow-up of 3–6 months after receiving their final dose.
Materials and methods
Patients Thirty-five patients with GEP tumours were studied. All patients had tumour uptake on the octreoscan preceding the therapy that was at least as high as the uptake in the normal liver tissue. None of the patients had received prior treatment with other radiolabelled somatostatin analogues. Eight patients used Sandostatin s.c.; this medication was discontinued from 1 day before to 1 day after the treatment. Prerequisites for treatment were: Hb ≥6 mmol/l, WBC ≥2×109/l, platelets ≥80×109/l, creatinine ≤150 μmol/l and Karnofsky Performance Score ≥50.
All patients gave written informed consent to participate in the study, which was approved by the medical ethical committee of the hospital.
Methods [DOTA0,Tyr3]octreotate was obtained from Mallinckrodt, St Louis, Mo., USA. 177LuCl3 was obtained from NRG, Petten, the Netherlands and Missouri University Research Reactor (MURR), Missouri, Mo., USA, and was distributed by IDB, Baarle-Nassau, the Netherlands. 177Lu-octreotate was prepared as described previously [11].
Granisetron 3 mg was injected i.v. and an infusion of amino acids (lysine 2.5%, arginine 2.5% in 1 l 0.9% NaCl; 250 ml/h) was started 30 min before the administration of the radiopharmaceutical and lasted up to 3.5 h afterwards. The radiopharmaceutical was co-administered via a second pump system. The treatment doses of 100 mCi were injected in 20 min and those of 150 and 200 mCi were injected in 30 min. The interval between treatments was 6–9 weeks. Patients were treated up to a cumulative dose of 750–800 mCi (27.8–29.6 GBq) (corresponding to a radiation dose to the bone marrow of 2 Gy) [11], unless dosimetric calculations indicated that the radiation dose to the kidneys would then exceed 23 Gy; in these cases the cumulative dose was reduced to 600–700 mCi.
Routine haematology, liver and kidney function tests, hormone measurements and serum tumour markers were measured 1 week prior to each therapy, as well as at follow-up visits. EORTC quality of life forms (QLQ-C30) [12] were filled out by the patients at each visit.
CT or MRI scanning was done within 3 months before the first therapy, and 6–8 weeks, 3 months and 6 months after the last treatment.
In vivo measurements. The tumours on the CT or MRI scans were measured and scored according to the WHO solid tumour response criteria. The uptake on the pretreatment octreoscans was scored visually on planar images on a 4-point scale: grade 1, lower than normal liver tissue uptake; grade 2, equal to normal liver tissue uptake; grade 3, greater than normal liver tissue uptake; grade 4, higher than normal spleen/kidney uptake.
Statistics Analysis of variance (ANOVA), paired t tests and chi-square tests were used. P values <0.05 were considered significant.
Results
The study population comprised 14 men and 21 women with a mean age of 54 years(range 19–78 years). Twelve had carcinoid tumour, 12 neuroendocrine pancreatic tumour, 8 neuroendocrine tumour of unknown origin and 3 gastrinoma. Twelve patients had been operated in the past, 1 had had radiotherapy, 3 had had chemotherapy and 14 had been treated with octreotide (Sandostatin). Sixteen of the 35 (46%) patients had documented progressive disease within 1 year before the start of the therapy. Cycle doses were 100 mCi in seven patients, 150 mCi in 14 and 200 mCi in the remaining 14. Higher dosages were not administered, since 200 mCi 177LuCl3 is typically bound to 180–300 μg [DOTA0,Tyr3]octreotate, and higher doses would result in a lower percentage tumour uptake owing to receptor saturation. In 30 patients, the final intended cumulative dose of 600–800 mCi was administered. Three of the five remaining patients had progressive disease and died before completing their treatment; the other two patients, who were both elderly, stopped their treatment after reaching a cumulative dose of 600 mCi because of the burden they felt the treatment to be.
Nausea and vomiting within the first 24 h after the administration were present in 30% and 14% of the administrations, respectively, and were independent of the administered dose. Some abdominal pain was noticed during 11% of the administrations, especially in patients with liver metastases. Increased, mild hair loss was present in 17 of the 35 patients; hair regrowth had occurred at follow-up 3 and 6 months after the final administration.
WHO toxicity grade 2 or 3 anaemia (Hb 4.95–6.2 or 4.0–4.9 mmol/l, respectively), leucocytopenia (WBC 2.0–2.9 or 1.0–1.9×109/l, respectively) and thrombocytopenia (platelets 50–74.9 or 25.0–49.9×109/l, respectively) occurred after 8% and 0%, 5% and 1%, and 3% and 1% of the administrations, respectively. Toxicity grade 2 or 3 leucocytopenia or thrombocytopenia occurred in two out of three (67%) patients who had had previous chemotherapy, as against seven out of 32 (22%) patients who had not. Mean haematological parameters rose again during the follow-up after the final administration. Serum creatinine, creatinine clearance and serum HbA1c did not change significantly (data not shown). In patients without thyroid hormone medication, serum TSH and fT4 levels did not change. In women, serum LH and FSH concentrations did not change significantly; in men, serum testosterone decreased and serum LH concentrations increased significantly. Also inhibin-B concentrations decreased and serum FSH levels increased significantly.
The effects of the therapy on tumour size were evaluable in 34 patients. Three months after the final administration (on average 9 months from the start of the treatment), a complete remission (CR), evaluated with CT scanning, MRI and somatostatin receptor imaging, was found in one patient (3%), partial remission (PR) in 12 (35%), stable disease (SD) in 14 (41%) and progressive disease (PD) in 7 (21%), including the three patients who died during the treatment period (Table 1) (Figs. 1, 2). Follow-up evaluation 6 months after the final therapy was available for 19 of the 34 patients. All seven patients who had PR after 3 months still had PR after 6 months; in 10 of the 12 patients with SD, the evaluation was unchanged, whereas one had a minimal response (MR) and one had PD.Table 1. Tumour responses
in 34 patients, 3 months after the final administration of 177Lu-octreotate. Three
patients with PD died before reaching their final doseTumour typeResponseTotalCRPRSDPDCarcinoid4 (33%)6 (50%)2 (17%)12NE pancreas1 (8%)1 (8%)7 (58%)3 (25%)12NE unknown origin4 (57%)1 (14%)2 (29%)7Gastrinoma3 (100%)3Total1 (3%)12 (35%)14 (41%)7 (21%)34Fig. 1.A–C  Planar scans of the abdomen, 3 days after the injection of 200 mCi 177Lu-octreotate in a patient with liver metastases of an operated neuroendocrine pancreatic tumour. A After the first treatment; B after the second treatment; C after the fourth treatment. Note the loss of intensity of uptake in the liver lesions (arrows in A). This sign virtually always indicates a tumour volume response. D, E CT scans of the same patient: before treatment (D) and 3 months after the last treatment (E). Tumour (arrows in D) is not demonstrated on E. Neither MRI nor octreoscan could demonstrate definite tumour deposits at that timeFig. 2. A, B MRI scans in a woman with hepatic metastases of a gastrinoma. Before treatment (A) she was inoperable because of the size and localization of the metastases. Three months after completion of her treatment with 800 mCi 177Lu-octreotate she had a PR (B). Note also that the hypertrophy of the gastric wall (arrow in A) had diminished. Concomitantly, serum concentrations of gastrin (closed dots) and chromogranin-A (open dots) had decreased markedly (C). At 6 months the MRI showed no further regression. The patient underwent left partial hepatectomy and the two right-sided lesions were drained and injected with alcohol. MRI 3 months after surgery showed further regression of the right-sided lesions
Tumour response (CR or PR) was significantly more frequent in patients whose tumours had a high uptake on the octreoscan [6/7 (86%) with grade 4 uptake vs. 7/27 (26%) with grade 2 or 3 uptake; chi-square test: P<0.05]. Five out of the seven patients (71%) with PD had hepatomegaly and diffuse liver metastases vs 6 out of 27 (22%) with CR, PR or SD (chi-square test: P<0.05). Tumour response was more frequent in patients with documented PD within 1 year before the start of the treatment (8/15; 53%) than in those without (5/19; 26%), although this difference was not statistically significant (P=0.11; chi-square test). Of the 15 patients with documented PD before the start of the treatment, 3 (20%) had SD at follow-up. Patients who had a Karnofsky Performance Score of less than 80 before the treatment more frequently had PD (5/8; 63%) than those who had higher scores (2/26; 8%) (P<0.01; chi-square test).
The serum tumour marker chromogranin-A was elevated in 29 patients. During the treatment and follow-up, there was a clear decrease in serum chromogranin-A concentrations in patients with PR or CR, whereas these concentrations were virtually unchanged in patients with SD, and initially showed an increase in patients with PD (Fig. 3).Fig. 3. Serum chromogranin-A concentrations during and after therapy in patients with PR, SD or PD. The reduction in the number of patients during the course of the follow-up was due to death or missing values. Note the logarithmic y-axis
The patient-assessed quality of life, according to the EORTC-QLQ30 questionnaire, was evaluated in 25 patients. Ten patients were excluded because of progressive disease/death (n=5) or missing forms during the follow-up period (n=5, foreign patients). Scores before the start of the treatment, after receiving 400–600 mCi 177Lu-octreotate and at follow-up 3 months after the final treatment were evaluated. There were no significant differences for functional scales or single symptom scales. The global health scale, on which patients were asked to assign marks regarding both their general health and their quality of life, and which ranged from 0 to 100, was judged as higher than 70 by nine patients (36%) before the start of the treatment, by 17 (68%) patients during the treatment and by 16 (64%) patients after the treatment (P<0.05; chi-square test).
Discussion
There are few treatment options for metastatic GEP tumours. The use of radiolabelled somatostatin analogues for tumour regression is a promising new development. With [90Y-DOTA0,Tyr3]octreotide, PR (or CR) has been reported in 10–25% of patients with neuroendocrine tumours [6, 7, 8]. In the present study we found objective tumour shrinkage in 38% of the patients, but PD before the start of the treatment was documented in only 46% of the patients. This last fact is important, because documented PD was present in more than 80% of patients in two of the reported series treated with [90Y-DOTA0,Tyr3]octreotide [6, 8]. As with chemotherapy, an objective tumour response after PRRT is more likely in patients with (fast) growing tumours. Indeed, in our series we also found a trend towards a more favourable treatment outcome in patients with documented PD before the start of the treatment. It may therefore be concluded that the percentage of patients with significant tumour shrinkage in our study might have been even higher had the percentage of patients with PD prior to treatment been comparable to that in studies with [90Y-DOTA0,Tyr3]octreotide. The side-effects of treatment with 177Lu-octreotate are few and mostly transient, with mild bone marrow depression as the most common finding. Neither renal nor pituitary function deteriorated in any of our patients. Other side-effects can be ascribed to the radiation dose to the testes in men. This dose leads to significantly lower serum testosterone and inhibin-B levels which in turn give rise to higher serum LH and FSH concentrations, thereby substantiating that the pituitary function is unimpaired.
The patient-assessed global health score improved in 30% of patients during the treatment and the follow-up period. This is an important finding which reflects the improvement in patient well-being and stresses the scarcity of side-effects as perceived by patients. The fact that other scores mainly addressing symptoms did not change significantly is likely due to the diversity of symptoms between patients, the use of Sandostatin by symptomatic patients and the small size of the patient group.
Many physicians adopt an expectant attitude when dealing with patients with metastatic GEP tumours owing to the low success rates of chemotherapy protocols. However, given that treatment with 177Lu-octreotate resulted in PR (or CR) in 38% of our patients, this attitude may be questioned. Although there was a (statistically non-significant) tendency towards more frequent tumour regression in patients who had documented PD before the start of the treatment, those of our patients who had an objective tumour response more frequently had a limited tumour load. This implies that waiting for tumour progression might place patients in a worse position, as PD during or after treatment was more frequent in patients with an extensive tumour load, especially in the liver. We therefore advocate treatment with177Lu-octreotate at an earlier stage of metastatic disease, when even CR may be possible. Another argument in favour of early treatment is that neuroendocrine tumours can dedifferentiate in the course of the disease, and lose their somatostatin receptors, making treatment with radiolabelled somatostatin analogues impossible. If serious side-effects of treatment with 177Lu-octreotate remain absent during longer patient follow-up, such treatment could also be considered in an adjuvant setting in patients with neuroendocrine tumours who are operated on with curative intent.
Lastly, as it has been shown in animal experiments that 90Y-labelled somatostatin analogues are more effective for larger tumours and 177Lu-labelled somatostatin analogues are more effective for smaller tumours [13, 14], combination therapy with 90Y-labelled and 177Lu-labelled octreotate may be tried in the near future. Such therapy might yield even better results than treatment with either of the radionuclides alone [15].

octreotate
somatostatin receptor
gep tumours
radionuclide therapy
lutetium-177