the neoplastic chondrogenesis of two rare cartilaginous tumours
Salvatore, R.
Citation
Salvatore, R. (2010, June 22). Chondroblastoma and chondromyxoid fibroma:
disentangling the neoplastic chondrogenesis of two rare cartilaginous tumours.
Retrieved from https://hdl.handle.net/1887/15712
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Chapter 1
General introduction
I. NON-NEOPLASTIC CHONDROGENESIS II. NEOPLASTIC CHONDROGENESIS
III. DIAGNOSIS AND THERAPY OF CHONDROBLASTOMA AND CHONDROMYXOID FIBROMA
IV. SCOPE OF THE STUDY AND OUTLINE OF THE THESIS
General introduction
The process regulating cartilage formation in tumours, referred to as neoplastic chondrogenesis, is poorly understood. In order to gain insight into this process, two rare benign cartilaginous tumours: chondroblastoma and chondromyxoid fibroma have been chosen for study. Despite their rare occurrence, and therefore the inherent difficulty in collecting a large number of observations, these two tumours provide a unique opportunity for this research. Since their morphology summarizes the spectrum of cartilaginous tumour differentiation,1 they represent a sort of in vivo model. The subject is introduced by a description of the mechanisms regulating normal cartilage, is followed by a discussion on the current knowledge of neoplastic chondrogenesis and ends with an outline of the thesis.
I Non-neoplastic cartilage
I.a. Features: composition, functions and location
Cartilage is a specialized connective tissue made up of a specific extracellular matrix and a unique cell type, i.e. the chondrocyte2, it is a paucicellular tissue in which cells constitute 2-5% of the tissue volume. Chondrocytes are located in lacunae surrounded by an intercellular matrix consisting of 72-75% water, 16%
collagens, 10% proteoglycans, 1.6% other glycoproteins and 0.5% minerals. Col- lagen type II is the major dry-weight component of cartilage. Other types of col- lagen, mainly found in cartilage, are collagen type IX, X and XI. Proteoglycans are made up of a core protein to which glycosaminoglycan (GAG) chains are attached .3 GAGs in cartilage consist of chondroitin sulphate, heparan sulphate, dermatan sulphate and hyaluronic acid. The most represented proteoglycan in cartilage is aggrecan. The distribution of GAGs is not uniform, with the highest concentration around the lacunae, or the so-called territorial matrix, and lower levels in the interterritorial matrix, i.e. the matrix distant from the cells.2
Blood and lymphatic vessels are absent in cartilaginous tissue and so nourishment and oxygen have to diffuse from nearby capillaries. Cartilage, bone and dentin are supporting connective tissues since their function is to give strong support to different organs. Besides biochemical composition, one important difference bet- ween cartilage and the other supporting connective tissues is the way the newly formed matrix is deposited. Bone matrix is only be secreted from the outside, through so-called appositional growth. Cartilage matrix can instead be secreted either from the outside, i.e. from the perichondrium, or from the inside, from the chondrocytes embedded in the tissue, i.e. through so-called interstitial growth.
There are three known types of cartilage characterized by the composition of the extracellular matrix: hyaline, elastic and fibrous cartilage. Hyaline cartilage is characterized by an homogeneous amorphous matrix. Macroscopically it has a glistening glassy appearance from which its name derives: the Greek term hyalos meaning glass. Hyaline cartilage is found lining articular surfaces and forms the backbone of the nasal septum, larynx, tracheal rings, costal cartilages and growth plate. Elastic cartilage contains elastin fibers and lamellae. Elastic cartilage is found in the ear and epiglottis. Fibrocartilage is rich in large bundles of collagen type I. Fibrocartilage is found in intervertebral discs, the pubic symphysis, the menisci of joints, and often where tendons and ligaments are joined to bones.
Hyaline cartilage has excellent weight-bearing performance and shock absorption
Fig.1: scheme recapitulating different steps in chondrogenesis, from left to right in chronological order we recognize migration, condensation and differentiation, for the explanation see the text.
in joints. Furthermore its ability to maintain these properties during interstitial growth is crucial for its role as bone template in skeletal development (enchondral ossification).
I.b. Developmental Chondrogenesis
Hyaline cartilage forms the template of the vertebral column, the pelvis and the extremity bones. This template is formed during embryogenesis and gradually replaced by bone, through a process called enchondral ossification. Postnatally it persists, as a growth plate and is responsible for the elongation of long bones and consequently for final height.4 Development of the cartilaginous template occurs in three consecutive steps: 1. migration of cells, 2. mesenchymal condensation and 3. differentiation (Fig.1).5
In limb formation, mesenchymal cells, covered by a layer of ectoderm, bud from the lateral plate mesoderm and become closely packed. They undergo cell-cell interactions and start to differentiate towards chondrocytes (pre-cartilage
condensation). The commitment towards cartilage formation is reflected in a synthetic switch from type I to type II collagen and by the production of sulphated proteoglycans. Cells subsequently become surrounded by a newly deposited extracellular matrix (interstitial growth). In the centre of this cartilaginous model (the so-called "anlage"), chondrocytes grow in size, start synthesizing type X collagen and differentiate into hypertrophic chondrocytes. In the mid-region of the model, the cartilage calcifies and periosteal capillaries grow into the calcified cartilage of the bone model, supplying its interior. These blood vessels, together with osteogenic cells, form the periosteal bud. The bone tissue replaces the calcified cartilage which is further remodeled by chondroclasts. The bony center expands due to ongoing bony replacement of the hypertrophic cartilage. Eventually the two cartilage areas, located at the extremities of the growing bony centre, become further and further apart.
The cartilage in the extremities - the growth plate - is responsible for the longitudinal growth of the bone. Structurally it is divided into three different zones: 1. the resting zone, where cells are spherical and little or no division is present, 2. the proliferating zone, where cells express type II collagen, producing a column of flattened cells and 3. the hypertrophic zone where cells become larger and synthesize type X collagen and the cartilage ECM is calcified and replaced by bone (Fig.2).
The time required for one cell to pass from the upper proliferative zone to the lowest part of the hypertrophic zone is around 3 days. As sexual maturity is reached, the growth plate is replaced by bone and growth ceases.
Chondrogenesis is a dynamic process characterized by gradual change in shape, proliferation activity and pattern of expression. This multifaceted process requires fine tuning, requiring several signalling molecules, acting at both short (paracrine/
autocrine secretion) and long range (endocrine secretion). Cell-cell interactions and cell-ECM interactions are also crucial for this process. Impairment of any of these mechanisms results in skeletal deformities and growth disorders.
I.c. Signalling regulating the growth plate
Most of our knowledge on signalling molecules that regulate chondrogenesis comes from studies on growth disorders. Several distinct disorders, involving different pathways and molecules, cause short and tall stature. These disorders can be divided into three subgroups: primary (defect in bone/cartilage), secondary (de- fect outside bone/cartilage), or idiopathic.6 Altered longitudinal bone growth occurs quite frequently and investigation of its causes furthers our understanding of growth plate regulation.
I.c.i. Systemic regulation
The main systemic hormones regulating longitudinal bone growth in childhood are growth hormone (GH) and insulin growth factor like I and II (IGF-I and IGF-II), thyroid hormone (T3 and T4) and glucocorticoids (GC), while sex steroids (androgens and estrogens) play a major role during puberty.
Postnatally, GH and IGF-I and IGF-II are the main players of the so-called hypothalamus-pituitary-growth plate axis. GH secretion from the pituitary is stimulated by GHRH and inhibited by somatostatin, both released by the hypothalamus. GH acts on its target tissue either directly or through two intermediates: IGF-I and IGF-II. Initially, they were called insulin-like growth factors due to their ability to stimulate glucose uptake; later their structure was also shown to be similar to pro-insulin.7;8 GH may either stimulate proliferation of resting
Fig.2 Growth plate zones organization: on the left a histological picture from human growth plate (10x original magnification), on the right schematic view of the different type of cells composing the growth plate
chondrocytes directly, upon binding to its receptor GHR9, or indirectly through the release of IGF-I. Circulating IGF-I is released by the liver; paracrine /autocrine IGF-I secretion is also present in the growth plate and is most responsible for promoting final chondrocyte differentiation. Furthermore IGF-II is produced by proliferative chondrocytes with a putative mitogenic effect. In sum, GH and IGFs have distinct, complementary effects on growth plate regulation both systemically and locally.10 Hypothyroidism in children, the clinical condition characterized by either low-level circulating T3 and T4 or impairment of their receptor, causes developmental bone abnormalities and growth arrest, while thyrotoxicosis accelerates growth rate and advances bone age.6;11 This assumes that thyroid hormones are important for normal bone maturation.12;13 Thyroid hormones may act directly on the growth plate and thyroid hormone receptors are indeed pre- sent in proliferating and resting zone chondrocytes in growth plates.12;13 Furthermore thyroid deiodinase, the enzyme regulating conversion of inactive T4 to T3, is also present in growth plates.12;13 In particular, T3 seems to stimulate hypertrophic differentiation of growth plate chondrocytes.6 The action of thyroid hormones on the growth plate is also indirect, since they can both influence GH release and increase IGF-I m-RNA in the growth plate.6
In young people, high glucocorticoid levels, be they endogenous (i.e. Cushing's syndrome) or due to prolonged therapeutic administration (i.e. juvenile rheumatoid arthritis, chronic asthma, etc.), cause growth retardation. Glucocorticoid receptors are present in the hypertrophic cells of the growth plate.6;14;15 Glucocorticoids most likely induce growth arrest and apoptosis of the hypertrophic chondrocytes 16. Glucocorticoids may also inhibit growth by interfering with either the GH-IGF- I pathway or the thyroid hormones.6;17-21
Sex steroids accelerate longitudinal growth in puberty. Specifically, the level of circulating hormones is hugely increased on reaching sexual maturation. Specific receptors for such hormones are found in the growth plate.22 Furthermore, administration of non-aromatizable androgens (that cannot be converted to estrogen by the aromatase enzyme), has been found to accelerate longitudinal growth in boys with constitutionally delayed growth.23 Androgens most likely directly influence growth plate processes and thus determine distinct skeletal regulation in males and females.6
I.c.ii Local regulation
Local regulation is mainly, but not exclusively, mediated by morphogens, which are molecules regulating the development and final architecture of the various tissues within a given organism. Another important feature of morphogens is that they work by diffusion along concentration gradients. In other words, specific cell types produce the morphogen which is then diffused to distant target cells. This section reviews the role of cartilage formation by morphogens - as Indian hedgehog (IHH), bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF- β) and Wnt proteins - and other growth factor and signalling molecules - as PTHLH and FGFs.
IHH binds to its receptor patched-1 (PTCH1), which releases smoothened protein (SMO), which is in turn transported via intraflagellar particles to the tip of primary cilia (Fig.3).24 In this location it abrogates GLI repressive processing by binding the SUppressor of FUsed (SUFU)24. The activated form of GLI is then transported back to the cytoplasm and translocates to the nucleus where it finally exerts its transcription factor activity.24
Fig3: HH signaling requires functional intraflagellary transport in primary cilium, in the absence of HH, its receptor patched is sequestrating smoothened and GLI is in its repressive (GLI R) form, no specific transcription is induced (left panel), upon HH binding (right panel) smoothened is released and transported into the primary cilium where it inhibits SUFU and GLI is activated (GLIA) and transported back to the nucleus where it can activate transcription (adapted from Mans et al., 2008, Biochim Biophys Acta)
IHH in the growth plate is produced and released by prehypertrophic cells. Its effect is either direct or indirect via PTHLH induction which binds to its receptor PTHR1 and activates a different pathway.6;25-29 The downstream molecules specific to these pathways are antiapoptotic molecules, as bcl2 upregulated by PTHLH, and cell- cycle promoting molecules, as cyclin D1 and cyclin E1, upregulated by PTHLH and IHH, respectively.6;25-29 The final effect of IHH stimulation is to delay the progression of chondrocytes towards hypertrophic chondrocytes therefore delaying bone growth.6;25-29 A negative feedback loop occurs between prehypertrophic chondrocytes and proliferating chondrocytes (Fig.4).6;25-29 TGF-β and BMPs are part of the TGF-β superfamily.30;31 BMPs, originally identified for their ability to induce ectopic bone formation 32, are widely expressed in cartilage and are instrumental for growth and differentiation. TGF-β/BMPs act both directly and indirectly via induction of other signaling molecules, i.e. PTHLH, or even as an intermediary of IHH signaling. In general TGF-β promotes chondrocyte proliferation and inhibits further differentiation towards hypertrophic chondrocytes. This may be either a direct effect or via induction of PTHLH.28;33;34 In addition, TGF-β2 seems to act as a signalling relay between IHH and PTHLH.33
In the developing vertebrate limb, the Wnt pathway, be it canonical or non- canonical, is also crucial for skeletogenesis (Fig. 5A).35-38 The canonical Wnt pathway controls gene expression by stabilizing β-catenin (Fig. 5A).35-38 The non-canonical Wnt pathway activates Ca2+-flux, JNK, and small and heterotrimeric G-proteins (Fig. 5A).35-38
Wnt signaling is operated by different Wnt homologues. Each molecule has several target cells with distinct and often opposite effects. Specifically, Wnt5a is released
Fig.4: A scheme summarizing the cross-talk among IHH, PTHLH (previously known as PTHrP), BMP, and FGF signaling to regulate growth plate organization. (Adapted from van der Eerden et al. 2003)
in the growth plate by proliferating chondrocytes, inhibiting transition from resting chondrocytes to proliferating chondrocytes. This inhibits SOX9 expression, but promotes chondrocyte hypertrophy by inhibiting cyclinD1 expression and inducing high levels of p130 expression (Fig.5B).38 Conversely, Wnt5b is released by the prehypertrophic chondrocytes and inhibits chondrocyte hypertrophy via cyclinD1 induction and p130 reduced expression.38 Lrp5 is expressed in the periosteum and trabecular bone and plays a critical role in determining bone mass by promoting osteoblast proliferation and terminal differentiation.38(Fig.5B) As regards FGF signaling in the growth plate, the main ligand FGF18 is released from the perichondrium and binds to different receptors in the different zones. In particular, FGFR3 is expressed in proliferating chondrocytes, while FGFR1 is expressed in prehypertrophic and hypertrophic chondrocytes.6;39 The activation of FGFR3 negatively regulates proliferation, either directly or by regulating IHH/PTHLH pathway expression.39-41 In prehypertrophic chondrocytes, FGFR1 activation may promote differentiation.6;39
All the above pathways require diffusion of the signalling molecules, notably the morphogen pathways, and efficient binding to the receptor. As elegantly shown in Drosophila modeling, efficient diffusion needs well-formed heparin sulphate proteoglycans.42-45 Heparan sulphate proteoglycan chain elongation in human is mediated by EXT genes. Remarkably these are mutated in osteochondroma, a benign cartilaginous tumour.1 It is therefore plausible that deranged signaling, in terms of altered diffusions and receptor binding, is instrumental in neoplastic cartilage formation.
I.d In vitro Chondrogenesis
In vitro models have further elucidated some characteristic features of
Fig.5: A) Scheme summarizing canonical and non/canonical WNT pathway, B) Wnt 5a and Wnt 5b have antithetic effect in regulating chondrocytes differentiation in growth plate (Adapted from Yang et al. 2003)
chondrocytes. Chondrocytes cultured at low density change their shape from round to elongated, to become spindle-like. This morphological switch is also reflected in a change in the pattern of expression: type II collagen and aggrecan are drastically reduced, while type I collagen is increased. This phenomenon is known as dedifferentiation - a term reflecting the loss of a differentiated phenotype to acquire a more primitive/ undifferentiated one. Dedifferentiation is reversible under the proper conditions, but chondrocyte cells definitively lose their cartilage-forming potential after long culturing, even if heavily pushed towards chondrogenic commitment.
Conditions promoting chondrogenesis include: loss of substrate interaction and induction of cell-cell contact in a three-dimensional constraint and presence of specific growth factors (i.e. TGF-β and BMP6). In these conditions dedifferentiated chondrocytes or mesenchymal stem cells begin to form cartilage. This process morphologically recapitulates cartilage development (Fig. 6). The cells become closely packed, as in condensation, and start producing a cartilaginous extracellular matrix, which is deposited interstitially. One important observation from the in vitro experiences is that dedifferentiated chondrocytes cultured in flasks and chondrocytes cultivated in 3D conditions respond differently to the same stimulation .46 On TGF-β stimulation, for instance, flask-cultivated chondrocytes increase their
proliferative activity and become dedifferentiated faster. In 3D pellet culturing, TGF-β stimulation promotes cartilaginous differentiation, presumably reducing proliferation.46 It is plausible to speculate that such opposing responses may be driven either by differences in the state of the cytoskeleton or by epigenetic modifications.47;48
II Neoplastic chondrogenesis
Cartilaginous tumours of bone have heterogeneous intratumoural features (the histological spectrum of benign cartilaginous bone tumours is reviewed in Chapter 2). For example, distinct morphological features are evident within the same tumour in terms of cell morphology and extracellular matrix appearance. It is possible to recognize areas that resemble and others that are morphologically distant from normal cartilage. The fact that this also occurs in benign lesions allows for some speculation. Most likely, the micro-environment leads to diversity in chondroid matrix formation. However, some questions remain. How plastic are neoplastic cells? What is the origin of cartilaginous tumours?
A sum of observations suggest that cartilaginous tumours may originate in mesenchymal stem cells.49 Almost no neoplastic transformation of normal cartilage has ever been described.49 Mesenchymal stem cells are present throughout the entire human life span and bone marrow is a rich reservoir of these cells. In response to damage, as during fracture repair, they are recruited and committed towards chondrogenesis. In addition in vitro experiments have largely proven that it is possible, under specific conditions (a sort of regulated artificial micro- environment), to induce de novo cartilage formation from mesenchymal stem cells. Hence the most likely hypothesis for the genesis of cartilaginous tumours is that they originate from mesenchymal stem cells. Furthermore the extreme plasticity of a putative mesenchymal staminal precursor, as intrinsic in the definition Fig.6: Scheme recapitulating different steps of in vitro chondrogenesis, from left to right:
cells grown in flask dedifferentiates and get an elongated shape, upon 3D pellet culturing cells atrt to get cell-cells interaction and finally diffentiate to cartilage with production of a specifc extracellular matrix. (Adapted from Romeo S et al. 2007)
of multi-potent cells, could contribute to the broad intra-tumoural morphological heterogeneity. The specific microenvironment, i.e. the physical and biochemical signalling network, determines a specific phenotype and pattern of expression.
The role of signalling pathways acting in normal cartilage formation has been investigated for central and peripheral chondrosarcoma and their possible benign precursor enchondroma and osteochondroma, respectively.50-53
In particular, IHH is active in enchondromas and central chondrosarcomas and pharmacological inhibition of this pathway reduces cell growth in chondrosarcomas .52;53 In peripheral cartilaginous tumours, IHH is active in osteochondromas but absent in malignant peripheral chondrosarcomas.50 Conversely, PTHLH is absent in osteochondromas but active in malignant peripheral chondrosarcomas. A higher expression of PTHLH and PTHR1 is found in higher grades peripheral chondrosarcomas. This observation implies that the IHH/PTHLH feedback loop normally found in the growth plate is not present in peripheral cartilaginous tumour.
In osteochondromas, IHH does not induce PTHLH and, vice versa, in malignant peripheral chondrosarcoma, PTHLH is not induced by IHH but most likely by TGF- β.33 PTHLH is indeed a known downstream molecule of TGF-β signaling and TGF-β signalling is upregulated in peripheral chondrosarcomas. In central tumours, PTHLH pathways are active in enchondromas and proportionally higher in high grade chondrosarcomas, with a trend remarkably similar to TGF-β, suggesting that this latter may also be responsible for high PTHLH expression in high grade central chondrosarcomas.54 -57 The role of WNT signalling is less clear. In peripheral tumours, canonical WNT signalling is active in OC and slowly disappears in chondrosarcoma, in proportion to the grade of the tumour. In central tumours it may play a role in malignant transformation: nuclear β catenin is lowly expressed in EC and highly expressed in low grade chondrosarcomas. However this signalling is not required for further malignant progression, as shown by its reduction in high grade central chondrosarcomas.50;53;58
Taken together, these data reinforce the concept that , despite their morphological similarities, peripheral and central cartilaginous tumours are distinct entities genetically and in terms of cell signalling.50;53;58;59 Furthermore, some pathways, such as IHH and WNT, seem to be important in the early events of tumour formation, while TGF-β and PTHLH, for example, seem more important for malignant progression. In agreement with this, Ho et al. 200960 showed, in conditional knock out mouse models, that overexpression of only GLI2 results in persistence of cartilage growth plate remnants and that an additional abrogation of p53 is needed for chondrosarcoma formation. A multistep model is hypothesized in which impaired signalling is required for benign cartilaginous tumour pathogenesis with additional genetic events leading to malignant transformation.61 Important additional events identified for malignant progression of cartilaginous tumours involve cell cycle/
apoptosis regulation. For instance, occurrence of TP53 mutations62;63, p16 downregulation64-67, HDM2 or CDK468 overexpression are increasingly more represented in high grade chondrosarcomas than in low grade chondrosarcomas.
While the multistep model is certainly useful for our understanding, it does not explain why not all benign cartilaginous tumours evolve to malignant cartilaginous tumours (i.e. chondroblastomas and chondromyxoid fibromas are not convincingly reported to progress to malignancy).69 Conversely not all malignant cartilaginous tumours originate from a benign precursor (i.e. no benign precursor is reported for mesenchymal chondrosarcomas or clear cell chondrosarcomas).69
Fig.7 scheme summarizing the signaling pathways in peripheral (left side) and central cartilaginous tumours (right side). IHH is active in osteochondromas (OC) and decreases in peripheral chondrosarcomas (CS) proportionally to the tumour grade. A steady level is observed in central cartilaginous tumours including enchondroma (EC) and conventional central chondrosarcomas (grade I to grade III). Active canonical WNT signalling is decreasing with grade in peripheral cartilaginous tumours while it seems to play a role in the progression from benign to malignant in the central cartilaginous tumours. TGF-β is increasing upon grade in both central and peripheral cartilaginous tumours. PTHLH is absent in OC and increasing with grade in peripheral CS, in central cartilaginous tumours a trend remarkably similar to the one of TGFB is observed. (Adapted from Schrage et al 2009)
III Diagnosis and treatment of chondromyxoid fibroma and chondroblastoma
The diagnosis of chondroblastoma and chondromyxoid fibroma is based on integrated evaluation of pathological, clinical and radiographic features. A correct diagnosis is crucial for appropriate treatment. Chapter 2 reviews the clinical, pathological and radiological features characterizing these two lesions and the possible differential diagnosis.
Chondroblastoma and chondromyxoid fibroma are two benign lesions prompting treatment as little invasive as possible: meticulous curettage with bone grafting is the preferred approach, sometimes combined with some form of local adjuvant treatment.70;71 However, this approach is hindered by a recurrence rate of 14- 18% and 15% in chondroblastoma and chondromyxoid fibromas, respectively.70;71 Particularly "difficult" locations for performing meticulous curettage (i.e. the skull for chondroblastomas and short tubular bones for chondromyxoid fibromas) are thwarted by high recurrence rates.70;71 The co-occurence of an aneurysmal bone cyst in chondroblastoma is also associated with a higher recurrence rate.70 Particular caution is required in chondroblastoma occurring in the epiphyses of young patients. Since the lesion is located close to the growth plate of a still growing individual, it is crucial to have as little effect as less as possible on this structure in order to avoid impairing bone elongation.
The importance of achieving a correct diagnosis of chondromyxoid fibroma and chondroblastoma is to avoid over-treatment. As concerns misdiagnosis of malignant lesions (i.e. clear cell chondrosarcomas instead of chondroblastomas or high grade central chondrosarcomas instead of chondromyxoid fibromas) patients may undergo unnecessary mutilating surgery. Finally, isolated reports in the literature warn of the existence of osteosarcomatous lesions simulating both chondroblastomas and chondromyxoid fibromas.72;73
IV Scope of the study and outline of the thesis
The most relevant literature on benign cartilaginous tumours specifically addressing chondroblastomas and chondromyxoid fibromas is reviewed in Chapter 2. Salient clinical, radiological and pathological features are examined and combined with genetics. In particular, osteochondromas and enchondromas occur more frequently than chondroblastomas and chondromyxoid fibromas.1;69;74 Each benign cartilaginous tumour is characterized by a specific age of occurrence, localization within the skeleton and regions of individual bones, in addition to specific radiological and pathological features.1;69;74 This is in keeping with the acknowledged need to integrate clinical, radiological and pathological features for the final diagnosis of each bone tumour. This approach has recently been improved by incorporating molecular biology/cytogenetic findings. In particular, while osteochondromas are known to harbour EXT gene mutations, the genetics of chondroblastomas and chondromyxoid fibroma are more elusive1;69. Non-recurrent balanced and unbalanced rearrangements affecting several chromosomal regions have been reported in chondroblastomas.75-79 Conversely, recurrent involvement of 6p25, 6q13, 6q15, 6q23 and 6q25 has been described in chondromyxoid fibromas.80- 84. However the ultimate effects of these genetic changes are poorly understood.
Chondroblastomas occur mainly in prepubertal patients in the epiphysis of long bones.1;70 Before puberty the growth plate is still viable and has a close anatomical relationship with the epiphysis. At this age there is significant ongoing proliferation in the growth plate, which is well regulated by key signalling pathways6, prompting investigation of the role of growth plate signalling in chondroblastoma pathogenesis (Chapter 3). Special attention has been focused on whether different patterns of expression occur in morphologically different areas within the same tumour. Lastly, the pattern of expression in multinucleated giant cells has also been observed.
Morphologically different areas in cartilaginous tumours bear a striking resemblance to the differentiation stages of chondrogenesis: from less differentiated spindle- shaped precursors to well differentiated chondrocytes. These features are particularly evident in chondromyxoid fibromas. This prompted a comparison study between chondromyxoid fibromas and articular chondrocytes cultivated in 3D pellets (Chapter 4).The morphology of the constituting cells and the architecture of chondromyxoid fibromas have been evaluated and compared with the pellets from articular chondrocytes. The pattern of sulphated proteoglycan deposition has been observed and integrated with the pattern of expression of crucial molecules in chondrogenesis.
The spindle-shaped cells of chondromyxoid fibromas have been reported to express smooth muscle actin and to have ultrastructural evidence of myofilaments with focal densities.85;86 This raised the possibility of spindle-shaped cells transdifferentiating into the myogenic phenotype. Neither the spectrum of this myoid/myofibroblastic differentiation nor the driving mechanism have been studied in depth.85 In particular, de novo expression of smooth muscle actin in chondrocytes is induced by TGF-β1 signalling.87 The extent of myofibroblastic differentiation in chondromyxoid fibroma and a possible causative role for TGF-β1 signalling have been studied (Chapter 5).
Substantially different histological features characterize chondroblastoma and chondromyxoid fibromas. Specifically, there is no clear-cut cartilaginous matrix in chondroblastoma and the pattern of signalling molecules and the quality of extracellular matrix have led to the hypothesis that the neoplastic cells are
committed to bone/hypertrophic chondrocyte differentiation.88;89 Within the histology of chondromyxoid fibromas, the different steps of in vitro chondrogenesis are reflected by the presence of round cells more similar to mature chondrocytes, and of less differentiated spindle cell precursors, showing partial myofibroblastic transdifferentiation90;91. Furthermore the polygonal - atypical cells in chondromyxoid fibroma sometimes closely resemble the cells of high-grade central chondrosarcoma .92 This can make the differential diagnosis difficult in clinical practice, especially in biopsy specimens. A scan approach to genome-wide expression has been used to further our knowledge on the molecules driving different morphology in chondromyxoid fibroma and chondroblastoma and to identify possible markers for differential diagnosis of chondromyxoid fibroma versus high-grade central chondrosarcoma (Chapter 6).
The distinct epiphyseal occurrence of chondroblastomas in prepubertal patients suggests a role for growth plate signalling in the pathogenesis of this lesion. Sex steroids play an important role in pubertal growth via their action on the growth plate.93 Furthermore, both in vivo expression of oestrogen receptors and in vitro oestrogen-induced neoplastic cell proliferation/survival have previously been demonstrated in cartilaginous tumours.94;95 An index case has been identified with a balanced translocation t(5;17) with breakpoints mapping close to the carbonic anhydrase 10 (CA10) and steroid reductase 5 alpha 1 (SRD5A1) genes. The involvement of candidate regions/genes have also been further investigated with particular attention to the pattern of expression of molecules involved in sex- steroid signalling (Chapter 7).
Chondromyxoid fibroma is associated with recurrent rearrangements of chromosome bands 6p23-25, 6q12-15 and 6q23-27.80-84 The majority of changes have been complex rearrangements and delineation of the breakpoints has been performed mainly at the resolution level of conventional cytogenetics, with spectral karyotyping utilized in a few cases, and FISH mapping used in one case.77;80-82;84;96
The genetic changes involving chromosome 6, recurrent breakpoints and possible candidate target genes have been identified and analyzed in a large group of chondromyxoid fibromas (Chapter 8).
Finally the achievements of the study are summarised and possible future directions of research are indicated in Chapter 9.
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