Neuroimaging and clinical trials with stem cells in amyotrophic lateral sclerosis: Present and future perspectives

Radiología. 2019;61(3):183---190

www.elsevier.es/rx

RADIOLOGY TODAY

Neuroimaging and clinical trials with stem cells in amyotrophic lateral sclerosis: Present and future perspectives夽 J.M. García Santos a,b,∗ , M. García Martínez-Lozano b , C. Vázquez Olmos a , M. Blanquer c a

Servicio de Radiología, Hospital General Universitario Morales Meseguer, Murcia, Spain Facultad de Medicina, Universidad de Murcia, Murcia, Spain c Unidad de Trasplante Hematopoyético y Terapia Celular, Servicio de Hematología, Hospital Universitario Virgen de la Arrixaca, Instituto Murciano de Investigación Biomédica (IMIB), Campus Mare Nostrum, Universidad de Murcia, Murcia, Spain b

Received 5 September 2018; accepted 17 November 2018 Available online 31 December 2018

KEYWORDS Central nervous system; Amyotrophic lateral sclerosis; Adult stem cells; Research with stem cells; Surgical interventions; Spinal cord; Magnetic resonance imaging; Neuroimaging; Analyzing patient outcomes

Abstract Amyotrophic lateral sclerosis is a rare neurodegenerative disease with a rapid fatal course. The absence of effective treatments has led to new lines of research, some of which are based on stem cells. Surgical injection into the spinal cord, the most common route of administration of stem cells, has proven safe in trials to test the safety of the procedure. Nevertheless, challenges remain, such as determining the best route of administration or the way of checking the survival of the cells and their interaction with the therapeutic target. To date, the mission of neuroimaging techniques has been to detect lesions and complications in the spine and spinal cord, but neuroimaging also has the potential to supplant histologic study in analyzing the relations between the implanted cells and the therapeutic target, and as biomarkers of the disease, by measuring morphological and functional changes after treatment. These developments would increase the role of radiologists in the clinical management of patients with amyotrophic lateral sclerosis. © 2018 SERAM. Published by Elsevier Espa˜ na, S.L.U. All rights reserved.

夽 Please cite this article as: García Santos JM, García Martínez-Lozano M, Vázquez Olmos C, Blanquer M. Neuroimagen y ensayos clínicos con células madre en la esclerosis lateral amiotrófica: perspectivas de presente y futuro. Radiología. 2019;61:183---190. ∗ Corresponding author. E-mail address: [email protected] (J.M. García Santos).

2173-5107/© 2018 SERAM. Published by Elsevier Espa˜ na, S.L.U. All rights reserved.

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PALABRAS CLAVE Sistema nervioso central; Esclerosis lateral amiotrófica; Células madre adultas; Investigación con células madre; Intervenciones quirúrgicas; Médula espinal; Imagen por resonancia magnética; Neuroimagen; Análisis del resultado en el paciente

J.M. García Santos et al.

Neuroimagen y ensayos clínicos con células madre en la esclerosis lateral amiotrófica: perspectivas de presente y futuro Resumen La esclerosis lateral amiotrófica es una enfermedad neurodegenerativa rara con un curso rápido y fatal. La ausencia de tratamientos efectivos ha hecho surgir nuevas líneas de investigación, entre ellas las basadas en células madre. La inyección quirúrgica intramedular, que ha sido la principal vía de administración, ha demostrado ser segura en los ensayos de seguridad del procedimiento. Sin embargo, persisten desafíos como la mejor vía de administración o el modo de comprobar la supervivencia de las células y su interacción con la diana terapéutica. La misión de las técnicas de neuroimagen ha sido hasta ahora la detección de lesiones y complicaciones espinales y medulares, pero tienen potencial para sustituir al estudio anatomopatológico, analizando la relación de las células implantadas con la diana terapéutica, y como biomarcadores de la enfermedad, midiendo cambios morfológicos y funcionales postratamiento, lo que implicará más a los radiólogos en el manejo clínico de estos enfermos. © 2018 SERAM. Publicado por Elsevier Espa˜ na, S.L.U. Todos los derechos reservados.

Introduction With an incidence of 1---2 cases per 100,000 population/year and a prevalence of 3---6 cases per 100,000 population,1,2 amyotrophic lateral sclerosis (ALS) is considered as a rare disease.3 However, along with Alzheimer’s disease and Parkinson’s disease, it is one of the main neurodegenerative diseases.1 Most cases are sporadic, although 5---10% of patients have a family history.1,2 Although the term ALS refers specifically to the disease with signs of degeneration of the first and second motor neurons (Fig. 1), it is a heterogeneous disease with different clinical variants depending on the neuron affected1 in which both pathology and neuroimaging studies have shown non-motor areas of the central nervous system (CNS) to be involved (Fig. 1).4 One clinical example of this is the known association of ALS with frontotemporal dementia in 15---20% of patients.2 The clinical course of ALS is progressive until death, usually from respiratory failure, three to five years on average after the onset of the disease.2 In terms of treatment, only Riluzole and Edaravone have been approved as useful drugs by the United States Food and Drug Administration2,5 and only Riluzole has achieved a slight increase of around three months in life expectancy.6 The main reasons for the poverty of therapeutic resources after five years of research are the biological and clinical heterogeneity of the disease and the difficulty in establishing the best therapeutic targets on which to act and measure the results.7 These challenges, along with the scant effect of current treatments on the progression of the disease, have recently led to lines of research based on molecular, genetic and stem cell therapies.8---10 As the origin of the disease has multiple sources (environmental, genetic, epigenetic and neuronal microenvironment; neuroinflammation and immune response in particular), the main focus of interest has been on stem cells, as on the one hand they could potentially replace damaged cells and, on the other, exert paracrine effects which would produce neurotrophic effects and modulate the immune and

inflammatory responses.10,11 That interest has led to phase I and I-II clinical trials in which neuroimaging tests are of particular importance for patient follow-up.10,11 With that in mind, the aim of this article is to set out the main challenges of treating ALS with stem cells and, in turn, discuss the challenges of neuroimaging in the field of research and the potential clinical application.

Challenges of treating amyotrophic lateral sclerosis with stem cells The treatment of ALS with stem cells has meant extra difficulties over and above those already posed by the heterogeneity of the disease. The areas of most interest for this article are, essentially, the multiple types and great physiological heterogeneity of stem cells, the best way to administer them, and how to check on the survival of the cells and their interaction with the therapeutic target.7,10,11 Stem cells can be obtained from a variety of different sources. Those potentially available include neural, mesenchymal, embryonic, human umbilical cord and induced pluripotent stem cells, and stem cells from the bone marrow and the olfactory glial envelope.10 Recent clinical trials have used neural and bone marrow (mesenchymal and mononuclear) stem cells.10,11 Neural stem cells, which have the potential to specifically develop neurons, astrocytes and oligodendrocytes, have the disadvantage of having their source in the foetal nervous system, which raises both technical and ethical problems for obtaining them.12 Recently, however, it has become possible to differentiate pluripotent cells into neural cells12 and these are the type of stem cells in the trials currently underway.13 Up until then, autologous mesenchymal stem cells were the most used in clinical trials for the ease of obtaining them (which avoided ethical conflict), plus their ability to differentiate into neural cells in an adequate environment with good functional results in animal models.10,14 However, these autologous cells can share

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Figure 1 (A) Diagrams of amyotrophic lateral sclerosis (ALS) morphological changes according to involvement of the first motor neuron (violet circles: upper motor neuron) or the second motor neuron (blue circles: lower motor neuron). In the upper diagram, as a characteristic of the lesion of the upper motor neuron (motor cortex), the atrophy of the pyramidal tract (arrowhead) can be seen, which can be manifested by a decrease in the volume of the bulbar pyramid (arrow) and the lateral pyramid of the spinal cord (double arrow), which defines the disease (lateral sclerosis). In the lower diagram we can see, as a characteristic of the lower motor neuron (anterior horn of the spinal cord), atrophy of the cranial and peripheral nerves (arrowheads), and striated muscle (arrows). (B) Conventional magnetic resonance imaging (MRI) images in patients diagnosed with ALS. The disease can cause cortical and subcortical changes (1), characterised by T2 hypointensity (black arrow) and subcortical T2 hyperintensity (arrowhead). Another sign is subcortical hypointensity in T2 Flair (white arrow). The degeneration of the pyramidal tract (2) can manifest with hyperintensities on T2 and T2 Flair (arrowhead). The atrophy of the pyramidal tract (3) can be seen where the corticospinal tract makes an imprint on the surface of the CNS, as happens in the bulbar pyramid (arrowhead). The main manifestation of the second motor neuron lesion (4) is muscle atrophy (asterisk) and increased fat tissue. (C) MRI diffusion tensor. Tract-Based Spatial Statistics (TBSS). The colours red and yellow mark the voxels with a significant negative correlation between the fractional anisotropy and the progression of the disease (left) and between the N-acetyl-aspartate/myo-inositol ratio and the mean diffusivity (right). The involvement of the white matter in the frontal and parietal lobes, as well as the corpus callosum, goes beyond localised involvement in the motor pathway.

the epigenetic characteristics of the patients, in which case they lose some of their therapeutic interest.13 The options for getting the stem cells to the CNS are intrathecal, intraspinal, intraventricular, intramuscular and intravenous injection.10,13 Of these, intraspinal (surgical implantation of stem cells in the spinal cord) has been the method of choice in most of the clinical trials.11,12 By injecting them directly into the marrow, near the anterior horns, the stem cells are placed next to their therapeutic targets, thus avoiding the blood-brain barrier.11,12 However, the site of injection remains a matter of debate as, by the time the cells are being implanted locally, the disease is already affecting many areas of the brain and spinal cord.12 The

choice of injection site in the first trials was related to the critical areas of respiratory control and to the stability of the spine, and so the procedures were performed at the first levels of the thoracic spinal cord.12,15 However, subsequent trials have increased the doses injected and demonstrated the potential for implanting the cells in other locations and, importantly, in multiple segments at the same time.12 In any event, the most significant findings were the fact that, after the follow-up periods, all the trials were shown to be safe and the patients did not suffer from undesirable effects or significant adverse reactions.16---29 Consequently, we are able to continue to follow this therapeutic research pathway.11

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Figure 2 Physiological bases of the treatment of amyotrophic lateral sclerosis (ALS) with intramedullary stem cell injection. The difference between the neuronal density of the healthy subject and the patient with ALS is represented in the anterior horn of the marrow in the diagrams (arrowheads) and in the density of violet lines representing the corticospinal tracts. The aim of treatment with stem cells injected into the spinal cord (represented in blue inside white box A) is that they migrate towards the anterior horn of the spinal cord (light blue curved arrows) and there exert trophic effects on the second motor neuron (box B) and, possibly, retrogradely, also on the first motor neuron (ascending red arrows). The microscopic pathology image corresponds to boxes A and B of the ALS diagram. (A) Marker of peripheral blood cells that highlights in black the stem cells (arrow) inside the posterior horn (ph) of the spinal cord. (B) CD90 mesenchymal and acetylcholinesterase (ChAT) marker. The injected mononuclear cells (in red) have migrated after the injection and appear surrounding neurons of the anterior horn of the spinal cord (n).

Future trials will aim to assess the therapeutic effect of stem cells in ALS. Although the clinical results so far have not shown conclusive benefits, this is partly due to the size and heterogeneity of the samples used, and it will therefore be necessary to adjust the inclusion and exclusion criteria and significantly increase the number of patients in multicentre studies.11,28 Researchers will also need to ensure that the cells reach the therapeutic target, interact with it and produce a measurable effect.7,10 In the particular case of the previous clinical trials in ALS, it has never been certain that these three conditions were met and failure to do so will increase the risk of clinical failure.7 Although in some cases pathology studies have shown implanted stem cells interacting with motor neurons locally (Fig. 2),22 this option is not valid for clinical application, and other follow-up options therefore need to be developed, essentially based on neuroimaging.10

Challenges for neuroimaging when treating amyotrophic lateral sclerosis with stem cells As outlined above, the challenges of neuroimaging in patients treated with stem cell implants in the spinal cord are to ensure the safety of the procedure, analyse the interaction of the stem cells with the therapeutic target and study the physiological effects of the treatment.

A. Safety of the procedure Until now, the fundamental role of neuroimaging after injecting stem cells into the spinal cord of patients with ALS has been to detect postoperative lesions and complications.11 In general, in the trials with bone marrow stem cells17,19,21,22 and neural stem cells24,26,27,29 no significant spinal changes after the surgical procedure have been reported. However, while focusing on the clinical aspects of the disease and treatment, these publications have paid very little attention to the spinal imaging findings and there are even inconsistent descriptions in publications from the same groups. In general, the reported changes can be classified as affecting the spine or the spinal cord. Among those involving the spine, the most commonly described lesions are minimal and transient extradural fluid collections19,24,29 ; and one of the most recent trials reported an isolated case of a kyphotic deformity.25 Among the spinal cord changes, the most common are sporadic changes in T2 signal in the spinal cord implant area, also minimal and transient,19,27 which have been related to the actual infusion material or oedema; only one of the studies reported acute (increase in volume) and chronic (deformity) morphological changes in the spinal cord.19 In one of the trials with bone marrow mesenchymal stem cells19 and in another with neural stem cells,29 diffusion tensor studies with magnetic resonance were also carried out. The authors described minimal changes in fractional anisotropy, which decreased

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Figure 3 Changes in the dorsal spinal cord after the surgical injection of mesenchymal stem cells. (A) Sagittal image of the upper dorsal spine with T1-weighted spin echo magnetic resonance imaging (MRI) in the immediate postoperative period. Hypointense extradural fluid collection (asterisk) compressing the dural sac and, slightly, the spinal cord. (B) Sagittal images of the upper dorsal spine with T2-weighted fast spin-echo magnetic resonance in the immediate postoperative period (left) and at 12 months (right). These images show the systematic changes in postoperative fluid collections which evolve from varying degrees of compression of the structures of the spinal canal (asterisk) to the retractable scar (arrowhead). (C) Sagittal image of the upper dorsal column with T2-weighted fast spin-echo MRI in the immediate postoperative period. The spinal cord has slightly increased in volume in the surgical area and shows an increase in signal (arrow). (D) Sagittal (left) and axial (right) images of the upper dorsal spine with T2-weighted fast spin-echo and T2*-weighted gradient echo MRI respectively, 12 months after the intervention. The images show the irregularity of the contours and the usual increase in the anteroposterior diameter of the spinal cord (white arrow) which, in this case, is particularly striking as it is longer than the length of the transverse diameter in the axial image. A hypointense focus (black arrow) can also be seen in the anterior margin of the cord in the gradient echo sequence which suggests haemorrhagic changes or deposit of chronic paramagnetic material related to the injected solution of stem cells.

transiently in both trials and, in the case of the mesenchymal cell trial, an increase, also transitory, in the apparent diffusion coefficient.19 Although one of the most important potential complications of implantation could be spinal tumours resulting from the uncontrolled proliferation of the cells,13 no cases have been reported to date in the patients included in the published clinical trials. Although there are these marginal descriptions included in the articles on the clinical trials, there is only one published article30 specifically dedicated to changes in the spine and spinal cord which describes the patients from one of the clinical trials where bone marrow mesenchymal stem cells were implanted in the spinal cord.22 The data from this publication,30 along with the not yet published data from a second trial by this group (García Martínez-Lozano and García Santos, unpublished results) using the same methods for qualitative analysis of magnetic resonance imaging (MRI) of the cervical and dorsal spinal cord (the interested reader can be informed in the material and methods section of literature reference 30) cover a total sample of 32 implanted patients, in whom the postoperative lesions and the degree of association with the clinical changes were analysed as they evolved. In our experience, injuries to the spine and spinal cord, far from being anecdotal, are actually common. They can be classified as acute (visible immediately

after the intervention, whether transient or with permanent sequelae) or chronic (appearing late and persisting over time) (Fig. 3): 1. Acute, usually transient, lesions. • In all the patients we found post-surgical extradural fluid collections in the spine, most of them with compression of the dural sac and occasionally with compression of the spinal cord. All of the fluid collections and their effects remitted quickly and compression was negligible three months later. • In more than half of the patients we detected a signal change in the T2-weighted sequences of the spinal cord, probably reflecting oedema and generally limited to the surgical implant area, although occasionally more extensive. In the vast majority of patients, the signal change was no longer visible three months after implantation. In a minority, the increased T2-weighted signal persisted over time and one patient developed a small residual medullary cyst in the implant area. 2. Acute lesions with chronic sequelae. Approximately one third of patients had paramagnetic foci. We rarely detect hyperintense lesions in T1 due to bleeding after implantation; the most common were very hypointense

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Figure 4 Studies in an animal model (transgenic rats) of Friedreich’s ataxia with a 7T magnetic resonance scanner with 11.5 cm gantry ring diameter (BioSpec 70/30v; Bruker Medical, Ettlingen, Germany), with a gradient coil of 675 mT/m (Bruker Medical, BGA 12-S) and a receive-only 1H phased-array rat head antenna (Bruker BioSpin MRI). Axial images of the lumbar spine with a T2weighted* multi-echo gradient sequence (repetition time: 1,500 ms; echo time: 3 ms; flip angle: 30◦ ; field of vision: 20 mm × 20 mm; slice thickness: 0.5 mm; matrix: 256 × 256; number of excitations: 2; acquisition time 12 min and 48 s in mice with intrathecal injection of stem cells without (A) and with (B) stem cells marked with superparamagnetic iron oxide (SPIO). In the second of the mice we can see the marked cells (arrows) immediately after the procedure (left) and 14 days later (right). Courtesy of Prof. Salvador Martínez, Institute of Neurosciences, Universidad Miguel Hernández (UMH-CSIC), San Juan, Alicante, Spain.

intramedullary foci in the T2-weighted gradient echo sequences. These foci are probably haemorrhagic and have persisted over time. 3. Late-onset chronic lesions. The distinctive lesion was the late deformity of the spinal cord, characterised by the irregular contour, the increase in anteroposterior diameter, which in some cases was longer than the transverse diameter, and posterior adherence to the dural sac. Virtually all the survivors at the one-year follow-up had this deformity. Despite the common nature of the lesions after surgical implantation of stem cells, their association with symptoms related to the procedure has never been statistically significant, except for an occasional correlation with transient neurological symptoms such as hypoaesthesia, paraesthesia and dysaesthesia (García Martínez-Lozano and García Santos, unpublished results). Moreover, the lesions described have never been related to acceleration of the disease.

B. Interaction of the stem cells with the therapeutic target Imaging techniques such as positron emission tomography (PET) and MRI may play a role as molecular biomarkers to replace the pathology analyses used in clinical trials in clinical practice.7,10 However, although the possibility of labelling stem cells with superparamagnetic iron oxide

(SPIO) particles in order to track them with MRI has been demonstrated (Fig. 4), it remains only a potential tool and clinical translation has yet to be achieved.31

C. Effects of the treatment Providing biomarkers that measure morphological and functional changes in these patients is one of the goals of future research (Fig. 5). Only one article has focused to date on neuroimaging with distant trophic changes in patients with a bone marrow stem cell implant in the spinal cord.32 Metabolic changes have been reported in the precentral frontal gyri with MR spectroscopy (increase in the ratio between N-acetyl-aspartate and creatine [NAA/Cr]) which may indicate trophic changes in the brain after spinal implantation. Moreover, the changes were inversely related to the progression of the disease and directly related to its duration.32 In a second trial (Vázquez Olmos and García Santos, unpublished results), the trends have been the same, although without reaching statistical significance, one reason for which may be a more heterogeneous sample due to less restrictive inclusion and exclusion criteria. In this same trial, analysis of the diffusion tensor data has shown evolutionary differences in the forceps major of the corpus callosum (increase in fractional anisotropy and decrease in mean diffusion) and in the internal capsule (increase in longitudinal diffusion 1 and mean diffusion) between patients with a stem cell implant and those not operated on.

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A NAA Cr

Preoperative

3 months

12 months

B

Figure 5 Follow-up of patients diagnosed with amyotrophic lateral sclerosis (ALS) after surgically injecting mesenchymal stem cells into the dorsal spinal cord. (A) Single voxel magnetic resonance (MR) spectroscopy focused on the left motor gyrus (repetition time 1,500 ms, echo time 35 ms) corresponding to immediate preoperative studies and at 3 and 12 months after the intervention. The images show a progressive increase of the N-acetyl-aspartate (NAA) peak with stable creatine (Cr), which may indicate a trophic effect of the injected stem cells on the first motor neuron. (B) MRI diffusion tensor. Tract-Based Spatial Statistics (TBSS). The voxels in red located in the left pyramidal tract represent those with significant variations between groups with different treatment (intramedullary injection of mesenchymal stem cells/intrathecal injection of mesenchymal stem cells/intrathecal placebo injection) in the diffusion parameters (fractional anisotropy, mean diffusivity and longitudinal diffusion 1), after 12 months of follow-up.

These trends reflect differences according to the treatment pending interpretation with all the clinical and epidemiological data for a small heterogeneous sample.

Conflicts of interest

Conclusion

References

To sum up, neuroimaging tests are essential in phase I and I-II clinical trials of new treatments for ALS. The challenges for imaging stem from the need to guarantee the safety of the procedure through the assessment of transient lesions and by studying the interaction of the stem cells with the therapeutic target and the physiological effects of the treatment.

Author’s contribution 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Responsible for the integrity of the study: JMGS. Study conception: JMGS. Study design: JMGS. Data acquisition: JMGS, MGML, CVO and MB. Analysis and interpretation of the data: JMGS, MGML, CVO and MB. Statistical processing: N/A. Literature search: JMGS and MGML. Drafting of the paper: JMGS and MGML. Critical review of the manuscript with relevant intellectual contributions: CVO and MB. Approval of the final version: JMGS, MGML, CVO and MB.

The authors declare that they have no conflicts of interest.

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