New Imaging Biomarkers for Muscular Diseases - Multispectral Optoacoustic Imaging in Spinal Muscular Atrophy
Phase: NA
Conditions: Muscular Diseases, Spinal Muscular Atrophy
Sponsor: University of Erlangen-Nürnberg Medical School

New Imaging Biomarkers for Muscular Diseases - Multispectral Optoacoustic Imaging 
in Spinal Muscular Atrophy (MSOT_SMA) 
 
 
 
Unique Protocol ID: 168_19B 
ClinicalTrials.gov Identifier: NCT04115475 
 
 
Sponsor:  
ELAN-Fonds, Interdisciplinary Center for Clinical Research (IZKF), Friedrich-Alexander-
University (FAU) Erlangen-Nürnberg 
and 
Department of Pediatrics and Adolescent Medicine 
University Hospital Erlangen 
Friedrich-Alexander-University (FAU) Erlangen-Nürnberg 
Pediatric Experimental and Translational Imaging Laboratory (PETI_Lab) 
Dr. med. Ferdinand Knieling 
Loschgestr. 15 
91054 Erlangen 
Germany 
Tel.: +49 9131 85-33118 
E-mail: ferdinand.knieling@uk-erlangen.de  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Protocol Version 2.3 – 07.11.2019 

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Study protocol 
 
 
 
MSOT_SMA 
 
Non-invasive molecular imaging of muscle degeneration in spinal muscular atrophy  

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1. Table of contents 
 
1. 
Table of contents .............................................................................................................. 2 
2. 
Study titel, version number, version date .......................................................................... 3 
3. 
Project summary ............................................................................................................... 4 
4. 
Responsabilities ................................................................................................................ 6 
5. 
Scientific background ........................................................................................................ 7 
6. 
Study objectives .............................................................................................................. 12 
7. 
Target parameters .......................................................................................................... 13 
8. 
Study design ................................................................................................................... 16 
9. 
Study population ............................................................................................................. 18 
10. 
Study course ............................................................................................................... 19 
11. 
Risk-benefit analysis ................................................................................................... 21 
12. 
Biometrics ................................................................................................................... 24 
13. 
Data management und and data protection ............................................................... 25 
14. 
Handling of biomaterials ............................................................................................. 26 
15. 
Insurance .................................................................................................................... 26 
16. 
Signatures ................................................................................................................... 27 
 
 
 

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2. Study titel, version number, version date 
 
Study title 
Non-invasive molecular imaging of muscle degeneration in spinal muscular atrophy 
 
Version number 
Version 2.3 
 
Version date 
7.11.2019 
 
 
Protocol versions 
Date 
Version 
Status 
Changes 
02.04.2019 
1.0 
Outline 
 
23.04.2019 
2.0 
Final application 
Adaptation formulations, 
correlation with 
ultrasound 
03.06.2019 
2.1 
Amendment 
Specification of ultrasonic 
measurement parameters 
02.10.2019 
2.2 
Amendment 
specification  
clinical testing, 
adjustment of muscle 
regions 
07.11.2019 
2.3 
Amendment 
Application specialist 
deleted 
 
 
 

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3. Project summary 
Neuromuscular diseases do often already present in the neonatal period and manifest with 
muscle hypotonia and weakness. Associated diseases are caused by numerous pathologies 
in the central nervous system (brain and spinal cord), the peripheral nervous system or the 
skeletal muscle. Spinal muscular atrophy (SMA) is a pathology of the central nervous system 
characterized by the degeneration of motor anterior horn nerve cells in the spinal cord or 
lower brain stem, which lead to progressive muscle weakness and atrophy. The clinical 
spectrum of all subtypes of the disease is broad, but in almost all cases the therapeutic 
options are limited to supportive therapy and palliative measures to prevent or alleviate 
complications such as spinal deformities or respiratory insufficiency. In the course of the 
disease, patients lose their motor neurons and thus their original skeletal muscle mass. In 
the muscle itself, a histological loss of the nerve and muscle fibers as well as a fatty 
remodelling can be observed. Most patients show normal cognition and consciously 
experience the course of their disease. In particular, the infantile development of SMA is 
lethal after only a few years of life. 
Since 2016 (USA) and 2017 (European Union), the drug Nusinersen (Spinraza®, Biogen) 
has been available for the treatment of children and adults with SMA. It contains an 
antisense oligonucleotide, which is supposed to increase the expression of the usually in 
SMA reduced "survival motor neuron protein". In the ENDEAR study, the study used for FDA 
approval, 40% of children in the treatment arm achieved significant improvements in the area 
of motor milestones, while no child in the control group achieved this. Further positive effects 
were also reported in older children and adolescents as a result of the CHERISH study. The 
drug is administered intrathecally (in the spinal fluid). The first three doses were administered 
at intervals of 14 days and the fourth at intervals of 30 days. Subsequently, maintenance 
therapy is administered at intervals of 4 months. According to the Joint Federal Committee, 
the annual therapy costs currently amount to 310,877.58 € to 310,942.95 € - internationally, 
Spinraza® ranks second among the most expensive drugs in the world. 
Whether patients benefit from this therapy can only be assessed clinically so far. To date, 
there are no prospective markers available that can predict a response or indicate 
therapeutic changes. Since 2017, a multispectral optoacoustic tomograph (MSOT) with an 
extended emission spectrum (660nm-1300nm) has been available to the University Hospital 
Erlangen through a DFG grant for large-scale equipment. MSOT imaging is similar to 
conventional sonography in that a transducer is placed on the skin and energy is supplied to 
the tissue by pulsed laser light instead of sound. On a macroscopic level, this leads to a 
constant change of minimal oscillations of individual tissue components. The resulting sound 
waves can then be detected by the same transducer. Previous studies have shown that the 

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quantitative determination of hemoglobin can be used to obtain information on blood 
circulation and inflammatory activity. In the extended spectrum, in contrast, not only 
hemoglobin and its oxygenation stages but also other biomarkers such as collagens and 
lipids can be detected. As already shown in preliminary work on Duchenne muscular 
dystophy, the method is very sensitive and correlates significantly with the corresponding 
clinic. In the light of progression of the spinal muscular atrophy, this method would for the 
first time allow a non-invasive and quantitative determination of the molecular composition of 
muscle tissue in affected patients. In this first pilot study, it will be investigated whether 
differences in muscular composition are also observed in spinal muscular atrophy. In the 
future, this could serve as a completely new method to provide information about the course 
of disease or response to therapy. 
 
 
 

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4. Responsabilities 
 
Study director  
Dr. Ferdinand Knieling 
Pediatric Experimental and Translational Imaging Laboratory (PETI_Lab) 
Department of Pediatrics  
University Hospital Erlangen  
Loschgestr. 15 
91054 Erlangen  
Germany 
Tel. 09131 8533118 
Mail: ferdinand.knieling@uk-erlangen.de  
 
Prof. Dr. Regina Trollmann 
Head of the Department of Neuropediatrics 
Department of Pediatrics  
University Hospital Erlangen  
Loschgestr. 15 
91054 Erlangen 
Germany 
Tel.: 09131 85 33753 
Mail: regina.trollmann@uk-erlangen.de 
 
Other facilities possibly involved (e.g. laboratory, imaging, etc.) 
Prof. Dr. Maximilian J. Waldner (technical realization) 
Deparment of Medicine 1  
University Hospital Erlangen 
Ulmenweg 18 
91054 Erlangen 
Germany  
Tel.: 09131 8545025 
Mail: maximilian.waldner@uk-erlangen.de 
 
Sponsoring  
Children’s Hospital Erlangen, Else-Kröner-Fresenius Foundation (Else-Kröner-Fresenius 
Memorial Scholarship Ferdinand Knieling) 
 
 

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5. Scientific background 
Spinal muscular atrophy (SMA) is an autosomal-recessive disorder, characterized by 
progressive muscle weakness and atrophy. Spinal muscle atrophy has an incidence of 
1/10,000 with an estimated carrier frequency of 1/50 (Sugarman 2012, McAndrew 1997, 
Pearn 1978). The condition is caused by a homozygous deletion or mutation in the survival 
motor neuron 1 (SMN1), resulting in reduced expression of the survival motor neuron (SMN) 
protein. This leads to the degeneration of motor neurons in the spinal cord and brain stem 
(Lefebvre 1995, Prior 2010). A nearby related gene, survival motor neuron 2 (SMN2), also 
produces the SMN protein, but due to aberrant splicing around 85-90% of translated protein 
are truncated and non-functional and rapidly degraded in the cell (Mailman 2002); remaining 
10-15% are functional (Butchbach 2016), partially compensating the loss of SMN1 by SMN2 
protein synthesis (Hsieh-Li 2000). Individuals with a higher copy number of SMN2 do in 
general have a milder phenotype (Mailman 2002, Butchbach 2016).  
The various forms of SMA are differentiated according to distribution pattern, onset of 
disease, disease severity and hereditary pattern and are generally designated according to 
the 
muscle 
groups 
mainly 
affected 
(Pearn 
1973, 
https://www.dgm.org/muskelerkrankungen/spinale-muskelatrophie). A large number of 
similar, much rarer forms are known that can be associated with additional dysfunctions. The 
non-proximal spinal muscle atrophies are also very rare and do usually not lead to significant 
limitations of vital functions at the onset of adulthood. However, the vast majority (approx. 
90%) of patients belong to the group of so-called proximal SMA, which is characterized by 
the onset of muscle weakness in muscle groups close to the trunk (above all thigh and hip 
muscles, later also arm and shoulder girdle involvement). Proximal SMA is divided into 
different subtypes, which are primarily defined according to the onset of the disease, the 
learned motor skills and life expectancy. 
Spinal muscular atrophy type 0, also called prenatal onset SMA, describes the most 
severe form of SMA as it presents before birth with decreased fetal movements. Neonates 
are born with severe hypotonia and weakness, areflexia, facial weakness, joint contractures, 
difficulty swallowing and respiratory failure on examination. Life-expectancy is greatly 
reduced, most patients die within the first 6 months of age (Dubowitz 1999, MacLeod 1999). 
Spinal muscular atrophy type 1, also called Werdnig-Hoffman disease or infantile SMA, is 
the most common type of SMA. Symptoms usually exhibit at birth or before 6 months of age. 
Infants present with hypotonia (proximal more than distal limbs, legs more affected than 
arms) with poor head control (“floppy baby”), reduced or absent tendon reflexes and are 
never able to sit unassisted. Patients may show a bell-shaped thorax, paradoxical breathing, 

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swallowing weakness and tongue fasciculations. Cognition is not affected. Disease is usually 
fatal prior to 2 years of life due to respiratory failure (Finkel 2014, Thomas 1994). 
Spinal muscular atrophy type 2, also called Dubowitz disease or intermediate SMA, 
presents usually between 6-18 months of age. This form tends to manifest with progressive 
leg weakness, which is worse than weakness in the arms, hypotonia and in a great 
percentage of patients areflexia. Comorbidities are related to muscular weakness and 
include scoliosis and joint contractures, as well as possible restrictive lung disease due to 
intercostal muscle weakness. Cognition is normal in affected patients (von Gontard 2002). 
Children with SMA type 2 are able to sit unassisted but are never able to walk independently. 
Life expectancy is reduced, but most patients live until adulthood (Fried 1971, Hausmanowa-
Petrusewicz 1985, Imai 1995). 
Spinal muscular atrophy type 3, also called Kugelberg–Welander disease or juvenile SMA, 
usually manifests after 12 months of age. Patients suffer from a proximal muscle weakness, 
which is more pronounced in the legs than in the arms. Individuals are able to walk 
unassisted, however at some point a wheelchair may be needed. Unlike SMA type 2, these 
patients do usually not suffer from orthopedic comorbidities or respiratory muscle weakness. 
Cognition and life expectancy are normal within this group. (Kugelberg 1956, Meadows 1969, 
Zerres 1997).  
Spinal muscular atrophy type 4, is the mildest form of disease with an onset in adulthood, 
often at age 30 or later. Patients present with mild muscle weakness in the legs, that may 
procced to the arms. A small number of patients may require wheelchair assistance. 
Cognition and life expectancy is normal (Pearn 1978, Tsukagoshi 1965, Piepers 2008). 
Extensive research has been done over the last two decades to develop an effective 
treatment strategy for SMA. Nusinersen (Spinraza®, Biogen), the first approved drug for the 
treatment of SMA, has been available since December 2016. This is an antisense 
oligonucleotide drug that leads to an increased production of full-fledged functional SMN 
protein. It is administered directly intrathecally as it does not cross the blood-brain barrier. 
Several studies showed improvements in motor function and survival in patients treated with 
Nusinersen, where early treatment was necessary to maximize the benefit of the drug. Long-
term data are still pending. Several other promising therapeutic approaches, including gene 
therapy for SMA, are currently under development. 
 
 

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There are currently no prospective markers available that can predict early muscle 
degeneration, disease progression, therapy response or therapeutic changes. Since 2017, 
the University Hospital Erlangen (Deparment of Medicine 1, Department of Pediatrics) has a 
multispectral optoacoustic tomograph (MSOT) funded by the DFG. This allows non-invasive, 
quantitative imaging of the composition of target tissues in the non-sedated child, 
comparable to sonography. This 
is of particular importance in this 
patient group. 
In MSOT, similar to conventional 
sonography, a transducer is 
placed on the skin and instead of 
sound, energy is supplied to the 
tissue by means of pulsed laser 
light. This leads to a constant 
change of minimal expansions 
and contractions (thermoelastic 
expansion) of individual tissue 
components or molecules. The 
resulting sound waves can then 
be 
detected 
by 
the 
same 
examination 
unit. 
Previous 
studies have shown that the 
quantitative 
determination 
of 
hemoglobin can be used to 
obtain information on blood flow 
and inflammatory activity in the 
intestine 
from 
patients 
with 
Crohn's disease (Waldner, Knieling 
et al. 2016, Knieling, Neufert et al. 2017) (Waldner 2016, Knieling 2017). In the newly 
configured device (Acuity Echo, iThera Medical GmbH, Munich, prototype) an extended 
spectrum of laser light can be used, which ultimately not only enables the detection of 
haemoglobin and its oxygenation stages, but also the detection of further markers such as 
collagen and lipid. This principle has already been successfully applied in a follow-up study 
on pediatric patients with Duchenne muscular dystrophy (Regensburger et al. submitted for 
publication, ESMI Young Investigator Award 2019). 
 
Figure 1 - MSOT principle (a), experimental preliminary work (b), and 
first time representation of collagen in vivo (c) 

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Figures 1 and 2 show exemplary MSOT 
images. In the light of the progression and 
the sometimes severe course of SMA, this 
method would for the first time allow a non-
invasive and quantitative determination of 
the molecular composition of muscle 
tissue. In this first pilot study in patients 
with SMA, it will now be investigated 
whether the differences in the muscle 
composition of healthy volunteers and 
SMA patients in the early stages can be 
quantified 
and 
whether 
this 
could 
simultaneously be used as marker during 
therapy with Nusinersen. In the future, this 
could generate a completely new, non-
invasive method to evaluate endogenous 
biomarkers for therapy response. 
 
Literature 
Sugarman EA, Nagan N, Zhu H, et al. Pan-ethnic carrier screening and prenatal diagnosis for spinal 
muscular atrophy: clinical laboratory analysis of >72,400 specimens. Eur J Hum Genet 2012;20:27-32 
 
McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, Prior TW, Burghes AHM. 
Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and 
SMNC gene copy number. Am J Hum Genet 1997; 60: 1411–1422. 
 
Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular 
atrophy. J Med Genet 1978; 15:409. 
 
Lefebvre S, Bürglen L, Reboullet S, et al. Identification and characterization of a spinal muscular 
atrophy-determining gene. Cell 1995; 80:155. 
 
Prior TW. Spinal muscular atrophy: a time for screening. Curr Opin Pediatr 2010;22:696-702. 
 
Mailman MD, Heinz JW, Papp AC, et al. Molecular analysis of spinal muscular atrophy and 
modification of the phenotype by SMN2. Genet Med 2002;4:20-26 
 
Butchbach ME. Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal 
Muscular Atrophy and Other Neurodegenerative Diseases. Front Mol Biosci 2016; 3:7. 
 
Hsieh-Li HM, Chang JG, Jong YJ, et al. A mouse model for spinal muscular atrophy. Nat Genet 2000; 
24:66. 
 
Pearn, J. K., Carter, C. O., Wilson, J. The genetic identity of acute infantile spinal muscular atrophy. 
Brain 96: 463-470, 1973. 
 
Figure 2 - Feasibility of 3D MSOT image (a) and diagnostic 
quality (b). 

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Dubowitz V. Very severe spinal muscular atrophy (SMA type 0): an expanding clinical phenotype. Eur 
J Paediatr Neurol. 1999;3(2):49–51. 
 
MacLeod MJ, et al. Prenatal onset spinal muscular atrophy. Eur J Paediatr Neurol. 1999;3(2):65–72. 
 
Finkel RS, et al. Observational study of spinal muscular atrophy type I and implications for clinical 
trials. Neurology. 2014;83(9):810–817. 
 
Thomas NH, Dubowitz V. The natural history of type I (severe) spinal muscular atrophy. Neuromuscul 
Disord. 1994;4(5–6):497–502. 
 
von Gontard A, et al. Intelligence and cognitive function in children and adolescents with spinal 
muscular atrophy. Neuromuscul Disord. 2002;12(2):130–136. 
 
Fried, K., Emery, A. E. H. Spinal muscular atrophy type II. A separate genetic and clinical entity from 
type I (Werdnig-Hoffmann disease) and type III (Kugelberg-Welander disease). Clin. Genet. 2: 203-
209, 1971. 
 
Hausmanowa-Petrusewicz, I., Zaremba, J., Borkowska, J. Chronic proximal spinal muscular atrophy of 
childhood and adolescence: problems of classification and genetic counselling. J. Med. Genet. 22: 
350-353, 1985. 
 
Imai, T., Matsuya, M., Matsumoto, H., Ishikawa, Y., Minami, R. Preservation of central motor 
conduction in patients with spinal muscular atrophy type II. Brain Dev. 17: 432-435, 1995. 
 
Kugelberg, E., Welander, L. Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. 
Arch. Neurol. Psychiat. 75: 500-509, 1956. 
 
Meadows, J. C., Marsden, C. D., Harriman, D. G. F. Chronic spinal muscular atrophy in adults. I. The 
Kugelberg-Welander syndrome. J. Neurol. Sci. 9: 527-550, 1969. 
 
Zerres K, et al. A collaborative study on the natural history of childhood and juvenile onset proximal 
spinal muscular atrophy (type II and III SMA): 569 patients. J Neurol Sci. 1997;146(1):67–72. 
 
Knieling F et al. Multispectral Optoacoustic Tomography for Assessment of Crohn's Disease Activity. 
N Engl J Med. 2017 Mar 30;376(13):1292-1294.  
 
Waldner MJ, Knieling F, et al. Multispectral Optoacoustic Tomography in Crohn's Disease: 
Noninvasive Imaging of Disease Activity. Gastroenterology. 2016 Aug;151(2):238-40. doi: 
10.1053/j.gastro.2016.05.047. Epub 2016 Jun 3.  
 
Regensburger A. Multispectral Optoacoustic Tomography in the Extended Near Infrared Range for 
Non-invasive in vivo Detection of Collagens in Duchenne Muscular Dystrophy. submitted for 
publication. 
 

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6. Study objectives  
 
To establish a spectral profile of muscle tissue based on multispectral optoacoustic 
tomography (MSOT) of patients with spinal muscular atrophy and healthy volunteers. 
Hypotheses: 
• 
The optoacoustic spectrum of patients with spinal muscular atrophy compared to 
healthy volunteers is different 
• 
The quantitative fraction of lipid signal in muscles determined by MSOT differs in 
patients with spinal muscular atrophy compared to healthy volunteers  
• 
The quantitative fraction of collagen signal in muscles determined by MSOT differs in 
patients with spinal muscular atrophy compared to healthy volunteers  
• 
The quantitative fraction of hemo-/myoglobin in muscles determined by MSOT differs 
in patients with spinal muscular atrophy compared to healthy volunteers 
• 
The quantitative fraction of oxygenated/deoxygenated hemoglobin in muscles 
determined by MSOT differs in patients with spinal muscular atrophy compared to 
healthy volunteers 
• 
There are no side differences in patients with SMA and healthy volunteers.  
 
Primary study objective: 
• 
Comparison of the optoacoustic spectrum determined by MSOT in patients with SMA 
and healthy volunteers 
 
Secondary study objectives: 
• 
Comparison of the quantitative lipid signal fraction determined by MSOT in patients 
with SMA and healthy volunteers 
• 
Comparison of the quantitative fraction of collagen signal determined by MSOT in 
patients with SMA and healthy volunteers 
• 
Comparison of the quantitative fraction of hemo-/myoglobin signal determined by 
MSOT in patients with SMA and healthy volunteers 
• 
Comparison of the quantitative fraction of oxygenated/deoxygenated hemoglobin 
determined by MSOT in patients with SMA and healthy volunteers 
• 
Correlation of lipid content determined with MSOT with disease duration/patient age 
• 
Correlation of collagen determined by MSOT with disease duration/patient age 

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• 
Correlation of haemoglobin/myoglobin content determined by MSOT with duration of 
disease/patient age 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined by MSOT with 
duration of disease / patient age 
• 
Correlation of lipid content determined with MSOT with age-related functional muscle 
tests (Hammersmith Infant Neurological Examination (HINE)/ The Children's Hospital 
of Philadelphia Infant Test of Neuromuscular Disorders (CHOP Intend)/expanded 
Hammersmith functional motor scale (HFMSE)/ Revised Upper Limb Module (RULM)/ 
&-Minute-Walk Test (6-MWT))) 
• 
Correlation of collagen determined with MSOT with age-dependent functional muscle 
tests (HINE/ CHOP Intend/ HFMSE/Revised Upper Limb Module/6-MWT) 
• 
Correlation of hemo-/myoglobin content determined with MSOT with age-dependent 
functional muscle tests (HINE/ CHOP Intend/ HFMSE/Revised Upper Limb Module/6-
MWT) 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined with MSOT with 
age-related functional muscle tests (HINE/ CHOP Intend/ HFMSE/Revised Upper 
Limb Module/6-MWT) 
• 
Measurement of signal differences in right / left comparison 
• 
Correlation of MSOT ultrasound image (RUCT) with standard sonography 
 
Study type 
Since no data exists so far to support the hypothesis of this study, it is an explorative study / 
pilot study. 
7. Target parameters 
All measurements with MSOT are performed over the proximal and distal limb muscles in a 
right-left comparison (leg proximal: Musculus quadriceps, distal: Musculus triceps surae; arm 
proximal: Musculus biceps, distal: Forearm flexors) in healthy subjects compared to patients 
with SMA. 
 
Primary target: 
Optoacoustic Absorption Spectrum of Muscle. 
This target is measured non-invasively by MSOT. 
 
Secondary targets: 
Quantitative lipid signal (in arbitrary units) 

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Quantitative collagen signal (in arbitrary units) 
Quantitative hemo/myoglobin signal (in arbitrary units) 
Muscle oxygenation (in %) 
Ratio of lipid to hemo/myoglobin signal or collagen to hemo/myoglobin signal 
These target values are collected non-invasively using MSOT. 
 
Diameter, number of nerve fibers (peripheral nerves, median nerve, ulnar nerve, ischiadic 
nerve) 
Muscle texture (Heckmatt score, echogenicity) 
These target values are determined by means of B-image sonography. 
 
Clinical scores for determining muscle strength consisting of: 
Patients < 2 years and patients ≥ 2 years with inability to sit: 
- 
Hammersmith Infant Neurological Examination (HINE)/ The Children's Hospital of 
Philadelphia Infant Test of Neuromuscular Disorders (CHOP Intend) 
 
at age ≥ 2 years and ability to sit in a wheelchair additionally: 
- 
Revised Upper Limb Module (RULM) 
 
Patients ≥ 2 years and sitting ability:  
- 
expanded Hammersmith functional motor scale (HFMSE)/ Revised Upper Limb 
Module (RULM)  
 
in case of ability to walk additionally: 
- 
6-minute walking test 
 
 

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< 2 years 
≥ 2 years with 
inability to sit 
≥ 2 years and 
ability to sit in 
a wheelchair 
≥ 2 years and 
ability to sit 
≥ 3 years and 
ability to walk 
HINE Section 2 
 
HINE Section 2 
 
HINE Section 2 
 
 
CHOP Intend 
CHOP Intend 
CHOP Intend 
 
(HFMSE) 
HFMSE 
HFMSE 
RULM 
RULM 
RULM 
 
6MWT 
 
These target values are clinically determined at presentation. 
Age 
Sex 
Weight 
Skin color 
Ethnic Background 
Disease duration 
Current medication 
 
These target values are either available in the electronic patient file or they are collected at 
presentation for the study. 
 
 
 

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8. Study design  
Monocentric / multicentric 
This is a monocentric study with matched collectives (age, gender). 
Study arms: intervention/control 
Interventions are not planned. A comparison is made between healthy volunteers 
and patients with SMA. The study procedure is identical for all volunteers and 
patients. 
Randomization 
Randomisation is not planned. The allocation to the groups is based on the known 
diagnosis (SMA) and controls (subjects). 
Blinding 
Blinding for the examination is not possible due to the often clear clinical appearance. 
Blinding takes place during the measurement and evaluation of the data. Blinding of 
the patients/test persons is not necessary. 
 
 
 

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Graphical presentation of the study design 
 
 
SMA patients 
N=10 
Healthy volunteers 
N=10 
Obtaining informed consent 
Checking inclusion criteria 
Inclusion of the participant 
Data collection 
Clinical examination 
B-Mode Ultrasound 
MSOT 

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9. Study population 
Inclusion and exclusion criteria 
Preliminary phase 
Inclusion criteria: 
SMA patients: 
• 
Genetically Confirmed SMA Type I-III 
• 
From birth 
• 
Independent from current therapy 
 
Healthy controls: 
• 
From birth, matched (age, gender) to SMA collective 
 
Exclusion criteria: 
SMA patients: 
• 
Pregnancy 
• 
Tattoo on skin to be examined 
 
Healthy controls: 
• 
anamnestic or other signs of myopathy 
• 
pregnancy 
• 
Tattoo on skin to be examined 
 
Patient/control number 
As this is a pilot study, an exact case number calculation is not possible. It is planned to 
study a total of 10 healthy volunteers and 10 patients with SMA.  
Recruitment routes and measures 
Patients (and parents) are informed about the possibility of participating in the study in the 
context of an elective presentation in the Department of Neuropediatrics in our pediatric 
Clinic. If the patient is willing to participate, he/she will be fully informed about the aims and 

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methods (especially about the scientific/explorative character of the study), benefit and risk 
and revocability of the study participation. Patients in childhood and adolescence will also be 
informed and educated about the study and its procedure according to their age.  
Healthy volunteers are recruited in the outpatient department of our Pediatric Clinic. Acutely 
ill or unstable patients are not recruited. In the preliminary phase, volunteers are parallelised 
with the SMA collective in terms of age and sex. 
10. 
Study course 
Procedure for informing about and obtaining consent 
Patients or test persons can only be included in the study after a written consent has been 
given. The written declaration of consent requires oral and written information of the 
patients/test persons as well as their parents or legal guardians about goals and methods 
(incl. scientific-explorative character of the study), benefit and risk as well as revocation of 
participation in the study. Children and adolescents are informed by means of age-
appropriate, comprehensible patient information sheets. By giving their written consent, the 
patients/test persons and their parents/guardians declare that they agree to the collection 
and storage of study-relevant data and their verification by monitoring or authorities. The 
study participant must be clearly informed that the declaration of consent can be withdrawn 
at any time and without any disadvantage. Furthermore, all study participants/test persons 
and parents/guardians are informed that this study is a purely scientific study without any 
current diagnostic or therapeutic benefit. 
The original of the declaration of consent will be kept in the study folder at the place of study. 
The patient/control and the parents/guardian receive a copy of the patient information and 
declaration of consent. The patient information and the consent form are attached to this 
protocol 
 
 
 
 
 
 

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Measurements 
After informing the patient / control and parent / guardian and 
obtaining consent, clinical scores are collected to assess 
muscle strength according to age (Hammersmith Infant 
Neurological Examination (HINE)/ expanded Hammersmith 
functional motor scale (HFMSE)/ The Children's Hospital of 
Philadelphia Infant Test of Neuromuscular Disorders (CHOP 
Intend)/ Revised upper Limb Module (RULM)/6-minute walking 
test (6-MWT)). Subsequently, MSOT imaging is performed on 4 
anatomical regions in all study participants: Upper/lower arm 
and upper/lower leg on predefined muscle groups. (Leg 
proximal: Musculus quandriceps, distal: Musculus triceps surae; 
arm proximal: Musculus biceps, distal: Forearm flexors). The 
examination 
is 
analogous 
to 
sonography 
over 
the 
corresponding skin layers without further invasive procedures. 
The anatomical region can be localized by means of built-in B-
image sonography; the corresponding optoacoustic signals can 
then be conducted. The duration per anatomical region is 
limited to 5 minutes; this corresponds to a maximum of 20 minutes for both upper extremities 
and 20 minutes for both lower extremities. Patients can remain in a relaxed posture during 
the examination, without the need for breathing maneuvers or similar assistance. 
Recording of target parameters 
• 
Clinical evaluation of muscle strength by HINE/HFMSE/CHOP Intend/RULM/6-MWT 
• 
Non-invasive in-vivo measurement of lipid, collagen, myo/hemoglobin content and 
oxygenation by MSOT 
• 
Determination of routine data (duration of disease, current medication) from electronic 
patient file in Soarian 
Time schedule and study duration for the individual patient/control 
For the individual patient, the duration of the study participation is 130 minutes. 
Approximately 30 minutes are spent on education for study participants and 
parents/guardians, 60 minutes on clinical (routine) testing of muscle strength, and 40 minutes 
for the actual examination.  
Total duration of study 
Depending on the number of patients, the expected total duration of the study up to the 
inclusion of the last patient is approximately 12 months. 
Presentation 

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11. 
Risk-benefit analysis 
All study-related risks  
Based on the classification criteria for medical devices (Directive 93/42/EEC, Annex IX), the 
optoacoustic system of iThera Medical corresponds to Class IIa: 
• 
Active diagnostic device 
• 
non-invasive 
• 
Temporary use (<60 min) 
No CE certification is available for this research device (current type designation according to 
imprint: Acuity Echo). A conformity assessment procedure in the sense of the MPG is not 
intended or planned by the manufacturer at the present time. It is therefore a purely scientific 
pilot study. There is no dependency on the manufacturer, all diagnostic and analytical 
procedures are available to the study directors on site. The cooperation with the company is 
regulated in a separate contract drawn up by the legal department before the start of the 
study. 
 
Adherence to energy levels  
The laser safety and maximum permitted radiation dose for irradiation with laser pulses is 
regulated in the laser standards ANSI and IEC 60825. The MSOT system meets these 
standards and therefore remains below the MPE (maximum permissible exposure) limits for 
skin irradiation and is therefore considered safe. 
 
Temperature increases due to MSOT in tissue 
Optoacoustic imaging does not result in any significant temperature increase in the tissue. 
The absorption of a laser pulse in the tissue results in a local transient temperature increase 
of a few millikelvin. Depending on the duration of the examination and the skin type of the 
patient, temperature increases occur typically in the range of less than one degree Kelvin. 
 
Histological changes in tissue 
Histological changes in the target tissue and surrounding structures are neither expected nor 
have they been observed in previous preclinical and clinical studies. 
 
Slight, reversible redness or warming might occur in very sensitive skin.  
 
Such side effects are to be noticed at any time by the test person or doctor; the examination 
can then be interrupted or aborted. In any case, no irreversible damage is to be expected. 
 

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In general, the near infrared light used in the MSOT can lead to retinal damage if the eye is 
irradiated. In order to prevent this, test participants and examiners will wear appropriate laser 
safety glasses during the examination. 
 
Since the data obtained is not used to interpret diagnostic results, there is no risk of possible 
misdiagnosis or incorrect display of data in this exploratory pilot study. 
 
No other risks exist for this study, nor have we described any risks based on our own 
preliminary data. 
 
Benefits associated with the study  
The data obtained in the studies may provide important insights into the pathomechanism of 
SMA. The possible quantitatively determinable differences could be used in the future as a 
monitoring method for a therapeutic response or prognosis of this disease. 
 
Termination criteria 
Termination criteria for the individual participant: 
Especially in light of the background of the inclusion of children and adolescents, 
participation in the study is discontinued if the skin becomes conspicuously warm or 
reddened. The examination time per anatomical region is limited to 5 minutes, so that these 
events are very unlikely. 
Due to the short duration of the study participation, no other discontinuation criteria are 
planned. 
Termination criteria for the whole study:  
A termination of the entire study is not planned. 
 
 
 

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Statement on medical justifiability  
Based on previous experience in children with Duchenne muscular dystophy and healthy 
controls, the risk of the occurrence of unwanted events is stratified as extremely low.  
So far, no serious event has been reported, neither at our site nor in the literature. The 
majority of the reported (predictable) problems related either to the use of ultrasound gel for 
examination or the need to wear eye protection. The use of filter glass also explains the 
reported redness. This phenomenon was reversible within seconds. Table 1 shows the 
reported events from our study (MSOT_DMD, 67_18 B). 
 
Table 1 – Adverse events  
 
In this study no central organs are examined, but only extremities are measured - this leads 
to a further significant reduction of a possible residual risk.  
Particularly in light of the background of completely new therapeutic approaches, we hope 
that this method will provide us with a child-friendly diagnostic tool for the treatment of these 
complex diseases. Since these diseases and their therapy are only relevant in childhood, no 
comparable adult collectives are available. 
 
 
 
Muscular dystrophy 
Duchenne N=10 
Healthy volunteer 
N=10 
Reversible adverse events- no.(%) 
 
 
     Pressure of safety goggles 
2 (20%) 
 
     Coolness of Ultrasound-gel 
 
4 (40%) 
     Red cast view 
 
1 (10%) 
Serious adverse events- no.(%) 
0 (0%) 
0 (0%) 

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12. 
Biometrics 
Explorative study: explanation of the statistical methodology, justification of the 
selected number of cases  
 
Case number calculation: 
As this is a pilot study and no information on the expected differences between the different 
groups is available yet, no case number calculation was performed. The number of cases 
given represents an estimate or is within the appropriate range for a pilot study.  
Statistical methods: 
Continuous variables are given as mean values with standard deviation, categorical variables 
as numbers with percentages if necessary. The MSOT parameters are compared using a 
two-sided, unpaired t-test with the same deviations. If the standard deviation is unequal, a 
correction according to Welch may be applied. Furthermore, ROC analyses (Receiver 
Operator Characteristics) between healthy/ill persons are planned. Genetics serve as the 
gold standard. Correlations are indicated by the Pearson coefficient. All statistical tests are 
performed on both sides and a p-value of <0.05 is considered statistically significant. All 
analyses are performed using GraphPad Prism (version 7.00 or later, GraphPad Software, 
La Jolla, CA, USA), RStudio (version 1.1.456 or later, RStudio Inc., Boston, MA, USA) or IBM 
SPSS Statistics (version 24 or later, IBM Corp., Armonk, NY, USA). 
 
 

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13. 
Data management und and data protection 
Data acquisition and storage  
All raw data, such as patient files, are source documents. Their availability is ensured for 
routine monitoring. The participation of the individual patients or test persons in the study is 
documented. The study leader maintains an independent list for the identification of the 
participating patients. This list containes the names and date of birth as well as the date of 
examination and pseudonymisation codes of the patients and subjects. The study leader is 
responsible for the quality of data collection and storage. The data storage (complete data) 
takes place on computers or specially designed network drives of the University Hospital 
Erlangen. The raw imaging data (no patient-related data) are stored on dedicated servers of 
iThera Medical GmbH. 
Pseudonymisation 
Prior to a scientific analysis of the materials and data of this study, all information will be 
pseudonymized according to the guidelines of the Federal Data Protection Act. 
Data transfer 
In this study, data transfer is only intended for the MSOT raw data. The company iThera 
Medical GmbH will work with this data to ensure adequate recording quality and to develop 
algorithms for evaluation. The data will only be passed on pseudonymously on encrypted 
physical drives. The data will not be used for later approval of the prototype used. The 
cooperation is explicitly regulated by the legal department before the start of the study (an 
additional agreement to the service contract can be found in the appendix). 
The study results can be published anonymously, whereby it will not be possible to draw 
conclusions about the identity of the participating persons. The data will be kept for 10 years 
and then destroyed.  
Revocation, data deletion 
If the declaration of consent is revoked, data collected up to this point can be taken into 
account. The patient has the right to demand that the data be destroyed, provided that legal 
provisions do not prevent such destruction. 
 
 

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14. 
Handling of biomaterials 
No biomaterials are obtained.  
15. 
Insurance 
The participants of the study are insured via the group contract of the CCS Erlangen. 
 
 

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16. 
Signatures 
 
 
 
Dr. med. Ferdinand Knieling 
Study director 
Prof. Dr. med. Regina Trollmann 
Study director 
 
 
 
 
 
Prof. Dr. med. Maximilian Waldner 
Technical implementation 
 
 
 
 
 
Prof. Dr. h.c. Dr. med. W. Rascher 
Clinic director