Hematopoietic stem cell transplantation-induced bone remodeling in autosomal recessive osteopetrosis: Interaction between skeleton and hematopoietic and sensory nervous systems

Journal Pre-proof Hematopoietic stem cell transplantation-induced bone remodeling in autosomal recessive osteopetrosis: Interaction between skeleton and hematopoietic and sensory nervous systems

Natalia Maximova, Floriana Zennaro, Massimo Gregori, Giulia Boz, Davide Zanon, Gabriel Mbalaviele PII:

S8756-3282(19)30438-7

DOI:

https://doi.org/10.1016/j.bone.2019.115144

Reference:

BON 115144

To appear in:

Bone

Received date:

18 July 2019

Revised date:

29 October 2019

Accepted date:

5 November 2019

Please cite this article as: N. Maximova, F. Zennaro, M. Gregori, et al., Hematopoietic stem cell transplantation-induced bone remodeling in autosomal recessive osteopetrosis: Interaction between skeleton and hematopoietic and sensory nervous systems, Bone(2018), https://doi.org/10.1016/j.bone.2019.115144

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© 2018 Published by Elsevier.

Journal Pre-proof

Title: “Hematopoietic stem cell transplantation-induced bone remodeling in autosomal recessive osteopetrosis: interaction between skeleton and hematopoietic and sensory nervous systems”. Natalia Maximova1, Floriana Zennaro2, Massimo Gregori1, Giulia Boz3, Davide Zanon1, Gabriel Mbalaviele4. 1. Natalia Maximova, Institute for Maternal and Child Health - IRCCS Burlo Garofolo, via

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dell’Istria 65/1, 34137 Trieste, Italy, [email protected]; 2. Floriana Zennaro, Hôpitaux Pédiatriques de Nice, CHU Lenval, 57 Avenue de la Californie, 06200 Nice, France, [email protected];

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1. Massimo Gregori, Institute for Maternal and Child Health - IRCCS Burlo Garofolo, via dell’Istria

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65/1, 34137 Trieste, Italy, [email protected];

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3. Giulia Boz, University of Cagliari, Cittadella Universitaria di Monserrato, S. P. Monserrato Sestu Km 0.700 CA, 09042 Monserrato, Cagliari, Italy, [email protected].

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1. Davide Zanon, Institute for Maternal and Child Health - IRCCS Burlo Garofolo, via dell’Istria

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65/1, 34137 Trieste, Italy, [email protected]; 4. Gabriel Mbalaviele, Division of Bone and Mineral Diseases, Department of Medicine,

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Washington University School of Medicine, 660 S. Euclid Ave, CB 8301, St. Louis, MO 63110, USA, [email protected]. Corresponding Author: Natalia Maximova, M.D. Bone Marrow Transplant Unit Institute for Maternal and Child Health - IRCCS Burlo Garofolo Via dell’Istria 65/1, 34134 Trieste, Italy. Phone: +39040378565; Fax: +390403785494 E-mail: [email protected]

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Journal Pre-proof ABSTRACT Objective: Autosomal recessive osteopetrosis (ARO) is a rare congenital disorder of defective bone resorption. The inability of osteoclasts to resorb bone compromises the development of bone marrow cavity, and ultimately, leads to defective hematopoiesis and death within the first decade. The only curative treatment currently available for certain forms of ARO is hematopoietic stem cell transplantation (HSCT). Infants over ten months of age suffering from ARO are defined as patients

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with advanced disease; HSCT to these patients is associated with high risk of transplant-related mortality (TRM). Because of the extreme variability of ARO clinical phenotypes, the most reliable predictive factor of TRM and graft failure risk is the residual bone marrow space volume.

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Case report: We report clinical and radiological outcomes of one patient affected by ARO and

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treated with HSCT at advance stage of the disease. We describe the anomalies in various tissues,

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including bone marrow and bones at the moment of the diagnosis and document their gradual disappearance after HSCT until their complete resolution based on magnetic resonance imaging

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(MRI) observations. We provided radiological images of the cranial vault bone structure

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modifications, correlating the radiological appearance of the optical canals and nerves and of the

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cerebellum with the neurological manifestations of the disease. Conclusions: Our results demonstrate that MRI is a highly sensitive technique that provides excellent images of bone marrow space before and after HSCT without exposing children to ionizing radiation. MRI also permits us to evaluate post-transplant skeletal remodeling and the deriving changes in the hematopoietic and sensory system.

Keywords: autosomal recessive osteopetrosis; hematopoietic stem cell transplantation; bone marrow space volume; magnetic resonance imaging.

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Journal Pre-proof 1. Introduction Autosomal recessive osteopetrosis (ARO) also known as malignant infantile osteopetrosis, represents a group of genetically and phenotypically heterogeneous rare inherited skeletal disorders, characterized by a marked increase in bone mass owing to defective bone resorption by the osteoclasts and bone fragility. The natural course of the disease is featured by severe bony abnormalities, progressive destruction

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of the medullary space by abnormal bone, gradual blindness due to cranial nerve compression, hematopoiesis failure and death within the first decade [1-4]. Several mutations are associated with ARO. Indeed, loss-of-function mutations in TCIRG1, CLCN7, OSTM1 or PLEKHM1 lead to

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osteoclast-rich osteopetrosis, in which osteoclasts are abundant but not functional [5-7]. Mutations

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in SNX10 are also linked to a form of ARO associated with defective osteoclast function [8]. By

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contrast, loss-of-function mutations of RANKL or RANK are associated with osteoclast-poor forms of ARO, in which osteoclastogenesis is impaired [9]. So far, hematopoietic stem cell transplantation

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(HSCT) is the only therapy available to treat ARO patients with osteoclast-intrinsic defects (e.g., RANK mutations) and without neurodegenerative complications [10-13]. HSCT is contraindicated

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for RANKL- osteoclast-poor form of ARO [14] because hematopoietic stem cells (HSCs) cannot

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restore cells producing RANKL, which arise from the mesenchymal stem cell lineage [15]. An early recognition of the disease and a prompt treatment performed in an experienced Transplant Center is crucial in determining the transplant outcome. Unfortunately, ARO remains often unrecognized and in most cases, the diagnosis is made at eight/nine months of life, when the disease is already advanced. Currently, infants over 10 months of age are defined as patients with advanced disease and HSCT in these patients is associated with high risk of transplant-related life-threatening complications such as graft rejection, graft failure, toxicity and infections [14]. However, ARO is characterized by an extreme variability of clinical phenotypes, and the age of the patient as such, could be an insufficient and unreliable factor to estimate the transplant-related mortality (TRM). Haploidentical transplant is needed in advanced disease because the donor is promptly available, 3

Journal Pre-proof while matched unrelated donor is preferred in younger patients with low risk of TRM. The most reliable predictive factor of the TRM is the residual bone marrow space volume, which allows to precisely define the graft failure risk [2]. For the patients with advanced disease who cannot wait for the search time of a matched unrelated donor (MUD) and don't have an HLA matched sibling, the haploidentical HSCT represent the most suitable therapeutic option, as it can be immediately performed and ensures the same outcome of HSCT from MUD or sibling. Bone marrow biopsy is considered as the gold standard examination to accurately evaluate the bone marrow space.

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However, it is not recommended in ARO patients because of the extremely high risk of fracture. In this research report, we describe the modifications induced by donor healthy HSCs to totally

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altered bone marrow microenvironment and bone structures, which led to a complete macroscopic

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resolution. A particular focus is on the patient with advanced disease stage who underwent frequent and specific post-transplant radiological imaging, which allowed us to richly document the

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remodelling of bone structures and subsequent modifications that occurred in other organs after the

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engraftment of donor HSCs. This patient was admitted to our Bone Marrow Transplant Unit 4 years ago, when he was 12 months-old and was diagnosed with ARO (Fig. 1). The detection of

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homozygous g.9909 G>A mutation in exon 14 of TCIRG1 gene confirmed the ARO form of the

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disease. The stage of the disease was already advanced as evidenced by the clinical examination showing bilateral exophthalmos associated with wandering nystagmus, skull bossing, closed occipital fontanelle and small and full anterior fontanelle, hepatosplenomegaly, short stature and delayed motor development; the infant could not sit, roll or crawl, and general movements were extremely limited. Visual evoked potentials showed a decrease in amplitude (7 μV) and an increase in P2 latency (146 ms). Blood investigations revealed anemia (8.2 g/dL haemoglobin; 9.5 - 13.5 g/dL normal range, n.r.), thrombocytopenia (75 x 109/L platelet count; 150 – 450x 109/L, n.r.), increased levels of alkaline phosphatase (848 U/L; 0 - 445 U/L, n.r.) and parathyroid hormone (201 pg/mL PTH; 12.0 - 95.0 pg/mL, n.r.), decreased levels of IGFBP3 (0.56 ng/mL; 12.0 - 95.0 pg/mL, n.r.), IGF1 (< 25.00 ng/mL; 55.00 – 327.00 ng/mL, n.r.), and ACTH (< 5.00 pg/mL; 7.00 - 10.00 4

Journal Pre-proof pg/mL, n.r.), and abnormal levels of HbF (4.11 gr/dL; < 2.0 gr/dL, n.r.). Urine calcium concentration was undetectable. An unfavourable prognosis of limited probability of success of HSCT and high risks of transplant related mortality associated with the advanced stage of disease was communicated to the parents. Still, a consent agreement form was signed by the parents, and haploidentical transplant was immediately performed. The patient underwent neurosurgery for suboccipital decompression and cervical laminectomy followed by the first haploidentical HSCT. An initial engraftment was achieved, but rejection

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occurred two months later. A second haploidentical transplant was performed ten weeks after rejection, which led to permanent engraftment of donor’s HSCs. The patient developed potentially

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fatal complications as veno-occlusive disease, sepsis, Ebstein-Barr virus (EBV)-related post-

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transplantation lymphoproliferative disease and hyperacute graft versus host disease (GVHD) following donor lymphocyte infusion for EBV proliferation. Fortunately, the patient overcame these

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complications; he is now 5 years old, and enjoys almost all normal life activities.

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2.1 Patient

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2. Materials and Methods

The patient underwent a single MRI-based pre-transplant bone evaluation and multiple post-

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transplant MRI-based bone assessments, every three weeks until complete replacement of recipient's hematopoietic system with functional donor's hematopoiesis was achieved, then every 3 months until the complete reconstruction of bone marrow space. CT scans were performed before HSCT and after 3, 13 and 18 months. 2.2 Control group Bone marrow space of the patient at the age of 11, 13, 15, 18, and 36 months was compared to bone marrow space of the healthy children of the same age and sex. The control group consisted of 28 pediatric patients, aged 11 to 36 months, who underwent pelvic MRI for orthopedic diseases, traumas, or before and after urogenital tract correction surgery. Skull of the Patient 1 at the age of

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Journal Pre-proof 11 months was compared with skull of 10 healthy children of the same age and sex who underwent cranial CT for trauma. 2.3 Transplantation Procedure Haploidentical HSCT was performed after myeloablative standard conditioning regimen based on treosulfan (30 g/m2 total dose) in the first HSCT and oral busulfan (480 mg/m2 total dose) in the second HSCT. Myeloablative conditioning was completed with high dose of a second alkylating agent as described previously [16]. Immunoablation therapy was performed with cyclophosphamide

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and antithymocyte globulin. High dose (> 6 x 108/kg) of donor's hematopoietic nuclear cells were infused. GVHD prophylaxis was performed with a calcineurin-inhibitor and mycophenolate

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2.4 Computed-tomography (CT) evaluation

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mofetil.

CT scans were performed using a Philips Brilliant 40, spiral machine, with pediatric age-adapted

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protocols for head, pelvis and low limbs, in 0.5 mm slices. 2D and 3D reconstructions were

analysis.

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obtained from coronal sequences. The obtained CT images were transferred to a workstation for

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2.5 MRI-based bone assessment

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MRI-based assessment of the pelvic bone was performed using a 1.5-Tesla Philips imager (Ingenia; Philips, Eindhoven, the Netherlands) with a body coil, according to a standard protocol based on Proton Density (PD), T2 Spectral Presaturation Inversion Recovery (SPIR) with a 3.5-mm slice thickness and T2 Short Tau Inversion Recovery (STIR) with a 4-mm slice thickness axial sequences. 2.6 Bone marrow space volume assessment Post-transplant pelvic bones MRI-based assessments were performed according to the standard protocol at regular and short time intervals. MRI sequences of the pelvic bones were analyzed to obtain the total bone volume and the total bone marrow space volume, using the same technique for the ARO patient and the control group. Direct comparison of the total pelvic bone marrow space 6

Journal Pre-proof between the ARO patient and the healthy control children of the same age can be biased by the well-known growth failure affecting ARO patients. Therefore, we compared the bone marrow space volume / bones volume ratio (BMSV/BV ratio) of the pelvic bones. A single senior radiologist, specialized in pediatric MRI, reviewed all the MR images. The region of interest was the pelvic bone. 3D bone and bone marrow reconstructions were performed using the open source Horos software (distributed under the LGPL license at Horosproject.org and sponsored by Nimble Co LLC d/b/a Purview in Annapolis, MD USA). The area of the bone marrow

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was manually traced on axial scans. The image analysis software calculated the segmental volume of each marrow slice and the total bone marrow volume was computed as the sum of the slice

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volumes. The total volume of pelvic bones was calculated with the same procedure.

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3. Case report

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We subjected ARO patient to diagnostic procedures to investigate the mechanisms underlying ARO symptoms and body changes induced by HSCT. We described all the structural anomalies found at

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complete resolution.

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the moment of the diagnosis, and monitored and documented their gradual disappearance until their

3.1 Pre-transplant residual hematopoiesis evaluation

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We, first of all, performed a CT of the hip bone to accurately evaluate the tissue morphology and to measure the residual bone marrow space of ARO patient. These data were necessary to estimate the probability of engraftment of donor’s HSCs. In the normal bone, CT distinguished the interior cancellous component from the exterior cortical bone (Fig. 2 a) of the healthy control, but not in our patient with advanced phases of ARO (Fig. 2 b). Therefore, we performed a MRI imaging of the pelvic bone to evaluate the residual bone marrow space. T2 STIR sequence of the normal bone structure showed a hypointense signal in cortical bone and a hyperintense signal in cancellous bone (Fig. 3 a). ARO coxal bone in the same MRI sequence revealed a completely subverted structure consisting of a central hypointense area surrounded by a

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Journal Pre-proof tissue with otherwise hyperintense signal (Fig. 3 b). MRI imaging shows the development of a pathological tissue that didn't have the radiological characteristics of normal bones. The absence of the residual bone marrow space suggested the occurrence of extramedullary hematopoiesis. The patient was therefore subjected to abdominal MRI to evaluate iron concentrations in soft tissues. The splenic parenchyma showed hypointense signal in T2 weighted star and T2 highly-weighted star sequences before HSCT (Fig. 4 a). This unexpected finding for a child who did not present hemoglobinopathy and never received blood transfusion suggested the

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presence of extramedullary hematopoiesis. Notably, normal bone marrow hematopoiesis was restored 6 months post-HSCT (Fig. 4 b), a view that was consistent with the appearance of the

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physiologic hyperintense splenic signal in T2 weighted star and T2 highly-weighted star sequences

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in the absence of any chelation treatment. This data is consistent with the hypothesis of antecedent splenic extramedullary hematopoiesis in the patient.

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3.2 Monitoring of the bone marrow space volume after HSCT

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The first post-transplant assessment was performed at day 24 after partial donor’s HSCT engraftment (WBC 330/mm3) was achieved. Total pelvic bone marrow space volume was 1.37

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mm3, corresponding to a BMSV/BV ratio of 2.6% for the patient compared to a ratio of 53.6% for

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the control group. Concomitant molecular analysis of chimerism performed on whole peripheral blood showed 88% of cells originate from the donor. The second post-transplant MRI-based assessment was performed at day 37, when WBC exceeded 1000/mm3 and showed a marrow space volume of 4.2 mm3 (BMSV/BV ratio of 8.3% versus 53.6% for the control group). Concomitant molecular analysis demonstrated the presence of an unstable mixed chimerism as only 80% of donor cells were detected. Follow up assessments of bone marrow volume, velocity of bone marrow space enlargement, and chimerism were performed every three weeks. A progressive decrease of the velocity of bone marrow space enlargement associated with an increase in the percentage of recipient cells was observed. At day +79 after HSCT, the percentage of donor cells had dropped to 7%, and the total marrow volume was 8.6 mm3 (BMSV/BV ratio of 15.1% versus 56.3% for the 8

Journal Pre-proof control group). At that moment, the child received the second haploidentical HSCT, which led to a rapid engraftment, with full-donor chimerism achieved in all three lineages, and a sudden acceleration of the velocity of the bone marrow space enlargement. Twenty-one months after the second HSCT, BMSV/BV ratio of the ARO patient was comparable to that of the control group (53.0% versus 57.0% respectively), with a total bone marrow volume of 52.1 mm3 (Fig. 5 a-l). The modifications of BMSV/BV ratio of ARO patient related to donor chimerism and total leukocyte count, and comparison with the control group of the same age are displayed in Table 1.

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3.3 Remodelling of appendicular skeleton after transplantation

We evaluated the remodeling of the appendicular skeleton in ARO patient and noticed that most

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notable outcomes occurred in the femur. Indeed, pre-transplant CT evaluation showed the femurs as

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a homogeneous structure composed of compact bone with a high density core, virtually occupying the whole medullary cavity. What remained of the medullary cavity was an extremely thin

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semicircular hypodense line, dividing the outer cortical bone from the abnormal hyperdense inner

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compact bone. The signal density was higher in the central area, suggesting a centrifugal growth of pathological compact tissue (Fig. 6 a). The first post- transplant CT-based evaluation of the femurs,

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performed three months after the first HSCT showed the appearance of a central hypodense area of

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2 mm in diameter, corresponding to an initial medullary cavity surrounded by compact bone, 5.1 mm thick (Fig. 6 b). Thirteen months after the first HSCT, the medullary cavity was obvious; the diameter reached 5.3 mm with the trabecular bone structure easily identifiable, and compact bone thickness reduced to 4.2 mm (Fig. 6 c). The last CT-based evaluation performed 18 months after the first HSCT showed apparently normal bones with a normal cortical bone/medullary cavity ratio for the age (Fig. 6 d). MRI-based assessment of the hips and femurs revealed radical changes of bone architectures. Resorption and remodeling of the excessive cortical bone led to progressive restoration of the anatomical balance between cortical and trabecular bone, normalization of the ossification center in the femoral head, and the appearance of the ossification center in the trochanter (Fig. 7 a-c). 9

Journal Pre-proof Further, we analyzed the markers of bone turnover and mineral metabolism. The period of intense bone resorption was characterized by marked hypercalcemia (> 11.5 mg/dL; n.r. 8.50 - 10.50 mg/dL) and hypercalciuria (Ca/creatinine ratio > 2 mg/mgcrea; n.r. < 0.11 mg/mgcrea), high serum levels of C-terminal cross-linking telopeptide (CTX) with serum values > 2.0 ng/mL (n.r. 0.12 – 0.75 ng/mL). While, the bone formation markers, as osteocalcin and bone alkaline phosphatase, were normal. 3.4 Remodelling of axial skeleton after transplantation

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Here, we document all remodelling phases of the skulls following HSCT based on CT imaging, three-dimensional (3D) reconstructions, and MRI investigations. Pre-transplant evaluation of the

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calvaria of ARO patient showed evidence of pansynostosis and fused sutures, opened anterior

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fontanelle and frontal bossing (Figure 8 a). Three-year after transplant the calvaria evaluation showed convolutional markings in parietal regions described as a copper beaten skull appearance,

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as a result of the increased intracranial pressure (Figure 8 b). Moreover, CT-based assessment the

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skull roof of ARO patient at baseline (Fig. 9 a) revealed significant bone thickening and complete loss of the normal architecture, consisting of the inner and outer tables of compact bone, separated

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by the diploe (cancellous bone). Post-transplant evaluation performed 30 months later, showed a

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marked and irregular thinning of the calvaria (Fig. 9 b). The two cortical tables were clearly distinguishable from the diploe. In some areas, though, only the outer table was evident, as a result of the forces exerted by the dura mater on the cranial vault bones. Sagittal sequences based on baseline MRI assessment showed a spherical cranial shape (Fig. 10 b), with a disproportion between cranial height (CC) and anteroposterior diameter (AP). The ARO patient’s AP was 15.2 cm whereas the AP of the control group was 15.4 (±0.6) cm; ARO patient’s CC was 13.0 cm versus 11.8 (±0.5) cm of control group; and the ARO patient presented a modified cranial index (CI) of 85.41 cm, versus 76.8 (±2.9) cm of control group (Fig. 10 a). At the posttransplant assessment, performed 3 years later, the skullcap shape had become oblong and the modified CI normal (77.43 cm). The thickness of the frontal and occipital bones appeared to be 10

Journal Pre-proof normal. The parietal bone in the vertex region presented a reduced thinning process, probably because of the reduced force exerted by the meninges along the craniocaudal axis (Fig. 10 c). 3.5 Visual impairment To understand the nature of the visual impairment affecting ARO patients, we studied the optic canals by analyzing axial CT sequences with bone window setting (Fig. 11 a,b). 3D reconstruction of the diameter of the optic canals of ARO patient was based on manual measurements (Fig. 11 c). Measurements were performed before HSCT and 12 months after HSCT (Fig. 11 d,e). The

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diameters obtained were 5.3/5.3 mm and 5.2/5.1 mm, and corresponded to normal values. No significant difference between pre- and post-transplant measurement was evident. This data

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suggested that blindness in ARO is not due to stenosis of the optic canals, but probably caused by

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previous prolonged nerve elongation. 3.6 Wandering Nystagmus

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One of the first clinical sign of ARO is nystagmus, which derives from the vestibulocerebellar

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injury caused by the Chiari malformation type 1. The vestibulocerebellum corresponds to the flocculonodular lobe of the cerebellum and is responsible for eye movement control. The pre-

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transplant MRI brain evaluation of ARO patient showed cerebellar compression and tonsillar

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herniation (Fig. 10 b).When compared to a healthy control child (Fig. 10 a) the cerebellum presented poor folia representation and narrowed lateral and posterior cerebrospinal fluid spaces. We found a correlation between the radiological imaging and the clinical manifestations of nystagmus in ARO patient. Before transplantation, when nystagmus was clinically evident, based on MRI evaluation the flocculonodular lobe was not identifiable, while tonsillar herniation was marked (Fig. 12 a,b). Post-transplant MRI-based evaluations performed 6 and 30 months after HSCT when nystagmus had disappeared, and tonsillar herniation was resolved, we noticed the presence of two small wispy appendages in the posterior region of the cerebellum, known as flocculi, which with the nodulus compose the flocculonodular lobe (Fig. 12 c-f). 4. Discussion 11

Journal Pre-proof Bone is a highly dynamic tissue whose quality after birth is maintained through the removal of old bone (bone resorption) carried out by the osteoclasts followed by de novo bone formation by the osteoblasts [17]. However, excessive bone resorption at the expenses of bone formation underlies the pathogenesis of osteoporosis whereas severe defective bone resorption relative to bone formation causes osteopetrosis [18]. Impaired osteoclast differentiation causes osteoclast-poor osteopetrosis whereas defective osteoclast bone-resorbing activity is responsible for osteoclast-rich osteopetrosis [5, 18]. Histological analysis of bone biopsies for confirmation of the osteoclast-rich

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osteopetrosis diagnosis was not performed in this study because of concerns of enhancing fracture and infection risk to ARO patients. Despite this shortcoming, the identification of G>A substitution

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in TCIRG1 combined with the successful outcomes following HSCT strongly support the

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proposition that our patients are affected by osteoclast-rich osteopetrosis. Indeed, inactivatingmutations in TCIRG1 are linked to osteoclast-rich osteopetrosis in which osteoclasts are abundant,

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but not functional due to defective function of TCIRG1. TCIRG1 encodes vacuolar (V)-ATPase 116

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kDa isoform a3, a subunit of the osteoclast vacuolar proton pump contained in vesicles that fuse with the ruffled border and mediates the secretion of H+ to the resorption lacuna. Acidification of

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this compartment results in bone demineralization, a process that enables osteoclast-derived

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proteases such as cathpesin K to have access to and digest the organic phase of the bone matrix. Lining cells, stromal osteoblasts and osteocytes are important sources of RANKL, the obligatory cytokine for osteoclast differentiation. We speculate that transplanted HSCs are exposed to a host bone marrow microenvironment characterized by high RANKL/osteoprotegerin (OPG) ratio. Overstimulation of transplanted HSCs leads to a robust differentiation of normal and active osteoclasts, and ultimately, a robust bone resorption. Obviously, better outcomes are expected with ARO patients subjected to HSCT in their first year of life, a period when the skeleton is genetically programmed to quickly change in size and shape to ensure growth. Osteoclast activity culminates in the increase of bone marrow space. The velocity of bone marrow reconstruction is mainly influenced by two factors: the grade of the donor-recipient chimerism and 12

Journal Pre-proof the inflammatory state of the patient. Higher percentages of donor’s HSCs is associated with higher number of osteoclasts and, thus, with a more rapid bone resorption. The patient presents an abrupt acceleration in the bone marrow space enlargement after the second HSCT. This acceleration is documented during the acute systemic III grade GVHD associated with very high levels of TNF- and IL-6, cytokines that are known to cause exaggerated osteoclastogenesis [19,20]. Notably, untreated ARO patients have high levels of PTH, perhaps as a result of the organism attempts to stimulate bone resorption and calcium reabsorption [21].

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In advanced stages of ARO, defective hematopoietic niches in the bone marrow environment trigger extramedullary hematopoiesis [22-24]. Because the splenic microenvironment is

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characterized by hypoxic and acidic conditions with the presence of numerous macrophages that are

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harsh and inhospitable for HSCs, extramedullary hematopoiesis usually occurs within the red pulp of spleen [25]. Sinus endothelial cells of red pulp express CXCL12, a marker of the bone marrow

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niche-constituting cells, which contributes to the attachment and recruitment of circulating

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hematopoietic precursor cells and formation of bone marrow niche-like islands of extramedullary hematopoiesis [26]. When the physiologic medullary space is restored, splenic hematopoiesis

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ceases and the spleen reacquires its physiologic hyperintense signal in T2 weighted star and T2

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highly-weighted star MRI sequences.

In advanced phase of ARO, and only in this phase, the bone structure is so altered that it is impossible to distinguish cortical and medullar bone. In our study we noticed that together with the donor’s hematopoiesis attachment, hyperintense in PD, SPIR and STIR sequences red marrow islands appeared in the central region of the ilium. The volume of this hyperintense areas increased together with the percentage of the donor’s hematopoiesis. Accordingly, even in case of unstable chimerism during rejection, the small number of active osteoclasts continue to resorb cortical bone [10]. HSCT also leads to important structural modifications to the axial skeleton. At birth, the normal cranial vault consists of unilaminar tables (flat bones) conjoint by flexible fibrous tissue (sutures) 13

Journal Pre-proof and separated in precise points by rather wide open spaces (fontanelles) [27]. One year after birth, all fontanelles are closed, but the bones remain separated by a thin periosteum line suture, which remain until adult age [28]. The normally functioning sutures, acting as areas of growth and adjustment allows calvarium remodeling that accommodates rapid brain growth [27]. In patients with ARO, the cranial vault consists of a unilaminar sheet of abnormal thickness with a bone bridge between the fused sutures. Despite the premature closure of the sutures, unlike the patients with craniosynostosis, where the skull appears harmonic and not deformed. We speculate that skull

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expansion in ARO follows the same pattern of the expansion of the femur, with an initial prevalence of bone resorption of the inner face of the vault, followed by the bone deposition in the

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outer face.

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The remodeling of any bone follows the Wolf’s Law [29]. In the cranial vault, the stimulus arises primarily from the expanding brain, sending signals by means of the dura mater [30]. The normal

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brain is mainly distributed along the antero-posterior axis and the neurocranium development

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follows the same axis. By contrast, the neurocranium in ARO patients presents a spherical shape and the brain is subjected to a higher compression along the sagittal axis. Consequently, after

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HSCT, the parietal bone acquires a normal thickness in close proximity of the frontal, occipital and

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squamous margin, but remains thickened at the skull vertex as a consequence of the lower force exerted by the dura mater along the craniocaudal axis. The optic canal stenosis is considered the only cause of visual impairment in ARO patients [31-33]. On the other hand, some authors have demonstrated that the correlation between visual impairment and optic canal size is unreliable [34,35]. Moreover, the visual function recovery after HSCT is rare [36], even if it is performed early [11]. In our study, the pre-transplant evaluation of the optical canals of ARO patient showed normal bilateral diameters according to age, in spite of a severe visual impairment. Post-transplant measurements of the optic canals performed on all the three patients after normalization of the bone structure showed outcomes that are similar to physiological values. We argue that in the case described in our study, the pre-transplant optic nerve elongation could be responsible for the visual 14

Journal Pre-proof impairment. The wandering nystagmus also represents a typical characteristic of these patients. It derives from the compression of the cerebellum, particularly of the flocculonodular lobe (vestibulocerebellum), which is responsible for the oculomotor control [37,38]. The volume of the posterior cranial fossa is extremely reduced in ARO patients, a defect that can lead to compression of the cerebellum, particularly the flocculonodular lobe. Because of its anatomical position, this lobe is compressed and is pushed forward to the brainstem. In the pre-transplant MRI sequences, the flocculonodular lobe and brainstem appear indistinguishable, but after HSCT, this lobe becomes

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evident when the nystagmus disappears.

Since 1977 HSCT had been successfully applied for ARO treatment. In 1994, 69 ARO patients

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were retrospectively analyzed showing an overall survival (OS) of 43,5% with a significantly better

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outcome for patients receiving HSCT from an HLA-identical donor [11]. A retrospective analysis of 122 ARO patients who received an HSCT between 1980 and 2001 showed the OS of 46% with

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functioning osteoclasts at last evaluation. Also in this case, the disease free survival according to the

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type of donor was significantly better in patients receiving an HLA identical graft [39]. The goal of HSCT in ARO is to repopulate the residual bone marrow spaces with hematopoietic

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stem cells producing osteoclasts, to rapidly restore the physiologic medullary environment and,

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ultimately, the effective and definitive hematopoiesis. This goal is achieved in this study. MRI of the pelvis provides excellent images of bone marrow spaces and is a highly sensitive technique for evaluating residual bone marrow space before HSCT. STIR imaging represents a powerful tool for the evaluation of bone marrow abnormalities [40]. Only one sequence is enough to get all the information on the bone details, minimizing the time of sedation. It is also an ideal technique for monitoring any modifications after HSCT without exposing children to ionizing radiation.

15

Journal Pre-proof Acknowledgements and Conflict of interests: Dr. Mbalaviele is supported by NIH/NIAMS AR064755 and AR068972 grants. He is consultant for Aclaris Therapeutics, Inc, and holds stocks from this company. Dr Maximova, Dr Zennaro, Dr Gregori, Dr Boz and Dr Zanon declare no potential conflict of interest.

CRediT author statement: Natalia Maximova: Conceptualization, Writing - Original draft preparation. Floriana Zennaro:

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Methodology, Investigation, Software. Massimo Gregori: Data collection, Investigation. Giulia Boz: Data curation, Data interpretation. Davide Zanon: Methodology, Software, Validation.

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Gabriel Mbalaviele: Writing- Reviewing and Editing, Supervision.

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38. Wcstheimer G, Blair SM. Functional Organization of Primate Oculomotor System Revealed by Cerebellectomy. Exp Brain Res. 1974; 21. 39. Driessen GJ, Gerritsen EJ, Fischer A, Fasth A, Hop WC, Veys P, et al. Long-term outcome of haematopoietic stem cell transplantation in autosomal recessive osteopetrosis: an EBMT report. Bone Marrow Transplant. 2003 Oct;32(7):657-63. 40. Jones KM, Unger EC, Granstrom P, Seeger JF, Carmody RF, Yoshino M. Bone marrow imaging using STIR at 0.5 and 1.5 T. Magn Reson Imaging. 1992;10(2):169-76. 41. Waitzman AA, Posnick JC, Armstrong DC, Pron GE. Craniofacial skeletal measurements based on computer tomography—part II: normal values and growth trends. The Cleft Palate19

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FIGURE LEGENDS Figure 1: Pre-transplant axial skeleton and skull X-ray of ARO patient showing a significant increase in density and thickness of all skeletal segments and early bone deformities (stubby long

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bones and ribs, thickened orbital rims, creating the typical “Batman sign”). Figure 2: (a) Axial CT of the pelvis bone of a 11 months-old healthy child showing the normal differentiation between the compact (white arrow) and the cancellous bone (black arrow); (b) Pre-

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transplant axial CT of the pelvis bone of ARO patient showing the absence of differentiation

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between the compact and the cancellous bone (black arrow). The slices were selected at the level of

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the sacroiliac joints.

Figure 3: (a) Axial pelvic T2 STIR MRI sequence of a 11 months-old healthy control child

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showing the central hyperintense (black arrow) cancellous bone surronded by the external

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hypointense (white arrow) cortical bone; (b) Pre-transplant axial pelvic T2 STIR MRI sequence of ARO patient confirms the absence of differentiation between the compact and the cancellous bone

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of the os coxae with "inversion" of the signal intensity (white arrow) from the bony center to external surface. The slices were selected at the level of the sacroiliac joints. Figure 4: (a) Pre-tranplant MRI-based assessment of ARO patient: T2-weighted axial sequence showing a markedly hypointense splenic signal (white arrow), compatible with iron overload, and suggesting extramedullary hematopoiesis; (b) Post-transplant MRI-based assessment of ARO patient performed 6 months after HSCT: the same T2-weighted axial sequence showing the physiologic hyperintense splenic signal (black arrow). Figure 5: 3D draw of the evolution of the bone marrow space reconstruction (brown) of ARO patient (a) 11-month-old (before HSCT), (b) 12-month-old (day +24 after 1° HSCT), (c) 15-month20

Journal Pre-proof old (day +79 after HSCT), (d) 18-month-old (day +88 after 2° HSCT) and (e) 36-month-old (month +21 after 2° HSCT); (f-l) bone marrow space volume of healthy control children of the same age. Volumes were calculated on MRI sequences. Figure 6: CT-based assessment of the femur of ARO patient: axial images showing the evolution of the bone structure morphology: (a) pre-transplant: no differentiation between compact and trabecular bone; (b) 3 months after HSCT: no evident differentiation between compact and

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trabecular bone, a bud of medullary cavity appears; (c) 13 months after HSCT: appearance of a thin initial medullary cavity; (d) 18 months after HSCT: appearance of a normal bone structure. The measurements displayed in white correspond to the diameter of the medullary cavity. The

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measurements displayed in black correspond to the thickness of the compact bone.

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Figure 7: (a) Pre-transplant MRI-based assessment of the hips of ARO patient: coronal PD fat sat

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sequence showing the absence of the hyperintense signal of the medullary space in femur neck (white arrow), and the presence of a markedly hypointense signal (black arrow) of the ossification

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center of the femoral head; (b,c) signal normalization of the medullary space and of the ossification

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(white arrow).

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center of the femoral head (black arrow); (c) appearance of the ossification center of the trochanter

Figure 8: CT-based 3D reconstruction of the skull: (a) ARO patient pre-transplant evaluation: the skull shows pansynostosis of all major sutures with bone bridging of fused sutures; (b) ARO patient three-years post-transplant evaluation: the internal table is beaten in copper in harmony with the raised intracranial pressure. Figure 9: skull CT imaging of ARO patient: (a) At baseline: marked thickening and loss of differentiation between the cortical tables and the diploe; (b) 30 months after HSCT: evident differentiation between cortical tables (white arrow) and diploe (black arrow), increased but irregular thickness of the vault bone, resulting from the compression by the growing brain.

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Journal Pre-proof Figure 10: Sagittal T1-weighted MRI sequences of an 11 months-old healthy control child (a), of ARO patient before HSCT (b) and 30 months after HSCT (c). Measurements were performed as follows: cephalic length or anteroposterior (AP) size between the most anterior and most posterior point of the inner table of the calvaria (white dotted line), cephalic height or craniocaudal (CC) size on the plane perpendicular to the bicallosal plane, and CC size as the length between the point at the basion parallel to the bicallosal line and the touch point of the calvarial inner table (white continuous line). Double-headed arrows (b,c) shows the reduction of the thickness of the parietal

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bone. The modified cephalic index (CI) was calculated according to the following equation: (cephalic height/cephalic length) x 100 [41]; (c) show a slow and progressive normalization of the

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neurocranium shape, with shorter cranial height (CC) and longer cephalic length (AP).

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Figure 11: Coronal, axial cranium CT scan and CT-based 3D reconstruction at the level of the optic

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canals of ARO patient before HSCT (a-c). The diameters of optic canals before HSCT (d) and 12 months later of HSCT (e) correspond to normal values.

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Figure 12: Axial and sagittal brain MRI, T1- weighted sequence, of ARO patient: pre-transplant

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MRI-based evaluation (a) flocculonodular lobe undetectable, compressed by other brain structures, at the pre-transplant MRI evaluation and (b) tonsillar herniation is evident; MRI-based evaluations

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performed 6 (c,d) and 30 months (e,f) after HSCT presence of the flocculi (white arrows) in the posterior region of cerebellum at post-transplant MRI evaluations, tonsillar herniation disappears.

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Table 1: Patient 1 relationship between reconstruction of bone marrow space and percentage of donor chimerism. TIME FROM HSCT

Baseline

BONE MARROW SPACE VOLUME (mm3) / BONES VOLUME (mm3) RATIO (%) (PATIENT 1)

BONE MARROW SPACE VOLUME (mm3) / BONES VOLUME (mm3) RATIO (%)

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0/52.49 = 0

AGE (months)

TOTAL WBC/mm3

DONOR CHIMERISM (%)

25.02/46.16 = 54.2

2060

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(CONTROL GROUP)

+ 24

12

1.37/52.54 = 2.6

24.78/46.2 = 53.6

330

88

Days

+ 37

13

4.42/52.90 = 8.3

24.78/46.2 = 53.6

1070

80

Days

+ 58

14

7.92/53.90 = 14.7

29.57/53.46 = 55.3

1240

53

Days

+ 79

15

8.59/56.76 = 15.1

34.46/61.17 = 56.3

620

7

39.96/69.81 = 57.2

2230

100

39.96/69.81 = 57.2

4210

100

Second HSCT

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Days

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First HSCT

+ 36

16

16.93/57.81 = 29.3

Days

+ 58

17

22.65/57.93 = 39.1

Days

+ 88

18

28.23/58.32 = 48.4

45.67/78.86 = 57.9

5040

100

Months

+6

21

35.05/70.25 = 49.9

53.66/91.52 = 58.6

5620

100

Months

+9

24

43.26/83.98 = 51.5

62.86/106.43 = 59.1

4990

100

Months

+ 12

27

50.15/96.02 = 52.2

69.54/117.41= 59.2

5700

100

Months

+ 21

36

52.14/98.43 = 53.0

71.99/121.50 = 59.3

6010

100

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Days

HSCT – hematopoietic stem cell transplantation; WBC – white blood cells.

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