Multispectral Optoacoustic Tomography for Translational Molecular Imaging in Pompe Disease
Phase: NA
Conditions: Pompe Disease, Pompe Disease (Late-onset), Pompe's Disease Juvenile Onset, Pompe Disease Infantile-Onset
Sponsor: University of Erlangen-Nürnberg Medical School

Version 1.4 – MSOT_Pompe 
 
 
1 
Content is confidential.  
                Study protocol 
 
 
 
SPOT_PD 
 
Multispectral Optoacoustic Tomography for Translational Molecular Imaging in Pompe 
Disease 
 
 

Version 1.4 – MSOT_Pompe 
 
 
2 
Content is confidential.  
1. Table of contents 
 
1. 
Table of contents .............................................................................................................. 2 
2. 
Study titel, version number, version date ......................................................................... 3 
3. 
Project summary .............................................................................................................. 4 
4. 
Project summary (German) .............................................................................................. 5 
5. 
Responsabilities ............................................................................................................... 6 
6. 
Scientific background ....................................................................................................... 7 
7. 
Study objective ............................................................................................................... 12 
8. 
Endpoint parameters ...................................................................................................... 15 
9. 
Study design ................................................................................................................... 18 
10. Study population ............................................................................................................. 20 
11. Study flow ....................................................................................................................... 21 
12. Benefit-risk-assessment ................................................................................................. 24 
13. Biometrics ....................................................................................................................... 27 
14. Data management and protection .................................................................................. 27 
15. Biomaterial handling ....................................................................................................... 28 
16. Individual paticipant insurance ....................................................................................... 28 
17. Signatures ...................................................................................................................... 29 
 
 
 

Version 1.4 – MSOT_Pompe 
 
 
3 
Content is confidential.  
2. Study titel, version number, version date 
 
Study title 
Multispectral Optoacoustic Tomography for Translational Molecular Imaging in Pompe 
Disease 
 
Version number 
Version 1.3 
 
Version date 
30.05.2023 
 
 
Protocol versions 
Date 
Version 
Status 
Changes 
03.01.2020 
1.0 
Outline 
 
11.06.2021 
1.1 
Final 
 
05.08.2021 
1.2 
Revision 
Minor arm excluded 
02.03.2022 
1.3 
Amendment 
MRI, UGAP included 
30.05.2023 
1.4 
Amendment 
DMD arm canceled 
 
 
 

Version 1.4 – MSOT_Pompe 
 
 
4 
Content is confidential.  
3. Project summary 
In patients with Pompe disease (PD) a progressive abnormal lysosomal glycogen storage in 
muscle tissue leads to impaired muscle function and to degeneration of muscle fibers. Children 
and adults with PD present with limb-girdle muscular weakness, diaphragm weakness and 
impaired breathing ability. Further, patients with classic infantile PD suffer from hypertrophic 
cardiomyopathy. To date, the muscle pathology and the extent of the disease can be assessed 
using invasive techniques (e.g., muscle biopsies) or imaging (e.g., MRI). These techniques are 
time consuming, and especially in young patients, require anesthesia, which increases the 
acute risk of respiratory failure. 
Multispectral optoacoustic tomography (MSOT) allows the detection of specific endogenous 
chromophores like collagen, myoglobin or hemoglobin by using a non-invasive approach 
comparable to conventional ultrasound. Instead of sound waves, MSOT illuminates tissue with 
near-infrared light of transient energy, which is absorbed and results in thermo-elastic 
expansion of certain molecules. This expansion generates ultrasound waves that are detected 
by the same device. Multispectral illumination and unmixing then allows the precise localisation 
and quantification of muscle-specific subcellular structures. MSOT has already been 
demonstrated the potential to visualize the muscular structure and the clinical extent of 
muscular disease in patients with Duchenne muscle dystrophy and differentiates those 
patients from healthy volunteers. 
The aim of the study is to establish glycogen as a novel PD-specific imaging target using 
MSOT-imaging. Furthermore, we will apply MSOT-imaging to identify a PD-specific muscle 
pathology-signature by quantification of already established targets (collagen, myoglobin, 
hemoglobin, glycogen if applicable). This signature will aid in differentiating PD from other 
muscular pathologies and healthy volunteers and will ultimately serve as a potential non-
invasive monitoring biomarker.  
 
 

Version 1.4 – MSOT_Pompe 
 
 
5 
Content is confidential.  
4. Project summary (German) 
Bei Patienten mit Morbus Pompe (PD) führt eine fortschreitende abnorme lysosomale 
Glykogenspeicherung im Muskelgewebe zu einer Beeinträchtigung der Muskelfunktion und 
zur Degeneration von Muskelfasern. Kinder und Erwachsene mit Morbus Pompe zeigen eine 
Schwäche der Gliedergürtelmuskulatur, eine Zwerchfellschwäche und eine Beeinträchtigung 
der Atemfunktion. Außerdem leiden Patienten mit klassischer infantiler PD an einer 
hypertrophen Kardiomyopathie. Bislang können die Muskelpathologie und das Ausmaß der 
Erkrankung mit invasiven Techniken (z. B. Muskelbiopsien) oder bildgebenden Verfahren (z. 
B. MRT) beurteilt werden. Diese Techniken sind zeitaufwendig und erfordern insbesondere bei 
jungen Patienten eine Anästhesie, was das akute Risiko eines Atemversagens erhöht. 
 
Die multispektrale optoakustische Tomographie (MSOT) ermöglicht den Nachweis 
spezifischer körpereigener Chromophore wie Kollagen, Myoglobin oder Hämoglobin durch 
einen nicht-invasiven Ansatz, der mit konventionellem Ultraschall vergleichbar ist. Anstelle von 
Schallwellen beleuchtet MSOT das Gewebe mit Nahinfrarotlicht von transienter Energie, das 
absorbiert wird und zu einer thermoelastischen Ausdehnung bestimmter Moleküle führt. Diese 
Ausdehnung erzeugt Ultraschallwellen, die von demselben Gerät detektiert werden. Die 
multispektrale Beleuchtung und Entmischung ermöglichen dann die präzise Lokalisierung und 
Quantifizierung von muskelspezifischen subzellulären Strukturen. MSOT hat bereits das 
Potential gezeigt, die Muskelstruktur und das klinische Ausmaß der Muskelerkrankung bei 
Patienten mit Duchenne-Muskeldystrophie sichtbar zu machen und diese Patienten von 
gesunden Freiwilligen zu unterscheiden. 
 
Ziel der Studie ist es, Glykogen als neuartiges PD-spezifisches Bildgebungsziel mittels MSOT-
Bildgebung zu etablieren. Darüber hinaus werden wir MSOT anwenden, um eine PD-
spezifische Muskelpathologie-Signatur durch Quantifizierung bereits etablierter Targets 
(Kollagen, Myoglobin, Hämoglobin, Glykogen, falls zutreffend) zu identifizieren. Diese Signatur 
wird helfen, PD von anderen muskulären Pathologien und gesunden Probanden zu 
unterscheiden und wird letztendlich möglicherweise als potenzieller nicht-invasiver 
Überwachungs-Biomarker dienen.  
 
 
 

Version 1.4 – MSOT_Pompe 
 
 
6 
Content is confidential.  
5. 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 
 
Sponsoring  
Investigator-initated trial, funding by Sanofi Genzyme 
 
 

Version 1.4 – MSOT_Pompe 
 
 
7 
Content is confidential.  
6. Scientific background 
Pompe disease (PD) is a rare, autosomal-recessive disorder caused by deficiency of the 
lysosomal acid alpha-glucosidase enzyme (GAA), leading to generalized build-up of glycogen, 
especially in the heart, muscle, liver and nervous system (Pompe, 1932, Hers, 1963). Among 
the glycogen storage diseases, PD is the only one with a defect in lysosomal metabolism.  
PD is considered as a progressive disease with variation by age of onset, severity of organ 
involvement and degree of myopathy. This great phenotypic variability has led to the creation 
of types based on the age of onset and degree of organ involvement. They all have in common, 
that symptoms of affected patients are expected to worsen over time if left untreated. The 
classification is generally based on the age of onset as infantile (infantile onset Pompe disease, 
IOPD) when it presents during the first 12 months of life and late-onset (LOPD) when first 
symptoms appear after 12 months of age. If cardiomyopathy is present, IODP is generally 
referred to as classic Pompe disease (however there may be variably classification in the 
literature with the infantile or childhood forms). Clinically, infants with classic PD present during 
the first few months of life with rapidly progressive disease characterized by prominent 
hypertrophic cardiomyopathy, hepatomegaly, hypotonia, generalized muscle weakness, 
macroglossia, feeding difficulties and respiratory insufficiency. Mortality rate is high by one 
year of age if untreated (Kishnani 2004, 2006). Patients with non-classic PD will usually present 
within the first year of life with motor developmental delay and weakness, but without clinically 
relevant cardiac involvement. The rate of clinical progression is slower in these children and 
without treatment, death will usually occur in childhood as a result of respiratory insufficiency 
(Hers 1963, Slonim 2000). LODP include childhood and adult-onset PD. These patients 
generally present with slowly progressive limb girdle type weakness and respiratory 
insufficiency without significant cardiomyopathy (Cupler 2012, Preisler 2013). The diagnosis 
of PD is usually established by the typical clinical presentation, followed by confirmation of 
GAA deficiency in dried blood spots, e.g. through new-born screening (Bodamer, 2017). 
Further (confirmatory) methods include GAA activity measurement in lymphocytes, muscle or 
skin fibroblasts, as well as GAA mutation testing (Van den Hout, 2003, Case, 2012). All of them 
are invasive techniques. Early identification is important as it will likely significantly improve the 
outcome for all patients with PD as treatment can be initiated earlier. Treating the underlying 
cause of PD involves the replacement of the missing enzyme GAA via enzyme replacement 
therapy (ERT) with alglucosidase alfa (recombinant human GAA, rhGAA). Currently, this is the 
only specific treatment approved for PD. In classical IOPD, treatment significantly lengthens 
survival and improves motor development and respiratory and cardiac function (Kishnani, 
2007). The sooner ERT begins, the better are the results (Chien, 2013). With ERT being one 

Version 1.4 – MSOT_Pompe 
 
 
8 
Content is confidential.  
very important aspect of care, patients will also need a multidisciplinary approach to ensure 
that all aspects of the disease are addressed.  
Regardless of age of onset and severity, all patients with PD should be monitored prospectively 
(Kishnani, 2006, Bembi, 2008).   However, there is lack of standardization across centers.  A 
variety of clinical evaluations and tests are currently used for monitoring Pompe’s disease, 
which may include laboratory tests including CK, AST, ALT, and LDH, cardiologic tests 
including electrocardiogram and echocardiogram and respiratory tests including sleep studies 
and breathing tests to measure lung capacity. To quantify muscle involvement 
electromyography is an option as well as clinical tests including 6 minutes walking test or timed 
to up and go test.  Muscle MRI of affected patients often show fatty degeneration of muscles. 
One study showed that muscle MRI correlates with muscle function in patients with adult-onset 
Pompe disease (Figueroa-Bonaparte, 2016). Another study suggested that muscle imaging 
data in late-onset Pompe disease reveal a correlation between the pre-existing degree of 
lipomatous muscle alterations and the efficacy of long-term enzyme replacement therapy 
(Gruhn, 2015). For small children, however, there is always a need for sedation for MRI’s, 
limiting its use.  Therefore, ultrasound is another option to examine children’s muscles. 
At the moment there are no prospective biomarkers available to detect muscle degeneration 
at an early age and/or to follow up disease progression or ERT-treated patients.  Within the 
last years our multidisciplinary research team (Medical Department 1, Department of Pediatrics 
and Adolescent Medicine, University Hospital Erlangen) published a novel non-invasive 
imaging modality to be able to detect subcellular tissue composition in vivo. Multi-spectral 
optoacoustic tomography (MSOT), an imaging technology comparable to ultrasound, allows 
quantitative imaging in patients of all ages (including the non-sedated child).  

Version 1.4 – MSOT_Pompe 
 
 
9 
Content is confidential.  
For 
MSOT, 
similar 
to 
a 
conventional 
ultrasound, 
an 
ultrasound transducer is positioned 
on the patient’s skin. Instead of 
sound waves, MSOT illuminates 
tissue with light of transient energy, 
typically near-infrared laser light 
pulses, which are absorbed by the 
tissue, resulting in thermo-elastic 
expansion. This expansion gives 
rise to ultrasound waves that are 
detected by the same device. 
Studies have already shown that 
MSOT-based 
assessment 
of 
hemoglobin levels in the intestinal 
wall 
has 
the 
potential 
for 
Assessment of Crohn’s Disease 
Activity (Knieling 2017, Waldner 
2016). With a newly configurated 
device 
(Acuity 
Echo, 
iThera 
Medical GmbH, Munich, custom-
built 
platform) 
an 
extended 
spectrum of laser light can be used, 
allowing the detection of not only hemoglobin but also further biomarkers like collagen or lipid. 
In our recent study, we were able to show the molecular composition of muscles in Duchenne 
muscle dystrophy via MSOT suggesting non-invasive measured collagen content as a novel 
biomarker for disease severity (Regensburger, 2019). In this study we want to establish 
glycogen as a novel PD-specific imaging target using MSOT-imaging.  
 
 
 
Figure 1 - MSOT principle (a), experimental preliminary work (b), and first 
time representation of collagen in vivo (c) 

Version 1.4 – MSOT_Pompe 
 
 
10 
Content is confidential.  
Figures 1 and 2 show exemplary MSOT 
images. In the light of the progression and 
the sometimes severe course of PD, 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 PD, it will now be investigated whether 
the differences in the muscle composition 
of healthy volunteers and PD patients can 
be quantified and whether this could 
simultaneously be used as marker during 
ERT-therapy. In the future, this could 
generate a completely new, non-invasive 
method 
to 
evaluate 
endogenous 
biomarkers for therapy response. 
 
Literature 
Pompe JC. Over idiopatische hypertrophie van het hart. Ned Tijdschr Geneeskd. 1932; 76; 304-12.  
 
Hers HG. Alpha-Glucsidase deficiency in generalized glycogen storage diease (Pompe’s disease), 
Biochem J. 1963, 86, 11-6.  
 
Kishnani PS, Howell RR. Pompe disease in infants and children. J Pediatr. 2004;144(Suppl 5):S35-43. 
 
Kishnani PS, Hwu WL, Mandel H, Nicolino M, Yong F, Corzo D. A retrospective, multinational, 
multicenter study on the natural history of infantile-onset Pompe disease. J Pediatr. 2006;148(5): 671-
6. 
 
Hers HG. Alpha-Glucosidase deficiency in generalized glycogen storage disease (Pompe’s disease) 
Biochem J. 1963;86:11–16. 
 
Slonim AE, Bulone L, Ritz S, Goldberg T, et al. Identification of two subtypes of infantile acid maltase 
deficiency. J Pediatr. 2000;137:283–285. 
 
Cupler EJ, Consensus treatment recommendations for late-onset Pompe disease. Muscle Nerve. 2012, 
45:319-33.  
 
Preisler N. Late-onset Pompe disease is prevalent in unclassified limb-girdl muscular dystrophies. Mol 
Genet Metab. 2013, 110:287-9. 
 
Bodamer OA. Newborn Screening for Pompe Disease. Pediatrics, 2017. Jul; 140 (Suppl 1) S4-S13 
 
Van den Hout HM. The natural course of infantile Pompe’s disease: 20 original cases compared with 
133 cases from the literature. Pediatrics. 2003, 112.332-40.  
Case LE. Infantile Pompe disease on ERT. Update on clinical presentation, musculoskeletal 
management and exercise considerations. Am J Med Genet C Semin Med Genet. 2012. 160C: 69-79.  
Figure 2 - Feasibility of 3D MSOT image (a) and diagnostic 
quality (b). 
a 

Version 1.4 – MSOT_Pompe 
 
 
11 
Content is confidential.  
 
Lumyzyme (alglucosidase alfa) for injection, for intravenous use (prescribing information). Cambridge, 
MA: Genzyme Corporation; 2014.  
 
Myozyme (alglucosidase alfa) for injection, for intravenous use (prescribing information). Cambridge, 
MA: Genzyme Corporation; 2014. 
 
Kishani PS, Recombinant human Recombinant human acid [alpha]-glucosidase: major clinical benefits 
in infantile-onset Pompe disease. Neurology, 2007, Jan 9; 68 82); 99-109. 
 
Chien YH, Hwu WL, Lee NC. Pompe disease: early diagnosis and early treatment make a difference. 
Pediatr Neonatol. 2013;54(4):219–227 
 
Kishnani PS, Steiner RD, Bali D,et al. Pompe disease diagnosis and management guideline [published 
correction appears in Genet Med. 2006;8(6):382]. Genet Med. 2006;8(5):267–288 
 
Bembi B, Cerini E, Danesino C,et al. Management and treatment of glycogenosis type II. Neurology. 
2008;71(23 suppl 2):S12–S36 
 
Figueroa-Bonaparte S, Muscle MRI Findings in Childhood/Adult Onset Pompe Disease Correlate with 
Muscle Function, PLoS One. 2016; 11(10): e0163493. 
 
Gruhn KM, Muscle imaging data in late-onset Pompe disease reveal a correlation between the pre-
existing degree of lipomatous muscle alterations and the efficacy of long-term enzyme replacement 
therapy. Mol Genet Metab Rep. 2015 Jun; 3: 58–64. 
 
Knieling F. Multispectral Optoacoustic Tomography for Assessment of Crohn's Disease Activity. N Engl 
J Med. 2017 Mar 30;376(13):1292-1294.  
 
Knieling F. Light and sound - emerging imaging techniques for inflammatory bowel disease. World J 
Gastroenterol. 2016 Jul 7;22(25):5642-54.  
 
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.  
 
Regensburger A. et al. Detection of collagens by multispectral optoacoustic tomography as an imaging 
biomarker for Duchenne muscular dystrophy. Nat Med. 2019 Dec 11 14:9809-9821.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Version 1.4 – MSOT_Pompe 
 
 
12 
Content is confidential.  
7. Study objective  
 
Primary study objective: 
Comparison of the optoacoustic spectrum determined by MSOT in patients with PD compared 
to healthy volunteers, generating a new biomarker for disease monitoring in PD. 
Secondary study objectives: 
• 
Comparison of the quantitative glycogen signal fraction determined by MSOT in 
patients with PD compared to healthy volunteers 
• 
Comparison of the quantitative lipid signal fraction determined by MSOT in patients 
with PD compared to healthy volunteers 
• 
Comparison of the quantitative fraction of collagen signal determined by MSOT in 
patients with PD compared to healthy volunteers 
• 
Comparison of the quantitative fraction of hemo-/myoglobin signal determined by 
MSOT in patients with PD compared to healthy volunteers 
• 
Comparison of the quantitative fraction of oxygenated/deoxygenated hemoglobin 
determined by MSOT in patients with PD compared to healthy volunteers 
 
• 
Correlation of glycogen content determined with MSOT with disease duration/patient 
age 
• 
Correlation of lipid content determined with MSOT with disease duration/patient age 
• 
Correlation of collagen determined by MSOT with disease duration/patient age 
• 
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 glycogen content determined with MSOT with R-Pact scale 
• 
Correlation of lipid content determined with MSOT with R-PAct scale 
• 
Correlation of collagen determined by MSOT with R-PAct scale 
• 
Correlation of haemoglobin/myoglobin content determined by MSOT with R-PAct scale 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined by MSOT with R-
PAct scale 
 
• 
Correlation of glycogen content determined with MSOT with age-related functional 
muscle tests (Hammersmith Infant Neurological Examination (HINE)/The Children's 

Version 1.4 – MSOT_Pompe 
 
 
13 
Content is confidential.  
Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP 
Intend)/expanded Hammersmith functional motor scale (HFMSE)/ Revised Upper Limb 
Module (RULM)/6-Minute-Walk Test (6-MWT)/Time-to-go-up-and-go-test/MRC Muscle 
Strength Grades) 
• 
Correlation of lipid determined with MSOT with age-dependent functional muscle tests 
(RULM /6-MWT/Time-to-get-up-and-go/MRC) 
• 
Correlation of collagen determined with MSOT with age-dependent functional muscle 
tests (RULM/6-MWT/Time-to-get-up-and-go/MRC) 
• 
Correlation of hemo-/myoglobin content determined with MSOT with age-dependent 
functional muscle tests (RULM/6-MWT/Time-to-get-up-and-go/MRC) 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined with MSOT with age-
related functional muscle tests (RULM /6-MWT/Time-to-get-up-and-go/MRC) 
 
• 
Correlation of glycogen content determined with MSOT with B-mode ultrasound 
(Heckmatt scale/Echogenity/Gray Scale Level/UGAP) 
• 
Correlation of lipid determined with MSOT with B-mode ultrasound (Heckmatt 
scale/Echogenity/Gray Scale Level/UGAPl) 
• 
Correlation 
of 
collagen 
determined 
with 
B-mode 
ultrasound 
(Heckmatt 
scale/Echogenity/Gray Scale Level/UGAP) 
• 
Correlation of hemo-/myoglobin content determined with MSOT with B-mode 
ultrasound (Heckmatt scale/Echogenity/Gray Scale Level/UGAP) 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined with B-mode 
ultrasound (Heckmatt scale/Echogenity/Gray Scale Level/UGAP) 
 
• 
Correlation of glycogen content determined with MSOT with respiratory function tests 
(Spirometry) 
• 
Correlation of lipid content determined with MSOT with respiratory function tests 
(Spirometry) 
• 
Correlation of collagen determined by MSOT with respiratory function tests 
(Spirometry) 
• 
Correlation of haemoglobin/myoglobin content determined by MSOT with respiratory 
function tests (Spirometry) 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined by MSOT with 
respiratory function tests (Spirometry) 
 
• 
Measurement of signal differences in right / left comparison 

Version 1.4 – MSOT_Pompe 
 
 
14 
Content is confidential.  
 
• 
Correlation of glycogen content determined with MSOT with (functional) magnetic 
resonance imaging parameters 
• 
Correlation of lipid determined with MSOT with (functional) magnetic resonance 
imaging parameters 
• 
Correlation of collagen determined with (functional) magnetic resonance imaging 
parameters 
• 
Correlation of hemo-/myoglobin content determined with (functional) magnetic 
resonance imaging parameters 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined with (functional) 
magnetic resonance imaging parameters 
 
Hypotheses: 
• 
The optoacoustic spectrum of muscles of patients with PD is different compared to 
healthy volunteers  
• 
The optoacoustic spectrum of the liver of patients with PD is different compared to 
healthy volunteers  
• 
The quantitative fraction of glycogen signal in muscles determined by MSOT differs in 
patients with PD compared to healthy volunteers 
• 
The quantitative fraction of lipid signal in muscles determined by MSOT differs in 
patients with PD compared to healthy volunteers  
• 
The quantitative fraction of collagen signal in muscles determined by MSOT differs in 
patients with PD compared to  healthy volunteers 
• 
The quantitative fraction of hemo-/myoglobin in muscles determined by MSOT differs 
in patients with PD compared to healthy volunteers 
• 
The quantitative fraction of oxygenated/deoxygenated hemoglobin in muscles 
determined by MSOT differs in patients with PD compared to healthy volunteers 
• 
There is a correlation between MSOT derived glycogen signal and clinical status of 
patients with PD 
• 
There are no side differences in patients with PD and healthy volunteers 
 
Study type 
Since no data exists so far to support the hypothesis of this study, it is an explorative study / 
pilot study. 

Version 1.4 – MSOT_Pompe 
 
 
15 
Content is confidential.  
8. Endpoint parameters 
All measurements with MSOT are performed over the paraspinal musculature, Musculus 
trapezius as well as 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 PD.  
Additionally, we will perform measurements of the liver with MSOT. 
 
Primary endpoint:  
Optoacoustic Absorption Spectrum of Muscle and liver in PD.  
This target is measured non-invasively by MSOT.  
 
Secondary endpoints:  
Quantitative glycogen signal (in arbitrary units)  
Quantitative lipid signal (in arbitrary units)  
Quantitative collagen signal (in arbitrary units)  
Quantitative hemo/myoglobin signal (in arbitrary units)  
Muscle oxygenation (in %)  
These target values are measured non-invasively by MSOT.  
B-mode ultrasound 
- 
Heckmatt scale 
- 
Echogenitiy 
- 
Gray Scale Level 
- 
Ultrasound-Guided Attentuation Parameter (UGAP) 
 
R-PaAt scale 
- 
Summed raw score 
 
Respiratory function test 
- 
FVC 
- 
FEV1 
 
Functional magnetic resonance imaging of lung. 
- 
Ventilation Defects / Non-defected 
- 
Perfusion Defects / Non-defected 
- 
Combined Defects / Non-defected 
 

Version 1.4 – MSOT_Pompe 
 
 
16 
Content is confidential.  
Magnetic resonance imaging of biceps muscle. 
 
Correlation of glycogen detected by MSOT with clinical scores for determining muscle strength 
consisting of: 
- 
Revised Upper Limb Module (RULM) 
- 
MRC Muscle Strength Grades 
in case of ability to walk additionally: 
- 
6-minute walking test 
- 
Time-to-get-up-and-go-test 
 
These target values are clinically determined at presentation (electronic patient record, 
physician letters): 
Age  
Sex 
Weight  
Skin color  
Ethnic Background  
Disease duration  
Current medication 
Results of last biopsy (if available) 
Results of last MRI (if available) 
 
 

Version 1.4 – MSOT_Pompe 
 
 
17 
Content is confidential.  
Study Parameters 
PD 
HV 
MSOT 
-‘Glycogen’ 
-Lipid  
-Collagen  
-Hemoglobin oxygenated 
-Hemoglobin deoxygenated 
X 
 X 
 X 
 X 
 X 
 X 
X 
 X 
 X 
 X 
 X 
 X 
Ultrasound 
-Heckmatt 
-Echogenity  
-Muscle texture 
-Grey scale level 
-UGAP 
X 
 X 
 X 
 X 
 X 
 X 
X 
 X 
 X 
 X 
 X 
 X 
Magnetic resonance 
imaging 
-Ventilation defects 
-Perfusion defects 
-Combined defects 
-Biceps muscle imaging 
X 
 X 
 X 
 X 
 x 
X 
 X 
 X 
 X  
R-Pact scale (≥16y) 
X 
X 
Respiratory function 
test 
-FVC 
-FEV1 
X 
 X 
 X 
X 
 X 
 X 
Muscle tests 
-RULM  
-6-MWT 
-Time-to-get-up-and-go 
-MRC 
X 
 X 
 X 
 X 
 X 
X 
 X 
 X 
 X 
 X 
Patient History  
Confirmed Disease  
X 
Type: 
Crim:  
X 
 
Diagnostics:  
-Genetics 
-Dried Blood Spot  
-Urinary Glc 4 
-Lymphozytes 
X 
 X 
 X 
 X 
 X 
 
Muscle biopsy 
X 
 
Neurological tests: 
-EMG 
-MRI 
-Sleep Study 
X 
 X 
 X 
 X 
 
Lab values  
-CK 
-Liver enzymes 
X 
 X 
 X 
 

Version 1.4 – MSOT_Pompe 
 
 
18 
Content is confidential.  
 
 
 
9. Study design 
Monocentric/Multicentric  
This is a multicentric study with matched collectives (age, gender)/  
Study arms: intervention/control  
Interventions are not planned. A comparison is made between patients with PD and healthy 
volunteers. The study procedure is identical for PD patients and healthy volunteers. Healthy 
volunteers are used as controls (no muscular pathology, no excessive intramuscular 
lipids/collagen, no excessive glykogen) versus PD patients (muscular pathology, 
intramuscular lipids/collagen, excessive glycogen). 
Randomization  
Randomization is not planned. The allocation to the groups is based on the known diagnosis 
(PD, other muscular pathology) and controls (subjects).  
Blinding  
Blinding for the examination is not possible due to the possible clear clinical appearance. 
Blinding takes place during the measurement and and evaluation of the data. Blinding of the 
patients/controls is not necessary.  
 
 
Sitting ability  
Walking ability  
X 
X 
 
Ventilation 
X 
 
Medication  
 
 

Version 1.4 – MSOT_Pompe 
 
 
19 
Content is confidential.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pompe disease 
N=10 
Healthy volunteers 
N=10 
Obtaining informed consent 
Checking inclusion criteria 
Inclusion of the participant 
Clinical data collection 
Clinical examination (muscle tests) 
R-Pact scale  
Respiratory function test 
US imaging 
MSOT imaging 
MRI Imaging 

Version 1.4 – MSOT_Pompe 
 
 
20 
Content is confidential.  
Study population 
Inclusion and exclusion criteria  
Inclusion criteria:  
PD patients (IOPD and LOPD) 
- 
Confirmed diagnosis of Pompe disease  
- 
From 18 years of Age 
- 
Independent from current therapy  
 
Healthy controls 
- 
From 18 years of Age, matched (age, gender) to PD collective  
 
Exclusion criteria 
PD patients  
- 
Pregnancy  
- 
Tattoo on skin to be examined  
 
Healthy controls  
- 
Anamnestic of other signs of myopathy or liver disease  
- 
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 patients with PD and 10 healthy controls. 
Recruitment routes and measures  
Patients (and parents) are informed about the possibility of participating in the study in the 
context of an elective presentation at the Clinic for Pediatrics and Adolescent Medicine 
(Neuropediatrics) and the Clinic for Neurology at the University Hospital in Erlangen, Germany 
as well as the the Clinic for Pediatrics and Adolescent Medicine (Neuropediatrics) in Gießen, 
Germany. Additional recruitment options include the Clinic Rummelsberg, Schwarzenbruck, 
Germany, the German Society for muscular diseases (DGM), the treatNMD network and the 
international Pompe Association. If the patient is willing to participate, he/she will be fully 

Version 1.4 – MSOT_Pompe 
 
 
21 
Content is confidential.  
informed about the aims and 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 departments of the Clinic for Neurology at 
the University Hospital in Erlangen, Germany.  
Acutely ill or unstable patients are not recruited. In the preliminary phase, volunteers are 
parallelised with the PD collective in terms of age and sex.  
10. 
Study flow 
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. 
  
Measurements  
After informing the patient / control and parent / guardian and obtaining consent, clinical scores 
are collected to assess muscle strength. R-Pact scale will be collected and respiratory function 
tests will be performed.  
 
Subsequently, MSOT imaging is performed on 6 anatomical regions in all study participants: 
Paraspinal muscles, Musculus trapezius, upper/lower arm and upper/lower leg on predefined 

Version 1.4 – MSOT_Pompe 
 
 
22 
Content is confidential.  
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 10 minutes for the parasinal 
muscles and Musculus trapezius, a maximum of 20 minutes for both upper extremities and 20 
minutes for both lower extremities and 10 minutes for the examination of the liver. Patients can 
remain in a relaxed posture during the examination, without the need for breathing maneuvers 
or similar assistance.   
 
At last, patients undergo MRI imaging of the lung and biceps muscle. Participants can be in a 
relaxed position during the examination. The time allotted for MRI is 30 minutes. 
 
Recording of target parameters  
• 
Determination of routine data (duration of disease, current medication)  
• 
Clinical evaluation of muscle strength by 6-MWT, Time-to-get-up-and-go, MRC muscle 
test 
• 
Assessment of R-Pact scale  
• 
Performance of respiratory function test  
• 
Non-invasive in-vivo measurement of glycogen, lipid, collagen, myo/hemoglobin 
content and oxygenation by MSOT  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Version 1.4 – MSOT_Pompe 
 
 
23 
Content is confidential.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Time schedule and study duration for the individual patient/control  
For the individual patient, the duration of the study participation is 220 minutes. Approximately 
30 minutes are spent on education for study participants and parents/guardians, 60 minutes 
on clinical (routine) testing of muscle strength, 10 minutes on R-Pact scale, 20min on 
Presentation 
Informed consent parents/guardian  
Check for in-/exclusion criteria  
(ca. 20min) 
Informed consent 
 (ca. 10min) 
Study inclusion 
Evaluation of clinical scores for muscle strength 
and clinicial routine data (ca. 60min) 
R-Pact scale (ca. 10min) 
Respiratory function test (ca. 20min) 
B-mode ultrasound (ca. 10min) 
Multispectral optoacoustic Tomography (MSOT) 
(ca. 60min) 
MRI imaging (Lung and Biceps muscle) 
(30 min) 
 

Version 1.4 – MSOT_Pompe 
 
 
24 
Content is confidential.  
respiratory function test, 10 minutes for B-mode ultrasound and 60 minutes for the MSOT 
examination. For MRI imaging 30 minutes are required. 
 
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 8 months.  
 
11. 
Benefit-risk-assessment 
All study-related risks 
Based on the classification criteria for medical devices (Directive 93/42/EEC, Annex IX), the 
iThera Medical optoacoustic system complies with Class IIa laser systems: 
- 
Active diagnostic device 
- 
Non-invasive 
- 
Temporary use (<60 min) 
 
The used system has CE-certification (TÜV Süd, 02.05.2021, type designation according to 
imprint: MSOT Acuity Echo). A conformity assessment procedure to expand the use of the 
system is not intended or planned by the manufacturer at this time. It is therefore a purely 
scientific pilot study. There is no dependency relationship with the manufacturer; all diagnostic 
and analytical procedures are available to the study directors on site. The cooperation with the 
company will be regulated in a separate contract drawn up by the legal department before the 
start of the study. 
Compliance with energy limits 
The laser safety and maximum permitted radiation dose for irradiation with laser pulses is 
regulated in the ANSI and IEC 60825 laser standards. The MSOT system complies with these 
standards and remains below the MPE (maximum permissible exposure) limits for skin 
irradiation and is therefore considered safe. 
Temperature increases in tissue 
Optoacoustic imaging does not result in a significant increase in tissue temperature. The 
absorption of a laser pulse in tissue results in a local transient temperature increase of a few 
millikelvin. Depending on the duration of the examination and the patient's skin type, there is 
typically a temperature increase in the range of less than one degree Kelvin. 
Histological changes in tissue 

Version 1.4 – MSOT_Pompe 
 
 
25 
Content is confidential.  
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 is only to be expected in sensitive skin. 
Such impairment of the patient can be noticed by the test person or physician at any time; the 
examination can then be interrupted or discontinued. In any case, no irreversible damage is to 
be expected. 
In principle, the near-infrared light used in the MSOT can lead to retinal damage if the eye is 
irradiated. To prevent this, the participants and examiners will wear appropriate laser safety 
glasses during the examination. 
Since the data obtained will not be used to interpret findings, there is no risk of possible 
misdiagnosis or incorrect display of data in this exploratory pilot study. 
There are no other risks within the scope of this study, nor have they been described based 
on our own preliminary data. 
Magnetic resonance imaging 
Unlike computed tomography, MRI does not use ionizing radiation, so no permanent side 
effects are to be expected. 
More than 1 million MRI examinations at higher field strengths (1.5/3T) are performed annually 
in Germany. Provided that the general contraindications for MRI examinations are observed, 
no serious side effects occur. MRI is therefore one of the safest examination procedures.  
The risks associated with an MRI examination emanate from the three main components of 
the MRI system. 
Static magnetic field 
The static magnetic field exerts forces and torques on ferromagnetic objects that can be so 
strong that the (mostly ferromagnetic) objects fly uncontrollably toward the magnet and can hit 
patients and staff (missile effect). The magnetic forces are proportional to the field strength B 
and the field change with location (dB/dz). These risks are lower with the low-field MRI system. 
Risks are further minimized by providing safety training to operators and excluding patients 
with ferromagnetic implants from the study. 
The gradient system 

Version 1.4 – MSOT_Pompe 
 
 
26 
Content is confidential.  
Gradient switching can cause the appearance of magnetic phosphenes and nerve and muscle 
cell stimulation. Rapidly switched gradients produce high magnetic field changes per time 
(dB/dt) that induce voltages in the body. If a current flows through the tissue via nerve endings, 
for example, this can result in so-called peripheral nerve stimulation. However, the 
manufacturer of the gradient system guarantees compliance with the limits for gradient 
switching times and amplitudes recommended in the IEC 60601-2-33 guidelines.  Thus, nerve 
stimulation effects need not be considered further in the risk assessment of this study. 
Another safety-relevant effect of gradient fields is noise caused by gradient switching due to 
current- and field-strength-dependent Lorentz forces in the gradient tube. These unpleasant 
loud knocking noises occur especially during fast imaging processes where high currents flow 
through the gradients. Noise levels can rise up to 115dB for 1.5T tomographs (background 
noise: approx. 78dB). Due to the lower magnetic forces at 0.55T, we expect lower noise levels. 
In addition, patients always wear hearing protection during the examination, so that the noise 
exposure remains well below the legal limit of 99 dB. 
The high-frequency system 
During the MR measurement, radiofrequency (RF) fields are sent into the human body, which 
are partially absorbed by the tissue and can lead to an increase in body temperature. The 
thermoregulatory response of human tissue to RF pulses has now been studied for 50 years. 
For example, using conservation of energy, it has been calculated that the body temperature 
of lightly clothed patients with undisturbed thermoregulation at room temperature increases by 
up to 0.6 °C with RF exposure of 4 W/kg (63 MHz, 1.5 Tesla). The assumed specific absorption 
rate (SAR) of 4 W/kg body tissue corresponds to the so-called "controlled mode first level" (IEC 
safety guideline), which is also used as an upper limit in routine clinical imaging. The magnitude 
of the actual temperature rise is generally smaller because skin cooling was not considered in 
the calculations (worst case scenario).  
The body's energy production at rest is about 1.2 W/kg - equivalent to the energy conserved 
when wearing a thin sweater. Most healthy people are capable of compensating for 15 times 
this resting energy, and only a minimal increase in core body temperature occurs. Studies at 
1.5 T have shown that RF absorption in humans leads only to the expected cardiac adaptation 
and does not cause adverse health effects. Theoretically, a 63 kg person is even capable of 
emitting 1296 W to the environment through the skin by cardiac adaptation (i.e., maximum 
increase in blood flow) - this would correspond to a SAR of 20.6 W/kg. 
The same limits are observed with the 0.55 Tesla MRI system. At 0.55 Tesla, the wavelength 
of the radio waves used is significantly longer, so that the spatial distribution of the energy 
emission is more homogeneous and thus the risks tend to be lower. 

Version 1.4 – MSOT_Pompe 
 
 
27 
Content is confidential.  
12. 
Biometrics 
Exploratory study: Explanation of the statistical methodology, justification of the chosen case 
number 
Case number calculation 
As this is a pilot study and no information is available on the expected differences between the 
different groups, no case number calculation was carried out. The number of cases given 
represents an estimate or is within reasonable limits for a pilot study.  
Statistical Methodology 
Continuous variables are given as mean values with standard deviation, categorical variables 
as numbers with percentages where appropriate. The MSOT parameters are compared using 
a two-sided, unpaired t-test with equal deviations. If the standard deviation is not equal, Welch 
correction is applied where appropriate. ROC (Receiver Operator Characteristics) analyses 
between healthy and sick persons are also planned. Genetics serves as the gold standard. 
Correlations are indicated by the Pearson coefficient. All statistical tests will be double-sided 
and a p-value of <0.05 is considered statistically significant. All analyses are performed with 
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). 
13. 
Data management and protection 
All raw data, such as patient records, represent 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 director maintains a separate list to identify the all screened and 
participating patients. This list contained the names and date of birth as well as the examination 
date and pseudonymization abbreviations of the patients and test persons. The study director 
is responsible for the quality of data collection and storage. The data storage (total data) is 
carried out on computers or specially designed network drives of the University Hospital 
Erlangen. The raw imaging data (no patient-related data) is stored on specially designated 
servers of the company iThera Medical GmbH. 
Pseudonymisation 
Prior to a scientific analysis of the materials and data of this study, all information will be 
pseudonymised in accordance with the guidelines of the Federal Data Protection Act. 
Data transfer 

Version 1.4 – MSOT_Pompe 
 
 
28 
Content is confidential.  
Data sharing in this study is intended solely 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 transferred pseudonymized on encrypted 
physical drives. The data will not be used for a later approval of the prototype used. The 
cooperation will be explicitly regulated by the legal department before the study starts (a 
supplementary agreement to the service contract can be found in the appendix). 
The study results can be published anonymously, but it will not be possible to draw conclusions 
about the identity of the participating persons. The data will be kept for 10 years and destroyed 
afterwards.  
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 their destruction, provided that legal provisions 
do not conflict with the destruction. 
 
14. 
Biomaterial handling 
No biomaterials are obtained. 
 
15. 
Individual paticipant insurance 
The participants of the study are insured through the group contract of the CCS Erlangen. 
 
 
 
 

Version 1.4 – MSOT_Pompe 
 
 
29 
Content is confidential.  
16. 
Signatures 
 
 
 
 
 
 
Dr. med. Ferdinand Knieling  
Study director  
 
 
 
 
 
 
Prof. Dr. med. Regina Trollmann  
Study director  
  
  
   
  
  
  
  
 
  
Prof. Dr. med. Joachim Wölfle 
Clinic director