3_Mesenchymal differentiation and pathology of bone.docx

**THEME 3: MESENCHYMAL DIFFERENTIATION AND PATHOLOGY OF BONE............................................................................** **Introduction** The skeletal system acts in the human body as mechanical scaffold for movement and body shape; it is also essential in mineral homeostasis. Mo...

**THEME 3: MESENCHYMAL DIFFERENTIATION AND PATHOLOGY OF BONE............................................................................** **Introduction** The skeletal system acts in the human body as mechanical scaffold for movement and body shape; it is also essential in mineral homeostasis. Moreover the skeletal system houses important parts of the hematopoietic system. These functions are the result of a delicate interplay both during development and adult life of the different cells which develop and differentiate during embryology until the terminally differentiated forms. These can easily be recognized in adult mature bone: osteoblasts, osteocytes, osteoclasts, and chondrocytes. In a number of pathological processes either as a reaction to exogenous injury or as a result of an intrinsic disease process there is a disturbance in this interplay resulting in metabolic abnormalities, growth abnormalities, or even neoplastic growth. Our knowledge and understanding of normal mesenchymal growth and differentiation vastly contributes to the understanding of these pathological processes. In year 1 of Biomedical Sciences, normal histology and homeostasis of bone already has been studied during the course \"Cellulaire Communicatie\". This knowledge is required in order to be able to understand pathological conditions of bone. Bone is a dynamic tissue in which (re)modeling occurs continuously. Normally the formation and resorption of bone are in balance. Defects in this sequence are underlying many pathological bone diseases (osteoporosis, Paget\'s disease, osteopetrosis, sclerosis etc.) In this theme we will recapitulate what has been learned about normal skeletal development and homeostasis in year 1, and focus on pathological conditions, especially abnormal (neoplastic) growth resulting in diseases such as reactive bone forming lesions with an emphasis on benign and malignant bone tumors. During this theme several clinical cases are studied. By looking at morphological changes in the affected body parts, both macroscopically as well as microscopically, we will study the pathological basis of disease by concentrating on the origin of a number of disturbed fundamental mechanisms of bone differentiation and growth. The effects of disturbed regulation of physiological mechanisms - resulting in pathology - will be shown and the observed alterations in structure and function will be related to the patient\'s clinical symptoms and signs. The progress in understanding bone tumor formation from basic science results will also be discussed in a journal club and in a PhD minisymposium. With the tour at the anatomical museum you will see the actual skeletal deviations that are presented in this module. Not all rare entities that are shown in the museum will be discussed at the lectures and therefore are no obligatory subjects for the exam, but they are a perfect illustration of the rich variation in morphological deviations, especially in the skeleton. **Theme-related objectives** *By the end of this theme you will be able to:* - Recognize, name and describe normal histology of bone and the physiological interplay between cells in the bone (1). (Repetition and extension of the course \"Cellulaire Communicatie\" year 1 Biomedical Sciences). - Describe the differentiation of the different cell types that constitute bone from primitive mesenchymal stem cells (MSCs) (1). - Classify the different types of bone neoplasms especially benign and malignant cartilage tumors, osteosarcoma, giant cell tumors of bone and small blue round cell tumors (2). - Recognize, name and describe tissue changes (morphological and structural alterations) in macroscopic and microscopic specimens of bone pathologies (3). - Describe how germline mutations in genes that regulate bone development can give rise to growth disorders (2,3) - Describe the application of knowledge from MSCs to clinical practice. (4) - Explain diagnostic methods indicated (4). - Indicate actual research subjects regarding the above described diseases (4). **Reading list and study guidelines:** Afbeelding met tekst, schermopname, Lettertype, nummer Automatisch gegenereerde beschrijving - Focus on Fig. 26-1 and 26-5. - Understand the macroscopic structure of bone. - Know the origin, location, and function of bone cells. - Understand the structural and functional organization of periosteum and endosteum. - Know the components of bone matrix. - Be able to compare and contrast the different types of bone. - Understand the process of intramembranous ossification and endochondral ossification. - Understand the differences between a bone growing in length and bone growing in width. - Recognize, name and describe normal histology of bone and the physiological interplay between these cells. - Distinguish benign and malignant primary bone tumor types and the molecular mechanisms that underlie their pathogenesis. **Normal histology and neo-plastic pathology of bone (09-04-2024)........................................................................................** **Goal:** - Recapitulation of normal histology of bone - Recapitulation of normal histology of cartilage - Knowledge on the cells in bone and the growth plate and their function - Knowledge on intramembranous ossification and endochondral ossification - Knowledge on tissue changes during fracture healing and in myositis ossificans → Basic knowledge to understand neoplastic and non-neoplastic pathology of bone **Function of bone:** - Mechanical support - Facilitates movement - Mineral homeostasis - Provides a niche for production of blood cells - Protection of viscera **Composition of bone:** **Extracellular matrix** - Organic component: osteoid (35%) -\> mainly collage type 1 - Anorganic component: minerals (65%) -\> mainly calcium hydroxyapatite (Ca~10~(PO~4~)~6~(OH)~2~) **Specialized cells that produce and maintain the matrix:** - **Osteoprogenitor cells** -\> stem cells that produce osteoblasts - **Osteoblasts** -\> synthesize matrix, regulate mineralization - **Osteocytes** -\> osteoblasts become osteocytes -\> mechanotransduction, control calcium/phosphate level - **Osteoclasts** -\> resorb bone **Interaction between osteoblast and osteoclast** - **RANK/RANK-L/OPG** - **M-CSF/M-CSFR** - **Wnt/b-catenin** - Activation leads to OPG synthesis - Can be inhibited by sclerostin, produced by osteocytes The interaction is complex and there are different pathways that play an important role in this: Osteoblast has the **RANK ligand**, this ligand binds to the **RANK receptor** on the osteoclast precursor cell. When this happens you get stimulation of the **NFkB pathway** which leads to *differentiation of the osteoclast precursor cell into an osteoclast* cell and as a result you get [bone absorption]. Also, the osteoblast can also produce **M-CSF** (macrophage colony stimulating factor). The osteoclast precursor has a **M-CSF receptor**, binding leads to stimulation of the **NFkB pathway** which leads to differentiation of the osteoclast precursor cell into an osteoclast cell and as a result you get bone absorption. The osteoblast can produce **OPG**, this is a protein that blocks the binding between RANK and RANK ligand and thereby inhibits the formation of osteoclasts -\> no bone resorption. Activation of the Wnt/B-catenin pathway results in OPG synthesis. There is a balance between bone production and bone resorption **RANK : OPG ratio** **Bone cells and their interrelated activities** *So after bone damage the osteoclast absorb the bone, this results in the release of growth factors. This lead to proliferation of the osteoprogenitor cells into the active osteoblasts (matrix synthesis). The active osteoblasts produce b-catenin, this leads to blockage of the differentiation (via OPG?) of the osteoclast progenitor cells into osteoclast cells.* **Bone cells involved in remodeling** - The osteocytes are in the bone - Osteoclasts are multinucleated giant cells that absorb the bone - Active osteoblasts produce bone  **Types of matrix** There are three types of matrix produced: 1. **Osteoid** (non-mineralized bone) 2. **Woven** **bone** -\> random deposition of collagen; weak; rapid; abnormal in adults 3. **Lamellar** **bone** -\> orderly deposition of collagen; strong; slow Lamellar bone woven bone Afbeelding met Kinderkunst, schermopname, verven, kunst Automatisch gegenereerde beschrijving **Cells in bone** **Cortical bone** - Osteoblasts, osteoclasts, and osteocytes **Sponges bone** - bone marrow and fat - Hematopoietic stem cells - Blood vessels - Adipocytes - Stromal cells - Osteoblast precursor cells Osteocytes are connected with each other and they interact via the cytoplasmatic processes -\> **Canaliculi**  Osteocytes are organized in a circle around the **haversian canal** (blood supply), this unit is called a **osteon**.  Afbeelding met tekening, schets, cirkel Automatisch gegenereerde beschrijving  **Bone formation during development** *Two ways of ossification:* 1. **Intramembranous ossification** 2. **Endochondral ossification** **Intramembranous ossification** Bones formed via intramembranous ossification: - Cranial vault - Facial bones - Clavicles - Cortical bone \> This leads to appositional bone growth (growth in width)  **Endochondral ossification** *The [chondrocytes] in the center of the model become active, they increase in size, which reduces the matrix to a series of small struts, and then [die], leaving an open space. [Blood vessels invade] this are and osteoblasts at the periphery lay down a superficial layer of bone. [Fibroblast] delivered by the blood vessels in the central region differentiate into [osteoblasts] and begin to produce spongy bone at what is called the [primary ossification center]. Over time, the primary ossification center undergoes remodeling that forms a medullary cavity. This process repeats at multiple [secondary ossification centers] located at the epiphyses. The epiphyses retain a thin layer of articular cartilage within the joint cavity. This layer will remain in the adult and it covers the ends of your bones today. The epiphyseal plate separates the epiphyses from the diaphysis.* *Lengthwise growth of the bone: Chondrocytes on the epiphyseal side of the plate continue to divide and enlarge while those in the diaphyseal side die. Osteoblasts migrate to this region and lengthen the bone by laying down more bone. At puberty, various hormones cause the epiphyseal plate to be fully replaced with bone. This epiphyseal closure signals the end of skeletal growth.* *So endochondral ossification is the process in which we see the early hyaline cartilage skeleton replaced with hard ossified bone. It begins in the diaphysis at a primary ossification center and proceeds at multiple secondary ossification centers in the epiphyses. Hyaline cartilage remains at the epiphyseal plate to regulate lengthwise growth of the bone until skeletal maturity is reached and the plate ossifies. Articular cartilage remains in the joint cavity covering the ends of long bones.*  Afbeelding met skelet Beschrijving automatisch gegenereerd met gemiddelde betrouwbaarheid Afbeelding met tekst, brief, roze Automatisch gegenereerde beschrijving  **Regulation of longitudinal growth** **Paracrine regulation (locally formed)** - Wnt - Bone morphogenic proteins (BMPs) - Indian hedgehog - VEGF - IGFs - FGFs - PTHrP **Systemic regulation (via bloodstream)** - Growth hormone, IGFs - Estrogen - Androgen - Vitamin D - Parathyroid hormone - Glucocorticoids **Endochondral bone formation** - Axial and appendicular skeleton (arm and legs) - Longitudinal (bone) growth - Joint cartilage - Fracture healing **Difference between articular and growth plate cartilage** Articular cartilage - Distal ends of bone - Joint formation and motility - Weight bearing - Resistant to resorption Growth plate cartilage - Entrapped between epiphyseal and metaphyseal bone - Longitudinal bone growth - Disappears at the end of puberty Afbeelding met schermopname Automatisch gegenereerde beschrijving **Non neoplastic pathology of the bone** - Fracture healing - Myositis ossificans **Bone fracture** **Bone fracture** = loss of bone integrity/interruption in the continuity due to mechanical injury and/or diminished bone strength. **Normal fracture**: due to acute trauma **Stress or fatigue fracture**: due to repetitive mechanical stress **Pathological fracture**: in weakened bone due to pre-existing lesion Fracture healing: **Callus** = hard mass of skeletal repair tissue, which unites the fractured bone ends \> Various proportions of haematoma, [osteoprogenitor cells], [fibrous tissue], [cartilage] and [bone] You will first have cartilage formation, and then that is replaces by bone \> Endochondral ossification  Early stage callus Fibroblastic proliferation Inflammatory cells New vessel formation soft callus: endochondral ossification you see the osteoblasts, and within the osteocytes Woven bone When all the cartilage is dissolved, you get woven bone produces by osteoblasts. after several months remodeling: lamellar bone ***\ *** **Myositis ossification** - Formation of bone within muscle - Can occur after hemorrhage; result of cell/tissue injury - Metaplasia: formation of tissue where normally these elements are not seen - Resul of reprogramming of stem cells; the precursor cells differentiate along anew pathway - It usually resolves within a year proliferation of fibroblasts infiltration of inflammatory cells  zonal architecture Afbeelding met kunst Beschrijving automatisch gegenereerd met lage betrouwbaarheid **Mesenchymal stem cell differentiation (09-04-2024)..............................................................................................................** **Outline:** - The mesenchymal stem cell MSC - Signal transduction pathways in mesenchymal differentiation - MSC in research and medicine - The dark side of the MSC **Bone fractures** The recruitment of new bone: MSC differentiation to bone **MSCs: Mesenchymal stem cells** - Multipotent progenitor cells - Reside in the bone marrow - Also present in adipose tissue and cord blood - Undifferentiated - Self-renewal (not unrestricted) - Can be distinguished by expression of specific protein (CD 73/90/105) (CD= cluster of differentiation) - Three lineage differentiation (bone, cartilage, adipose) - MSC is a single cell that is capable of generating a complete heterotopic bone or bone marrow organ (ossicle) in vivo. **Identification of MSC markers** - MSCs express CD105, CD90, CD73 - MSCs do not express CD34, this is for hematopoietic cells **Differentiating MSCs** MSCs can differentiate into osteocytes, chondrocytes and adipocytes in vivo:  **The postnatal bone marrow** MSCs can be found around the vessels in the bone marrow Afbeelding met tekst, schermopname, roze, brief Automatisch gegenereerde beschrijving  MSCs differentiate to mesoderm - Connective stromal cells - Cartilage cells - Fat cells - Bone cells MSCs can also trans-differentia, this is controversial in vivo - Ectoderm: epithelial cells; neurons - Endoderm: muscle cell; gut epithelial cell; lung cell **The cells that make the skeleton** - Osteoprogenitor cells (MSC derived) -\> membranous ossification - Osteoblasts (MSC derived) -\> membranous ossification - Bone surface lining cells (MSC derived) -\> membranous ossification - Osteocytes (MSC derived) -\> membranous ossification - Chondrocytes -\> endochondral ossification - Osteoclasts (monocyte derived, HSC) -\> bone resorption [You should know: ] RUNX2 Starting point of the osteoblastic lineage RANKL, produced by osteoblasts Starting point for the osteoclast maturation Afbeelding met tekst, schermopname, Lettertype, lijn Automatisch gegenereerde beschrijving **MSC to chondro- and osteocytes** BMP and WNT are important for bone development! IHH is important for chondro lineage! Wnt/b-catenin inhibits and promotes osteogenic differentiation WNT regulates OPD synthesis \> OPG blocks osteoclast differentiation  **CBFA1/RUNX2** CBFA1: Core binding factor alpha 1 RUNX2: runt-related transcription factor 2 RUNX2 blocks the differentiation into adipocytes RUNX2 plays a role in the further differentiation of chondrocytes CBFA1 also induces RANK ligand CBFA1 knockout: no production of bone, only cartilage **Fate of the osteoblast**  **WNT signaling** \> Defects in Wnt genes in several hereditary bone pathologies Destruction complex existing of APC, Axin, Gsk, this complex destruct b-catenin. If this complex is degraded then b-catenin can migrate to the nucleus. Wnt binding inactivates the destruction complex. - AXIN2 - Inactivation of LRP5 -\> osteoporosis - Activation of LRPp5 -\> osteopetrosis - SOST **BMP signaling** Mutations in ACVR1/ALK2, a bone morphogenic protein (BMP) type 1 receptor: - Pathway will be overactivated, muscles turn into bone - Fibrodysplasia ossificans progressive (FOP) - Autosomal dominant inheritance - Progressive heterotopic endochondral ossification **The chondrogenic program** Driven by Sox9 and taken over by CFBA1  **RANK/RANKL for osteoclast differentiation** Afbeelding met schermopname, diagram Automatisch gegenereerde beschrijving Osteoprotegerin (OPG): decoy receptor for RANKL RANK ligand: receptor activator of nuclear factor (NFkB) **MSC in research and therapy** - Present in bone marrow and cord blood - Easy to obtain - Differentiation can also occur in vitro -- use of the correct culture media - MSCs can be transfected with foreign DNA - Can be frozen for use later **Obtaining MSCs** Take bone marrow, ficoll to separate, culture the cells, you still have a mixture of HSC and MSC. But HCS cannot attach to the culture dish.  **Use of MSC** - Repair tissue in patients - Cartilage - Bone - Muscle - Heart muscle - Tendon - Immunosuppressive, inhibit alloreactive T-cell proliferation - Study differentiation pathways **Differentiation for research to make specific cell types** Afbeelding met tekst, diagram, patroon Automatisch gegenereerde beschrijving **MSCs can become sarcomas** This is a downside **Summery** - Bone consists of several cell types: - MSC can differentiate to various cell types, depending on gene expression, growth factors and environment - Signal transduction driven by a.o. WNT pathway, RUNX2 and RANK guide MSC differentiation - MSC are used for clinical applications and scientific research **Mesenchymal tumors: cartilage (09-04-2024)..........................................................................................................................** - Knowledge on classification of bone tumors - Knowledge on mechanisms involved in cartilage tumor formation - Understand these mechanisms in relation to normal bone development **Terminology: mesenchymal tumors**  Terminology regarding biologic behavior: - Benign - Intermediate - Locally aggressive - Rarely metastasizing - Malignant To determine how tumors will behave and how they should the treated. **Hallmarks of cancer** Afbeelding met tekst, klok, schermopname, cirkel Automatisch gegenereerde beschrijving **Clonal expansion**  **Multistep progression model for cancer** The multistep progression model for cancer also applies for bone tumors **CHONROGENIC TUMORS** **Cartilage tumors** Most of the benign tumors are cartilage tumors Afbeelding met tekst, schermopname, Lettertype Automatisch gegenereerde beschrijving **Benign cartilage tumors** **Osteochondroma** - Bone surface - Cartilage cap - Stalk continuous with underlying bone **Enchondroma** - In marrow of bone - Often in hands and feat **Malignant cartilage tumors** **Peripheral chondrosarcoma (\~15%)** - Bone surface - Secondary to osteochondroma **Central chondrosarcoma (75%)** - In marrow - Primary or secondary to enchondroma **Histological spectrum**  **Conventional chondrosarcoma: histological grading** Afbeelding met tekst, schermopname Automatisch gegenereerde beschrijving **Osteochondroma** - Almost always arise in long bone, adjacent of the growth plates. - Located at the bone surface, formation of cartilage cap - Marrow cavity continuous with that of underlying bone - 86% solitary (sporadic) - The peak age is in young patients, because of the open growth plate. Adults have a closed growth plate so they don't develop new osteochondromas osteochondroma looks similar to growth plate   **Multiple osteochondromas (MO)** - Hereditary disorder - Autosomal dominant - Can lead to deformation of the bones - Mutations in EXT1 (8q24) or EXT2 (11p) - 1-5% secondary peripheral chondrosarcoma [Normal EXT function ] - EXT genes are responsible for the sugar chain attachment to **heparan sulphate proteoglycans** (HSPG) - And also for the elongation of the sugar chains - HSPG is involved in the signaling of [FGF] and [VEGF], they function as a co-receptor - HSPG is involved in signaling of [Hedgehog, Wnt and TGFb/BMP] - These pathways are important for normal growth plate **Endochondral ossification** So endochondral ossification also occurs in osteochondroma Afbeelding met tekst, schermopname, Lettertype, diagram Automatisch gegenereerde beschrijving **IHH coordinates skeletal morphogenesis** Inactivated Hedgehog leads to smaller mice, and there is little bone formation occurring **IHH induces bone collar formation** Inactivation of hedgehog leads to lack of bone collar formation EXT1 knockout in the cartilaginous cell growth plate/ groove of Ranvier -\> develop osteochondroma Cartilage cap -\> there will be mutant and normal cells. EXT1 acts like a tumor supressor gene, you need to knockout both -\> biallelic inactivation **Osteochondroma formation** - First mutation in EXT occurs - Second hit in EXT occurs - heparan sulphate proteoglycans is no longer formed, this affects Hedgehog signaling - as a result the bony color is not properly formed so there comes a hole. - The mutant cells grow out - Together with some normal cells this forms an osteochondroma - An osteochondroma is not entirely clonal  **\ ** **Progression towards secondary peripheral Chondrosarcoma** Osteochondroma forms a niche in which (normal) cells are prone to acquire other mutations leading to malignancy. This leads to chondrosarcoma. Some of the chondrosarcoma cells do not have the EXT mutations anymore, probably normal cells that became malignant. \> Osteochondromas are not clonal \> Chondrosarcomas are clonal The other mutation is often a mutations that inactivated cell cycle genes For example INK4 or Trp53 Afbeelding met tekst, diagram, schermopname, kaart Automatisch gegenereerde beschrijving **Central cartilaginous tumors**  **Enchondromatosis: Ollier disease** - Unilateral predominance - Non hereditary - Rare (1:100.000) - Risk malignant transformation 40% It often only occurs at one site of the body -\> no not germline mutation -\> non hereditary Occurs during skeletal formation in young patients **\ ** **\ ** **IDH1 or IDH2 mutations in central cartilage tumors** \> These mutations are often found in Ollier, secondary CS, and primary central CS \> IDH mutants converge a-KG to D2HG \> D2HG is usually in low concentrations present, but with IDH mutations there is a high concentration \> The oncometabolite affects DNA methylation, it also affects histon modifications  Afbeelding met tekst, schermopname, diagram, lijn Automatisch gegenereerde beschrijving Inhibition of mutant IDH does not lead to less cell viability and migration So IDH mutation plays no role in progression **\ ** **Mesenchymal tumors: Ewing sarcoma and molecular diagnostics (10-04-2024).................................................................** **Goals:** Understand how a pathologist comes to a (differential) diagnosis: - Use of immunohistochemistry - Use of molecular testing In case of Ewing sarcoma **Classification of bone tumours** Bone tumours are difficult for pathologists: - Relatively rare - In total almost 60 entities: - Benign n=20 - intermediate n=10 - Malignant (sarcoma) n=28 - Considerable morphological overlap - Immunohistochemistry of limited value - Entities differ widely in treatment and outcomes **Pathological diagnosis** - Immunohistochemistry - Molecular analysis - Morphology - Clinical and radiological information **Sarcomagenesis** These are the three main tumors, they have a specific molecular pattern Ewing sarcoma -\> specific translocations  **Age specific incidence of bone sarcoma** Ewing sarcoma almost never occurs in older people Chondrosarcoma needs an accumulation of mutations and therefore occurs more in older people Afbeelding met tekst, lijn, Perceel, diagram Automatisch gegenereerde beschrijving **Routing of tissue specimens** A biopsy is taken  **Small blue round cell tumor in bone: differential diagnosis** - Ewing sarcoma - Rhabdomyosarcoma (striatal muscle differentiation) - Neuroblastoma - Non Hodgkin lymphoma/leukemia - Small cell osteosarcoma - Mesenchymal chondrosarcoma - Poorly differentiated monophasic synovial sarcoma - Other undifferentiated round cell sarcoma (BCOR (or CIC) rearranged) Additional tests: - Histochemical stains - Immunohistochemistry - Detection of antigens (proteins) in tissue sections using antibodies - To determine diagnosis: tumor classification, determine the line of differentiation of tumors - Determining prognosis - Prediction of therapeutic response **Immunohistochemistry to define the line of differentiation** Afbeelding met tekst, schermopname, Lettertype, nummer Automatisch gegenereerde beschrijving **Immunohistochemistry in small blue round cell tumors**  **Immunohistochemical profiles may overlap** So then you also need to do molecular testing Afbeelding met tekst, schermopname, patroon Automatisch gegenereerde beschrijving **Molecular alterations in small blue round cell tumors**  **Ewing sarcoma specific translocations** Afbeelding met tekst, Lettertype, schermopname, wit Automatisch gegenereerde beschrijving -\> EWSR1-ETS fusion proteins **Specific translocations in sarcomas** \> Translocation is early step in tumorigenesis \> Fusion product essential in tumor formation There are three different mechanisms in which translocation and fusion can lead to tumorigenesis - Chimeric gene -\> **transcriptional deregulation** (e.g. Ewing sarcoma) - Promoter swap -\> **deregulated signaling** (e.g. tGCT, nod fasciitis) - Truncation -\> **altered expression** (e.g. epithelioid hemangioma, osteoblastoma)  **Techniques for translocation detection** - Conventional cytogenetic (fresh tissue) - RT-PCR (frozen tissue, paraffin) -\> design primers at both ends of the fusion (no fusion no PCR product) - FISH (paraffin material, not decalcified!) - Immunohistochemistry -\> Now mainly with next generation sequencing **\ ** **Break in EWSR1 is not specific!** You need to know the fusion partner Afbeelding met tekst, schermopname, diagram, Lettertype Automatisch gegenereerde beschrijving **Future: genetic testing without the pathologist?** Why not only next generation sequencing? Some tumors share the same fusion but are entirely different  **New: antibodies against a chimeric protein: SS18-SSX**... **Case** 8 year old boy, tumour humerus - Immunohistochemistry: CD99, NKX2.2 positive, other markers negative - NGS analysis using Archer: EWSR1-FLI1 fusion - Diagnosis: Ewing sarcoma - Treatment: resection, chemotherapy, radiation, 5 year survival about 60-65% Ewing sarcoma is found in young people, in bone and soft tissue   **EWSR1-ETS target genes** EWSR1-ETS fusion protein, the chimeric protein acts as a transcription factor that orchestrates a variety of other genes, these can all be seen in the hallmarks of cancer. [Stimulation of cell proliferation] - Upregulation of PDGF-C, CCDN1, c-MYC [Evading growth inhibition] - Downregulation of cyclin-dependent kinase inhibitors (p21, p57) - Downregulation of TGF beta type II receptor [Escape from senescence ] - Upregulation of hTERT; increased telomerase activity [Escape from apoptosis ] - Repression of IGFBP-3 promotor [Angiogenesis ] - VEGF expression - Invasion and metastasis - MMP? **Syndromal growth disorders (10-04-2024)...............................................................................................................................** **Objectives** - Describe the definition of short stature and tall stature - Describe the difference between syndromic and non-syndromic growth disorders - Recognize and name the clinical features of the most common causes of syndromic short stature - Recognize and describe the relation between specific syndromes (covered during this lecture) and its molecular background - Reason what the inheritance pattern of a specific syndrome (covered during this lecture) means for the patient and his/her family **Tall stature** *Tall stature vs overgrowth*: Tall stature is when you are just tall, and overgrowth is when you are bigger in certain fields. Tall stature = Height \> 2 SDS (*standard deviation*) Tall stature = Height \> 1.6 SDS above target height (based on parent's height) **Short stature** Height versus length (length is when laying down, so just born child) Short stature = Height \< -2 SDS Short stature = Height \> 1.6 SDS under target height (based on parent's height) **Guideline for measuring** How to measure you patients - Heights - Weights - Head circumference - Armspan (from finger to finger, for adults this is the same as your height) - Sitting height (ratio between height and trunk, indication proportions) - Parents (only measuring when Armspan and sitting height are abnormal in child) **Height** Height = 60-80% genetic But feeding, health and medication are other important factors on height Look at mutations mainly high penetrance (large effect on height), not focus on the low penetrance (have a small effect on height). **Syndrome** Non syndromic short stature or syndromic short stature? **Syndrome** = A group of signs an symptoms that occur together and characterize a particular abnormality or condition Syndrome -\> pattern recognition **Cause of short stature** **Primary growth disorders** - Syndromes - SGA (small for gestational age) without catch-up growth - Skeletal dysplasia - Disorder of bone metabolism **Secondary growth disorders** (you have a conditions, and short height is a secondary response) - Malnutrition - Celiac disease - Hormonal disorder - Metabolic disorder **Idiopathic short stature** (cause is unknown) Syndromes with short stature: 1568 syndromes Syndromes with short stature and disproportion: 320 syndromes With disproportion there is almost always a genetic aspect The most common chromosomal cause of short stature is: Down syndrome -- trisomy 21 **Turner syndrome** - Tuner syndrome only occurs in girls - They have only one X chromosome, or when one X chromosome is a iso chromosome (misses short arm) - They often have a heart condition - They are often infertile - These children benefit from growth hormone therapy  **Noonan syndrome** *Caused by a gene mutation, occurs in both boys and girls* *Sometimes they get growth hormonal therapy* Short stature - Mean height male 169 cm - Mean heigh female 154 cm Developmental delay (mean IQ 86) Facial features: - Hypertelorism (eyes far away from each other), ptosis (hanging eyelids) - Low set ears - Broad neck Congenital cardiac anomaly (pulmonary stenosis) Pectus deformity (dimple in sternal bone) Lymphatic abnormalities Coagulation defect Cryptorchidism (testis are not descended) Genetics and Noonan syndrome: - Mutations in genes in the RAS-MAPK pathway Noonan gene panel (UMCN): \>14 genes **Skeletal dysplasia** Frequency - 1:3000 live births - 1:110 perinatal mortality (when a child dies before birth, it is in 1:110 a skeletal dysplasia) \> Often the limbs are short for the trunk \> Only in rare occasions is the trunk short for limbs Abnormalities of epiphysis, metaphysis and/or diaphysis Mostly causing disproportionate short stature  Afbeelding met schets, diagram, skelet Automatisch gegenereerde beschrijving - There are many different types - Heterogeneity (clinical and genetic) - Non-uniform terminology **Skeletal dysplasia: Achondroplasia** **Achondroplasia**: 1:15.000 -- 1:40.000 Clinical features of achondroplasia: - Short stature versus large head - Short arms and legs - Skin creases - Pronounced lumbar lordosis - Deep nasal bridge - Frontal bossing - Short hands with trident  Afbeelding met persoon, maag, Naaktfotografie, overdekt Automatisch gegenereerde beschrijving x-ras for achondroplasia: As you descend in the back the bones usually it become wider because of al the weight, but in achondroplasia it doesn't, sometimes it even becomes smaller. Other problems: - Narrow craniocervical junction - Delayed motor development - Obesity - Obstructive sleep apnea - Middle ear dysfunction - Stenosis L1-14 Treatment for achondroplasia: Treatment is symptomatic - Growth: growth hormone doesn't work, not effective/limb lengthening - Decompression spinal stenosis - Apnea - Spinal stenose - Psychosocial support Medical treatment for achondroplasia: - Vosoritide (has effect on growth) - Effect on growth and possibly positive effect on spinal stenosis - Tested in a few hundred patient **FGFR3 -\> gene responsible for achondroplasia** - Fibroblast growth factor receptor 3 gene, so a receptor for a growth factor - Located on chromosome 4 - Mutations that cause achondroplasia are located in the transmembrane -\> gain-of-function mutation Often a de novo mutation in ACH, 80-90%, so the parent don't have it. When one of the parents have it, there is a 50% change for the child of having it Mutations (approx. 98%): - Nucleotide 1138: G\>A or G\>C - Amino acid 380: glycine\>arginine - Notation: p.Gly380Arg or p.G380R  \> FGF binding to the receptor, and then activates the receptor. The normal effect of this is growth inhibition. \> When you have a mutation in the gene, the receptor is constantly active, so you have constant growth inhibition **Vosoritide** as a treatment for achondroplasia - The protein CNP inhibits the inhibiting effect of the receptor - Vosoritide is a CNP analogue - Approved for use in children with achondroplasia age \> 2 years until 12 years - Height gain approximately 1,5 cm a year - Side effects: low blood pressure - Not available in the Netherlands yet, negotiations on the costs are ongoing **FGFR3** New mutation in 80-90% - Paternal, if the father is older then you have a higher change for a new autosomal dominant mutations - (if the mother is older then there is a higher change for down syndrome in the child) Familial in 10-20% Autosomal dominant  When one parent has achondroplasia then there is 50% change for the child to have achondroplasia Note! What is both parents have achondroplasia? - If you have achondroplasia from both parents then you have a severe form (biallelic) -\> often lethal - But if you look at the children than would survive, then it is 66,7% Other forms of FGFR3 mutations:  TD = **thanatophoric dysplasia** (milder type of achondroplasia) HYP = **hypochondroplasia** (severe type, similar to biallelic achondroplasia) **Leri-Weil dyschondrosteosis** LWD clinical features: - Short stature (not as short as achondroplasia) - Short arms and legs - Madelung deformity (abnormality of the wrist) - Muscle hypertrophy - Short 4e metacarpal bone Girls -2,8 SDS Boys -2,4 SDS Growth hormonal therapy works LWD clinical: - Incomplete penetrance - More severe in girls - 2x mutation (biallelic) = langer mesomelic dysplasia Genetics of LWD: Mutations in the SHOX gene, located in Y and X chromosome Locates in the pseudo-autosomal region 1, so it escapes X-inactivation Afbeelding met tekst, schermopname, lijn, Parallel Automatisch gegenereerde beschrijving Pseudo-autosomal inheritance and high level of crossing-over: If the father is affected and the mutation is located in the Y chromosome, then you would expect it to be passed on to all the sons but not the daughters. But here you can see that the father passed in on to his two sons and the other is not affected. And in the next generation you see that the males pass in on to their daughters. This is because of the high level of crossing over.  LWD treatment: Growth hormone - Effect similar to Turner syndrome - Gain of 7-10 cm Wrist Conservative management - Splint, ergonomic devices - Reduce wrist discomfort Operative procedures **Summery, what is important to know?** For recognition of a syndromic growth disorder you need measurement - Describe the definition of short stature and tall stature - Describe the difference between syndromic and non-syndromic growth disorders - Recognize and name the clinical features of the most common causes of syndromic short stature DNA analysis is not always necessary in making a diagnosis - Recognize and describe the relation between specific syndromes and its molecular background Reason what the inheritance pattern of a specific syndrome means for the patient and his/her family **\ ** **Mesenchymal tumors: giant cell containing tumors of bone (10-04-2024)...........................................................................** **Goal:** - Knowledge on giant cells in bone tumors incl GCTB (giant cell containing tumors of bone) - Understand the biology of GCTB and thereby its therapy **Case** Female 35 years Lytic lesion, less mineral (less white) It reaches to the end of the bone We can see giant cells, and in between mononuclear cells Afbeelding met röntgenfilm, Medische beeldbewerking, radiologie, radiografie Automatisch gegenereerde beschrijving **Giant cell containing lesions in bone** When you see giant cells in the bone it is not necessarily 'giant cell tumor', there are many types of giant cell containing tumors. Giant cell tumor is intermediate because it is locally aggressive, and rarely metastasizing. \> Most giant cell containing tumours are not malignant \> Almost any kind of lesion in bone can contain giant cells, sometimes numerous The function of the giant cells (osteoclasts) is resorption of the bone  Afbeelding met tekst, Lettertype, schermopname, algebra Automatisch gegenereerde beschrijving **Non-malignant giant cell containing lesions of bone**  \> Group includes reactive, metabolic and neoplastic lesions **Location in bone** Some tumors are only in certain regions in the bone \> Giant cell tumor -\> in the end of the bone (in the metaphysis or epiphysis) Afbeelding met tekst, schermopname Automatisch gegenereerde beschrijving **Giant cell tumor of bone** \> Giant cell tumor of the bone mostly occur after closure of the growth plate \> In young adults \> Slight preference for female \> preferential location -\> knee aria, sacrum, distal radius, never in the skull!  **Examples** Afbeelding met röntgenfilm, Medische beeldbewerking, radiologie, radiografie Automatisch gegenereerde beschrijving  Afbeelding met röntgenfilm, Medische beeldbewerking, radiologie, radiografie Automatisch gegenereerde beschrijving **Giant cell containing lesions of bone** Histological evaluation: - Morphological criteria are given by the mononuclear cell compartment - Exception: size and distribution of the giant cells *When you do a histological evaluation you have to look at the cells surrounding the giant cell, because these are the tumor cells. But you also have to look a bit at the giant cells themselves, you look at the size and the distribution. In giant cell tumors, the giant cells are equally distributed!* **Central theme: osteoclast like giant cells** They can have more nuclei per cell  **Bone cells involved in the remodeling** *Osteoclast are normally involved in bone remodeling* - Osteoprogenitor cells (MSC) - Osteoblasts and lining cells - Osteocytes - Osteoclasts Afbeelding met kaart Automatisch gegenereerde beschrijving **Osteoclast function** The osteoclast absorbs the bone You have the **Howships lacuna**, and the you have deposition of H+, this creates an acid environment. This takes away the minerals of the bone  **Giant cell tumor of bone** There are three cell types: - Stromal cells - Mononuclear cells - Multinuclear cells You can sometimes also see spindle cell changes Afbeelding met Lila, paars, violet, stof Automatisch gegenereerde beschrijving You can sometimes also see bone deposition, metaplastic bone formation  **Malignancy in giant cell tumours** *Giant cell tumor of bone is intermediate, as small percentage is malignant.* *You can have primary or secondary* Primary: high grade sarcoma arising in/next to a giant cell tumor - Rare: \ end of chromosomes can stick together - Mutation: **H3f3A G34** -\> driver mutation -\> hallmark The mutation is used to distinguish GCTB from CB (chondroblastoma): Giant cell tumor of bone (GCTB) -\> **H3F3A, often position G34 G\>T** Chondroblastoma (CB) -\> **H3F3B, often position K36M A\>T** H3F3A and H3F2B both code for the histone H3.3 protein The histon genes are involved in histon modification, and they can regulate gene expression. 92% giant cell tumours H3F3A G34 mutations 95% chondroblastomas H3F3B K36M mutations Afbeelding met tekst, schermopname, diagram, lijn Automatisch gegenereerde beschrijving **Sensitivity in mutation detection depends on technique**  All other giant cell containing bone tumours are negative, none of them have these mutations There is an antibody for the mutant protein! -\> IHC [The antibody is specific for G34W (in H3F3A)] [Another antibody is specific for K36M] Other mutations are not picked up by this antibody. So if the IHC is negative, than it doesn't rule out GCTB or CB There are also antibodies for the rarer variants, but these are not used routinely H3F3A G34V in GCTB (5-6%) H3F3A G34R in GCTB (2-5%) The mononuclear cells will be positive, but the giant cells will be negative The giant cells are attracted to the tumor, but they are not tumor cells themselves **Solid ABC versus giant cell tumour** Solid aneurism bone cist is very similar to giant cell tumor, so molecular diagnostics can be used to distinguish them. Solid ABC GCTB \~60% USP6 rearrangement No USP6 rearrangements No H3F3A mutation 69-96% H3F3A mutations  **Giant cell tumour of bone: treatment** - Locally aggressive - Often local recurrences (40-60%) - Lung metastases (\ Curettage \> Excision **Metastases of giant cell tumor** - Rare (\ high grade conventional osteosarcoma **Osteosarcoma** *Osteosarcoma occurs most often around the knee, not in the knee.* *At the end of the long bones.* *It is characterized by osteoids, this is non mineralized bone matrix.* **Diagnosis of osteosarcoma** - Bone producing malignant cells - Painful mass in the bone (especially at night because of low level corticosteroid) - Mostly in the long bones of the [extremities], especially distal femur and proximal tibia - Conventional high grade is the most common and very aggressive - X-ray for first diagnosis  Both lytic and sclerotic Afbeelding met röntgenfilm, Medische beeldbewerking, radiologie, Medische radiografie Automatisch gegenereerde beschrijving Codman triangle The periost is lifted by the tumor in de bone.  **Histology** - Severe anaplasia and pleomorphism (different formed nuclei) - Eosinophilic cytoplasm (more pink) - Identification of neoplastic disorganized woven bone with lace like pattern - Unmineralized matrix \> **osteoid** - Subclassification according to matrix (some make cartilage, some bone, and some fibroblastic tissue) - No specific immune histochemical staining (**SATB2**) -\> this is a marker for bone differentiation.  **Classification** *This classification is based on severity* - Conventional osteosarcoma: 80% - Telangiectatic osteosarcoma: \~4% -\> a lot of blood vessels and blood - Small cell osteosarcoma: 1.5% -\> less aggressive form, less metastasis - Several other rare osteosarcoma subtypes Conventional OS Telangiectatic OS  **Clinical** - Most prevalent malignant primary bone tumor - Occurs mainly in adolescents/young adults - Bimodal age distribution (young adults, but also older people because of radiation or Paget's disease)  **Survival of osteosarcoma patients** Chemotherapy resistance: poor survival in 40% of the patients Afbeelding met tekst, diagram, lijn, Perceel Automatisch gegenereerde beschrijving **Treatment of osteosarcoma** \> Surgery, amputation or limb salvage \> But there are often metastases, especially to the lungs are the cause of poor survival [Neo-adjuvant chemotherapy (before surgery, less heavy surgery) ] - Doxorubicin - Cisplatin - Methotrexate [Surgery \> resection is studied response to chemotherapy ] - \>90% necrosis: considered as good response - Associated with good prognosis [Post surgery chemotherapy ] If the neo-adjuvant chemotherapy has a good response histologically then it will probably also help afterwards. Since introduction of adjuvant chemotherapy no improves survival \> fatal because of lung metastases **Issue that hamper osteosarcoma studies** 1. Relatively rare 2. Preoperative chemotherapy destroys tissue for research 3. No specific hereditary syndrome for OS 4. No benign precursor 5. Gross genetic instability, chromothripsis & kataegis (there are a lot of breaks in the chromosomes) **Immunotherapy for osteosarcoma** *Ongoing research* - High genetic instability \> neoantigens - There is also a high T-cell infiltrate - You need HLA expression - Expression of T-cell blocking proteins Blocking checkpoints: \> Avoiding immune destruction \> genome instability & mutations **Pathways in osteosarcoma** *Research* *There are two gene inactivated -\> RB and P53*  **Osteogenic tumours and hereditary disorders** These hereditary disorders can cause osteosarcoma Afbeelding met tekst, schermopname, Lettertype, nummer Automatisch gegenereerde beschrijving **Li-Fraumeni syndrome** P53 gene  - Li Fraumeni syndrome is autosomal dominant with high penetrance (93% before age 50) - TP53 gene is mostly mutates, sometimes CHK2 - Sarcoma is the index diagnosis - 25% of LFS tumors are sarcoma - Of these 40% are osteosarcoma Of note: TP53 mutations also occur in dog osteosarcoma **P53 and the cell cycle** *P53 detect molecular changes and stops the cell cycle.* *Loss of P53 leads to genetic instability* Afbeelding met tekst, schermopname, Lettertype, diagram Automatisch gegenereerde beschrijving **Knudson's two-hit hypothesis for tumor progressor gene inactivation**  **Retinoblastoma** - Malignant tumor of the retina (primary) - Birth/childhood - Unilateral/bilateral - Sporadic/hereditary - Second tumor: 60% sarcoma, mainly osteosarcoma **Rb mutation spectrum** RB1 is a huge gene Mutations occur on all parts of RB1 **RB and the cell cycle** *Rb is also involved in the cell cycle* Afbeelding met tekst, diagram, cirkel, schermopname Automatisch gegenereerde beschrijving Rb and osteosarcoma: 70% of primary osteosarcomas **Rb1/Tp53 mouse model** - Conditional knockout for Rb1 and Tp53 \> only osteoblastic precursor cells have Rb1 and Tp53 loss (Osx-Cre) - These mice get osteosarcoma - These osteosarcomas are mostly metastatic - Apparently there is synergy between Rb and Tp53, needs to occur both **Paget's disease of bone** - Increased bone resorption - Increase bone formation - Pain, fractures, deformities - Bone tissue of poor quality - Secondary tumors - Primary abnormality resides in [osteoclasts] (osteoclasts are to active) Dense inactive, largely acellular bone  **Osteosarcoma in Paget's disease** - Increased risk to get osteosarcoma (0.15-1%) - Age of onset is late - Characterized by giant multinucleated osteoclasts Genes involved in hereditary Paget's: - **SQSTM1** also known as TNFRSF11A - Regulate osteoclastogenesis - Not mutated in sporadic osteosarcoma **Concluding** - Osteosarcoma is a highly malignant tumor that occurs in young patients - Treatment options are sparce - Osteosarcoma is characterized by genetic instability - Genes involved in genetic stability may be mutated in hereditary and sporadic osteosarcoma - TP53 and RB1 are tumor suppressor genes and mutated in hereditary osteosarcoma as well as sporadic - Paget's disease of bone is associated with an increase risk for late onset osteosarcoma