Skin Tumor Biomarkers by Mass Spectrometry Imaging
Phase: NA
Conditions: Skin Cancer, Squamous Cell Carcinoma, Basal Cell Carcinoma, Non-melanoma Skin Cancer
Sponsor: Bispebjerg Hospital

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Study Protocol 
 
Project title: 
 
Skin tumor biomarkers by mass spectrometry imaging 
 
 
 
Sponsor and principal investigator 
Catharina M. Lerche, MSc Pharm, PhD 
Bispebjerg Hospital, University of Copenhagen 
Department of Dermatology, D92 
Nielsine Nielsensvej 17, Entrance 9 
DK-2400 Copenhagen NV 
Direct: + 45 21470444 
Cell: + 45 28207100 
Mail: catharina.margrethe.lerche@regionh.dk 
 
 

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Study Participants 
 
 
Martin Gluud, MD, PhD, Consultant 
Dermatologist, Bispebjerg Hospital, 
University of Copenhagen, Department 
of Dermatology, D42, Nielsine 
Nielsensvej 9, Entrance 4, DK-2400 
Copenhagen NV 
 
Professor Merete Hædersdal, MD, DMSc, 
PhD,  Bispebjerg Hospital, University of 
Copenhagen, Department of 
Dermatology, D42, Nielsine Nielsensvej 
9, Entrance 4, DK-2400 Copenhagen NV 
 
June Svendsen, Project nurse, Bispebjerg 
Hospital, University of Copenhagen, 
Department of Dermatology, D92, 
Nielsine Nielsensvej 17, Entrance 9 DK-
2400 Copenhagen NV 
 
 
Fernanda E. Pinto, MSc (Pharm), PhD                                          
Department of Pharmacy, University of 
Copenhagen, Universitetsparken 2, DK-
2100 Copenhagen Ø 
 
Christian Janfelt, MSc, PhD, Department 
of Pharmacy, University of Copenhagen, 
Universitetsparken 2, DK-2100 
Copenhagen Ø 
 
 
 
 
 
 
 
 
 
Mohs Surgeon 
 
 
 
 
 
Consultant Dermatologist 
 
 
 
 
Project Nurse 
 
 
 
 
Mass spectrometry imaging analyses 
 
 
Mass spectrometry imaging analyses 
 
 

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TABLE OF CONTENTS 
1. Title ............................................................................................................................................................. 4 
2. Aim .............................................................................................................................................................. 4 
2.1 
Problem definition ..................................................................................................................... 4 
2.2 
Hypothesis ...................................................................................................................................... 4 
2.3 
Outcome measures ..................................................................................................................... 4 
2.4 
Background .................................................................................................................................... 5 
3.1 
Study design for patients with BCC and SCCs ............................................................... 7 
3.2 
Study design for patients with actinic keratoses ....................................................... 8 
3.3 
Tissue microarray ...................................................................................................................... 8 
4. Statistical calculations ...................................................................................................................... 8 
5. Participants............................................................................................................................................. 9 
5.1 
Inclusion criteria ......................................................................................................................... 9 
5.2 
Exclusion criteria ..................................................................................................................... 10 
6. Potential risk and adverse effects ........................................................................................... 10 
6.1 
Punch biopsy .............................................................................................................................. 10 
7. Biobank .................................................................................................................................................. 10 
8. Information from patient journals .......................................................................................... 11 
9. Data protection, information storage & quality control .............................................. 11 
9.1 
Quality control ........................................................................................................................... 11 
9.2 
Data protection and storage .............................................................................................. 11 
10. Financial considerations............................................................................................................... 11 
11. Reimbursements ............................................................................................................................... 12 
12. Information to patients ................................................................................................................. 12 
13. Publication of results ...................................................................................................................... 12 
14. Ethics ....................................................................................................................................................... 13 
15. Insurance .............................................................................................................................................. 13 
16. References ............................................................................................................................................ 13 
 
 
 
 

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1. Title 
Dansk: Biomarkører i hudkræft og forstadier hertil ved hjælp af billeddannende 
massespektrometri  
Engelsk: Skin tumor biomarkers by mass spectrometry imaging 
 
2. Aim 
2.1 Problem definition 
More than 1 million patients were diagnosed with non-melanoma skin cancer (NMSC) 
worldwide in 2018 and this number could be underestimated due to the lack of registries or 
incomplete registration of this type of cancer [1]. Timely detection of microscopic tumors is of 
utmost importance in cancer diagnostics. We believe that mass spectrometry imaging (MSI) can 
successfully locate microscopic aggregates of a common skin cancer, basal cell carcinoma (BCC) 
and squamous cell carcinoma (SCC), and distinguish them from adjacent normal skin ex-vivo. 
MSI unveils an altered chemical profile in BCC and SCC region (lipid patterns) and does not rely 
on visual identification of histopathologic features.  
 
2.2 Hypothesis 
We hypothesize that NMSC cells exhibit specific, endogenous biomarkers (lipid patterns) that 
differ from healthy skin tissue in sections of skin biopsies. If that hypothesis is correct it will be 
possible in the future to develop real-time tissue diagnosis and treatment of NMSC using mass 
spectrometry guided surgery.  
 
2.3 Outcome measures 
• Identification of masses and distributions of selected lipid biomarkers in sections from 
removed BCC and SCC tissue from Mohs surgery. 
• Identification of masses and distributions of selected lipid biomarkers in sections from 
biopsies with actinic keratoses (pre-malignant lesions). 
 
 
 

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2.4 Background 
The incidence of non-melanoma skin cancer (NMSC) was of 1 million people in 2018, according 
to the Skin Cancer Report of 2019 by World Cancer Research Fund, being classified as the more 
commonly occurring cancer worldwide [1]. The NMSC englobes primarily basal cell carcinoma 
(BCC) and squamous cell carcinoma (SCC).   
In Denmark and other Western countries the occurrence of these tumors is also high [2], and 
Danish incidence continue to rise, amounting to 15.000 per year. The main risk factor to NMSC 
is exposure to ultraviolet radiation (UVR). Light skin color, light or red hair, light eye color and 
immunocompromised people are other established causes to NMSC [1,3]. 
The Danish population is predominantly fair-skinned, geographically mobile and exceedingly 
sun-loving, which increases the risks of developing skin cancer. Daily, ~44 Danes receive a 
NMSC diagnosis, consisting of either BCC or SCC, with 40% developing new tumors in the 
following years [4] 
NMSC has a low mortality, however it has a notable patient morbidity due to its high prevalence 
and recurrence [2], costing more than 19 million Euro yearly to Denmark, exceeding the cost of 
health management and health care costs of melanoma [5]. Care for NMSC is primarily by 
dermatologists, and multiple modalities can be used on the treatment of NMSC, such as Mohs 
micrographic surgery, excision, cryotherapy, and different topical modalities [2]. 
The lowest recurrence rates of Mohs micrographic surgery make this excisional surgery the 
first choice of treatment for high risk NMSC [6]. During this microscope-guided surgery, tissue 
is excised, sectioned, stained, and evaluated by a trained pathologist. The patient waits on the 
operating table while tissue margins are inspected for cancer cells, a finding that prompts 
further tissue removal from the treatment area. Accordingly, Mohs surgery is time-consuming 
and expensive, due to limited number of patients that can be treated per day and need for highly 
trained laboratory staff.  
For decades, laboratories have benefitted from the impressive sensitivity and specificity of 
mass spectrometry (MS). A well-established analytical technique, MS ionizes compounds and 
sorts them based on their molecular mass. MS imaging (MSI) visualizes the spatial distribution 
of molecules in a similar way. Scanning entire tissue sections (e.g. skin) and generating a mass 
spectrum for each unit area, a map of a compound’s tissue distribution is provided (Figure 1).  

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Figure 1. The tissue is sampled point by point with the laser (a). Desorbed material from each point 
is transfered to the mass spectrometer and a spectrum is recorded (b). Signal intensity of a 
component is then extracted from each mass spectrum and are transformed into gray scale values 
of a pixel image (c). In this way the distribution of all compound detected in the mass spectrum 
can be displayed [7] 
MSI can detect many endogenous molecular species simultaneously, including lipids, small 
proteins, as well as exogenous pharmaceutical drugs distributed in minute concentrations 
within analyzed tissue [8]. Figure 2 shows a MSI picture of bleomycin after laser-assisted drug 
delivery in skin [9]. 
 
 
Figure 2. Mass spectrometry imaging. MSI of bleomycin (m/z 1425.56323) with laser channels. 
(A) Vertically cut skin cryosection after 24 h of topical drug exposure. Laser channels are easily 

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seen with high concentrations of bleomycin (yellow) in the coagulation zones and drug 
dissemination into the surrounding tissue. 
Janfelt and colleagues [10] were able to distinguish between epidermis and dermis using MSI 
on phospholipids. We will use this technique to identify endogenous tumor biomarkers present 
in skin tumours but not present (or in different intensities) in adjacent normal skin. Integrating 
MSI into the diagnostic arsenal for NMSC, using the technology to differentiate in real-time, 
specific membrane lipid signatures of malignant and benign tissue. 
The long-term goal beyond this project, if it is possible to identify lipid patterns able to 
distinguish malignant and healthy skin, is to introduce Rapid Evaporation Ionization Mass 
Spectrometry (REIMS), also now as iKnife [11]. This method has the potential to be far more 
sensitive by directly identifying chemical tumor signatures rather than simple tissue anatomy, 
in addition to be less labor-intensive and time-consuming. REIMS technique consists of an 
electrosurgical handpiece connected to a mass spectrometer, capable of performing real-time 
tissue identification using the aerosolised tissue produced during electrosurgery. Tissue 
identification is achieved by automated matching of mass spectra recorded during surgery, 
with a preexisting “reference library”. The modality provides the surgeon with immediate 
information on what type of tissue is being cut, guiding treatment by delineating cancer 
margins with astounding precision. Lipidomic profiles showed variation between histological 
tumor types, enabling distinction of tissues by acting as biomarkers [12]. REIMS has also proven 
efficacious in other fields as a way of affirming product authenticity and quality. Thus, in the 
food industry, REIMS is used to distinguish between beef and horse meat in <3 seconds, 
boasting an accuracy of 100 % [13]. 
 
3. Method 
3.1 Study design for patients with BCC and SCCs 
When patients are referred for Mohs surgical procedure at the Department of Dermatology, 
Bispebjerg Hospital, BCCs or SCCs are removed and the tissue is embedded, sectioned and 
stained for the Mohs surgeon to evaluate whether more tissue is needed to be removed. Three 
skin sections (5-10 um thick) of this tissue there is already removed will be use in our study. 

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One section will be HE stained so we know exactly where the regions of interest are. Two 
sections will be used for MS analysis (MSI spectrum and REIMS spectrum).  
Currently, we are doing Mohs surgery on high-risk BCCs at Department of Dermatology, 
Bispebjerg Hospital, but Mohs Surgery of SCCs will be included within a year.  
 
3.2 Study design for patients with actinic keratoses  
When patients are referred for a procedure to have treated several actinic keratoses (grade 1, 
2 or 3) at Department of Dermatology, Bispebjerg Hospital we will take an extra punch biopsy 
(2-4 mm) depending on the size of the lesion. The biopsy is embedded and sectioned. We will 
use 3 skin sections (5-10 um thick). One section will be HE stained so we know exactly where 
the regions of interest are. Two sections will be used for MS analysis at Department of 
Pharmacy, University of Copenhagen. We will include actinic keratoses from up to 100 patients. 
 
3.3 Tissue microarray  
The above-mentioned sectioning for MSI will provide knowledge about a single tumor in 
comparison with the adjacent normal tissue. However, we also need to compare patients with 
each other and therefore we need to make a tissue microarray (Figure 3). 
 
Figure 3. Example of a tissue microarray made from artificial tissue. 
We will make the tissue microarray from the removed and embedded Aktinic 
keratoses/BCC/SCC tissue. One section will analysed by MSI with the same procedure as 
described above and extra sections will be HE stained for pathology evaluation and traditional 
immunohistochemistry markers as Ki67 and p53. 
 
 
4. Statistical calculations 
Multivariate statistical analysis will be performed on all mass spectra using Matlab or similar 
program. Linear discriminant analysis will be used to identify spectral differences between pre-
malignant, cancer and normal tissue. Classification performance will be recorded with a leave-

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one- patient- out cross- validation scheme. The number of samples is based on sample size 
calculation and previous publications on other cancer forms and estimated to be sufficient to 
generate a reference library. We are planning a study of a continuous response variable from 
matched pairs of study subjects.  Prior data indicate that the difference in the response of 
matched pairs is normally distributed with standard deviation 8.  If the true difference in the 
mean response of matched pairs is 3, we will need to study 58 pairs of subjects to be able to 
reject the null hypothesis that this response difference is zero with probability (power) 0,8.   
The Type I error probability associated with this test of this null hypothesis is 0,05. 
(programmed used for sample size calculation; Power and Sample Size Calculation, PS, 
Vanderbilt University; version 3.1.2). Based on these calculations will we recruit between 60 
and 100 patients with BCCs, AKs and SCCs. Minimum will be 180 patients and maximum 300 
patients.  
5. Participants 
In total maximum 300 patients will be recruited to enter the study. Men and women aged 18 
years or older who meet the inclusion and exclusion criteria are included. All potential patients 
will be recruited from the Department of Dermatology, Bispebjerg University Hospital. 
Interested patients will be invited to a preliminary information screening visit at which they 
will receive comprehensive oral information on the study by the primary investigator, 
Catharina Lerche, Mohs surgeon Martin Gluud or project nurse June Svendsen, and written 
information will be handed out. The meeting will take place in a separate office to ensure a safe 
and calm environment. The patients are specifically informed that they can bring one or more 
assessors to all meetings and interventions if they so choose. Written informed consent will be 
secured at inclusion on the surgical procedure day of which will take place at least 24 hours 
after the screening visit. 
Each patient will be evaluated by the investigator to assess the suitability of entering the study. 
To ensure a homogeneous group of optical comparison, recruitment will be performed 
according to the following criteria:  
 
5.1 Inclusion criteria 
• Patients above 18 years of age. 

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• Patients presenting with AKs, SCCs and BCCs. 
• Written informed consent obtained from patient. 
 
5.2 Exclusion criteria 
• Immunosuppressed patients 
 
6. Potential risk and adverse effects 
All patients will be informed both verbally and in written form about risks and possible side 
effects. 
 
6.1 Punch biopsy 
For patients with BCC and SCC we will only use tissue there is already removed by the standard 
Mohs Procedure. For patients with actinic keratoses, a biopsy will be obtained under local 
anesthesia, with 2, 3 or 4 mm in diameter and 3 mm in depth. Each biopsy will leave a round 
scar of 2 mm or 4 mm in diameter that hills within 2 weeks. Injection of local anesthesia may 
cause pain and a tingly/prickling sensation at the injection site. There is a minimal chance for 
infection, but we will treat that if it occurs. 
 
7. Biobank 
A research biobank will be established in accordance to the committee-law §2 nr. 13. All 
potential biopsies/sections will be kept in a locked -80 °C freezer at the Department of 
Dermatology, Bispebjerg University Hospital until analysis at the Department of Pharmacy, 
University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø. We cannot always do the 
analysis within 5-7 days because it takes up to 20 hours to make one MSI picture and if we have 
several BCC/SCC/Actinic keratoses patients in one day there will be a delay. When making the 
tissue microarray we will gather tissue from 6-8 patients before analysis. After MSI analysis the 
samples will be destroyed. Destruction of remaining parts of biopsy blocks and tissue 
microarrays blocks will be no latter than 1. January 2025. No tissue will be kept for unspecific 
research. 
 

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8. Information from patient journals 
Health personnel will use information regarding the pathology description of the suspected 
lesion from the patient journal when screening possible patients to be included in this study.   
After the informed consent is signed by the patient, information from the patient journal 
concerning description of tumor type, location, size and whether it is recurrent lesion will be 
used. 
 
9. Data protection, information storage & quality control 
9.1 Quality control 
The declaration of Helsinki II will be respected, as well the standards of good clinical research. 
Respect of privacy as well as physically and mentally integrity of patients will be maintained. 
The study will be registered with the Committee on Health Research Ethics and The Danish data 
Agency protection. 
 
9.2 Data protection and storage 
General Data Protection Regulation (Danish: Databeskyttelsesloven/Persondataforordningen) 
will be respected. Research using Capital Region of Denmark’s data is considered public 
research. Use and distribution of data collected in this study will be discussed with patients 
during the consent process. The project will be reported to the Danish Knowledge Center on 
Data Protection Compliance (Danish: Videncenter for Dataanmeldelse, Region Hovedstaden). 
List of screened and patients including names, study ID number, and date of birth will be 
devised. All data collected will be anonymized and protected by Danish law regarding 
management of personal information and the Danish Health Act (Danish: Sundhedsloven). Data 
will be registered and stored for 5 years after study termination at the Dermatological 
Department, Bispebjerg Hospital.  
 
10. Financial considerations  
The study is conceived by principle investigator Catharina M. Lerche. No financial support has 
been conceived by commercial companies. The department of Dermatology, Bispebjerg 
University Hospital will provide salary for the principal investigator Catharina M Lerche. Other 

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expenses related to project are covered by the Danish Cancer Society (Knæk Cancer midler 1.6 
mill DKK) and the Lundbeck Foundation (10 mill DKK). Principle investigator Catharina M. 
Lerche and the collaborators do not have any personal relation or economic stake in the Danish 
Cancer Society or the Lundbeck Foundation. 
 
11. Reimbursements 
There will be no reimbursements to the patients. 
 
12. Information to patients 
The primary investigator, Catharina M. Lerche, is responsible for giving clear verbal and writing 
information about aim, design and risks of the study, as stated in “Information og samtykke til 
deltagelse I sundhedsvidenskabelige forskningsprojekter” by Danish Ministry of Healthy 
(Danish: Sundheds- og Ældreministeriet). In an undisturbed setting, the participant will be 
made aware of their right to have an/or more assessor(s) present, that participant is voluntary, 
and that withdrawal is possible at any time during the study. Patients will be given adequate 
consideration time (minimum 24 hours). Patients will be asked to sign a consent form.  
The patient will be urged to read enclosed material on study participant’s right. No study 
procedures will be started before the participants has signed the informed consent. Additional 
questions from patients will be addressed by the contact person, Catharina M. Lerche 
 
13. Publication of results  
Positive, negative and inconclusive results will be published. The aim is to publish and present 
the results in a peer-reviewed international dermatology journal or/and at dermatological 
conferences. The intellectual property rights to the results belong to Bispebjerg University 
Hospital. Publications will be in accordance to the Vancouver guidelines. 
 

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14. Ethics 
The biopsies will be taken at Department of Dermatology, Bispebjerg University Hospital. 
Personal information and samples are treated under the Act on Personal Data and Health Act. 
The project is furthermore carried out with minimal health and safety risks to the participants. 
Surgical procedures are daily events at hospitals and clinics all over the world. This study of 
NMSC biomarkers and development of new treatment of skin cancer based on mass 
spectrometry has the potential to reduce tumor recurrence, morbidity and costs. A study of 
NMSC biomarkers and clinical implementation of REIMS in the treatment of skin cancer has not 
yet been performed. It is anticipated that the potential risks for adverse effects in this study are 
small and that there is a potential benefit of improvement in tumor recurrence, morbidity, costs, 
less labor-intensive and time-consuming. The potential of evidence -based future gains and the 
future perspectives this study may provide, should be held against the participants discomfort 
during the biopsy, and extra visit to Bispebjerg Hospital. Danish laws regarding patients’ rights 
and compensation will be followed. 
 
15. Insurance 
Bispebjerg Hospital 
 
16. References 
1. 
Diet , nutrition , physical activity and skin cancer. 2018. 
2. 
Lamberg, A. L. et al. The Danish nonmelanoma skin cancer dermatology database. Clin. 
Epidemiol. 8, 633–636 (2016). 
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Jensen, A. et al. Intake of alcohol may modify the risk for non-melanoma skin cancer: Results 
of a large danish prospective cohort study. J. Invest. Dermatol. 132, 2718–2726 (2012). 
4. 
Kyrgidis, A., Tzellos, T. G., Vahtsevanos, K. & Triaridis, S. New Concepts for Basal Cell 
Carcinoma. Demographic, Clinical, Histological Risk Factors, and Biomarkers. A Systematic 
Review of Evidence Regarding Risk for Tumor Development, Susceptibility for Second Primary 
and Recurrence. J. Surg. Res. 159, 545–556 (2010). 
5. 
Bentzen, J. et al. Costs of illness for melanoma and nonmelanoma skin cancer in Denmark. 
Eur. J. Cancer Prev. 22, 569–576 (2013). 
6. 
Smeets, N. W. J. et al. Mohs’ micrographic surgery for treatment of basal cell carcinoma of 
the face - Results of a retrospective study and review of the literature. Br. J. Dermatol. 151, 

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141–147 (2004). 
7. 
Römpp, A. & Spengler, B. Mass spectrometry imaging with high resolution in mass and space. 
Histochem Cell Biol 139, 759–783 (2013). 
8. 
Nilsson, A. et al. Mass spectrometry imaging in drug development. Anal. Chem. 87, 1437–
1455 (2015). 
9. 
Hendel, K. K. et al. Fractional laser-assisted topical delivery of bleomycin quantified by LC-MS 
and visualized by MALDI mass spectrometry imaging. Drug Deliv. 26, 244–251 (2019). 
10. 
Sørensen, I. S. et al. Combination of MALDI-MSI and cassette dosing for evaluation of drug 
distribution in human skin explant. Anal. Bioanal. Chem. 409, 4993–5005 (2017). 
11. 
Balog, J. et al. Identification of biological tissues by rapid evaporative ionization mass 
spectrometry. Anal. Chem. 82, 7343–7350 (2010). 
12. 
Balog, J. et al. Intraoperative tissue identification using rapid evaporative ionization mass 
spectrometry. Sci. Transl. Med. 5, (2013). 
13. 
Balog, J. et al. Identification of the Species of Origin for Meat Products by Rapid Evaporative 
Ionization Mass Spectrometry. J. Agric. Food Chem. 64, 4793–4800 (2016).