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Title:
Description of early/acute and late skin reaction (toxicity) after radiotherapy.
Background:
Year 2001 47 percent of patients with cancer in Sweden were treated with radiotherapy
sometime during their illness (1). Ionizing radiation used in radiotherapy causes damage to
normal living and malignant cells either indirectly, via free radicals, or directly by causing
DNA damage, alteration of proteins, lipids, carbohydrates and complex molecules. One of the
most common side effects of radiotherapy is skin reaction, which is a result of the cells
dividing rapidly and frequently (2, 3). Several studies show that radiotherapy destroys the skin
cell layers during the treatment, and may cause an atrophic epidermis (4). Irradiated skin can
become thinner and hypopigmented or hyperpigmented and is more sensitive to injury and
sunlight (5). Ionizing radiation damage starts a complex healing process that involves a large
number of interacting molecular signals, including cytokines, chemokines and growth factors.
The skin has three basic structures, epidermis, dermis and subcutis. Epidermis is a thin
membrane with three cell types, keratinocytes, melanocytes and Langerhans cells. Langerhans
cells have an immunologic function. Epidermis contains keratinocytes and forms a multilayered epithelium. The basal cells located on the basal membrane constitute the border
towards dermis and they divide relatively rapidly and frequently and are considered to be
radiosensitive. Melanocytes are also placed in the stratum basale and constitute about 10% of
the basal cells. The melanocytes contain melanosomes that create the dark pigment melanin.
The corneal thickness, pigmentation and the skin content of urokanacid will create the skin’s
natural sunblock. Dermis, corium, gives a mechanical protection and plays an important roll
for the nutrition of the epidermis. Dermis contains fibroblasts, mastcells, histiocytes,
macrophags, collagen, elastin, blood vessels, lymph-vessels and nerves (6, 7).
Factors that affect the degree of skin reaction are the angle of the beam, fractionation, total
dose, volume of treated tissue, the quality and energy of the beams, application of bolus,
concomitant use of chemotherapy, presence of skin folds, site of treatment, individual
variations in patients skin and possibly patients nutrition and patients tendency to follow
given advice.
The side effects of radiotherapy can be divided in to acute and late reactions. Acute skin
reactions develop within 2 to 3 weeks of irradiation and persist approximately 6 months after
treatment (5, 8, 9). The ability of the skin to maintain its integrity depends on the function of
mitotic reproduction of cells. Radiotherapy affects the cell reproduction rate. Even low doses
inhibit cell divisions of the basal cells, which will cause a temporary thinning of the
epithelium. More superficial cells that keratinize are more radio resistant. Irradiated skin
seems to be more sensitive for sun light, so called photosensitivation. This could be the result
of destroyed melanocytes and a slower melanin production that will decrease the skin’s ability
to protect the body from UV-radiation. UVB radiation has little effect on the dermis where
high energy ionizing radiation can cause severe skin alteration. The clinical picture of acute
reaction consists of erythemas, skin pigment changes, dry and/or moist desquamation and
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RESEARCH PLAN FOR: PER FESSÉ
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epilation. When the area has recovered from the acute reaction, the skin can become thinner,
smother, pinkish and dryer. In a previuos study (10) from our institution biopsies of the skin
were taken after completing the treatment (total dose =54Gy). It was shown that the epidermis
was thin and contained only one to three layers of keratinocytes and was enlarged with
abnormal cytology. Late side effects are mainly related to vascular and connective tissue
changes. Atrophy of the skin, telangiectasia, a changed pigmentation, fibrosis, in rare cases
ulceration and necrosis, are late skin reactions. Late reactions take months to years before
they appear. Late reactions are usually changes that are permanent and are more related to the
total dose that is given and volume of tissue that has been treated. Melanocytes are relatively
radiation resistant (5, 8).
However the whole complex processes behind acute and late skin reactions after radiotherapy
are not evidently described (4). The personnel also often advise the patients to protect the
irradiated skin against sunlight the clinical evidence for this recommendation is scarce and
should be investigated further.
Purpose:
Up to now most research in this field has focused on the keratinocyte toxicity. There is
virtually no data on the role of the melanocyte in this process. Therefore the aim of this
project is to evaluate the response of the melanocytes to irradiation and how it affects the
degree of skin toxicity after radiotherapy.
Study-plan/Method:
1) The aim of the project was to evaluate melanocytes if there is a dose range where lowdose hypersensitivity and an increased radioresistance occur. Another objective was to
compare the DNA damage response of ionizing radiation to that known for UVR.
Material that was investigated was 260 skin punch biopsies from 33 men treated for
prostate cancer with radiotherapy (11, 12). Biopsies were taken at 1 week and 6.5
weeks into the treatment course, from regions of the exposed to fraction doses 0.1, 0.2,
0.45 and 1.1 Gy. Control biopsies were collected before treatment. Sections from the
biopsies were stained with ∆Np63, eosin-PAS, MITF and Bcl-2. The total number of
identified cells per mm of the basal membrane was counted for all biopsies on three
separate sections for each immunostaining, as well as the morphological staining. All
counting was performed by one of the authors (PF) in a bright field microscope with a
100 x objective. A total of 3120 separate sections was counted and analyzed. Cells in
the melanocyte lineage are negative for ∆Np63. The numbers of ∆Np63-negative cells
were constant and independent of dose per fraction over the treatment and unchanged
from unexposed skin. Small but consistent increase of eosin-PAS, MITF and Bcl-2
stained melanocytes were observed. Repeated dose fraction over 0.05 Gy trigger
melanocytes to express higher levels of MITF and Bcl-2. During the research found a
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subpopulation of MITF negative cells that suggest that undifferentiated cells do exist
in unirradiated interfollicular epidermis. Upon low doses of radiation this subset
upregulates MITF and Bcl-2. The findings are compatible with an induction of
radioresistance and supports that the DNA damage response of melanocytes to
ionizing and UVR shares common main pathways. A draft manuscript has been
written and it will be submitted for publication during October or November 2010.
2) Aim of the second project is to evaluate melanocytes response during treatment and
after treatment. The investigated material is 147 skin punch biopsies from 15 breast
cancer patients that have received post-mastectomy radiotherapy 2 Gy 5 times a week
under a period of 5 weeks. Biopsies were taken once a week during treatment and up
to 5 weeks past treatment. Control biopsies were collected before treatment. Sections
from the biopsies were stained with ∆Np63, MITF and Bcl-2. The total number of
identified cells per mm of the basal membrane is counted for all biopsies on three
separate sections for each immunostaining, as well as the morphological staining. All
counting is performed by one of the authors (PF) in a bright field microscope with a
100 x objective. A total of 1323 separate sections and most of the sections are counted
and planned to be finished end of October 2010. All data will be analyzed under
November 2010. Cells in the melanocyte lineage are negative for ∆Np63. Preliminary
results follow same response during treatment as project 1, but 4 to 5 weeks past
treatment the number of stained melanocytes has stabilized to same amount as before
treatment. The subpopulation of MITF negative cells follow same pattern as project 1
and they also recover to same amount of cells as before treatment 4-5 weeks after
treatment. This supports the idea that melanocytes and undifferentiated melanocytes
need the keratinocytes to survive and recover. A draft manuscript is planned to be
written December 2010 and it will be submitted for publication during January or
February 2011.
3) Aim of the third project is to investigate if there is a keratinocyte dependency to
melanocytes survival and reaction and possible differences in the adverse effects in the
skin after radiotherapy. If the survivals of melanocytes end thereby late changes in
skin pigmentation is dependent on early damage to the keratinocytes.
The third project will use photographic pictures taken of the treatment area in women
that has received post-mastectomy radiotherapy (13). A picture was taken once a week
during treatment and at follow-up visits up to 5-10 years after treatment. There were 9
different fractionation schemes with about 25 women in each group. This makes a
total of about 225 women. Four schemes were 2 Gy 5 times a week for 20, 25, 30 or
35 fractions. Five schemes 4 Gy 2 times a week for 8, 10, 11, 12 or 14 fractions. Each
photograph is going to be scored visually 1-5 regarding skin reaction and pigmentation
up to the 5 year follow up. All the photographs are filed in Gothenburg, I will start
scoring the pictures in March and that will take approximately one week. The late
reactions, ie hypo- or hyperpigmentation will be compared with the degree of early
skin reaction. If a severe early skin reaction, ie keratinocyte damage, is correlated with
a late hypopigmentation in the skin, it will support the hypothesis that melanocytes
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depend on keratinocytes to survive ionizing radiation. All data will be analyzed under
March 2011. A draft manuscript is planned to be written March/April 2011 and it will
be submitted for publication during May or June 2011.
Importance:
This research can give fundamental knowledge about the melanocytes role and their
sensitivity during and after radiotherapy. If radiotherapy changes the skin character and
increases its sensitivity the patient’s way of life can be affected. Data of melanocytes and
undifferentiated melanocytes dependency of keratinocytes communication that influence
melanocytes and undifferentiated melanocytes ability to recover and defend their self’s for
survival can help us understand some of the side effects in the skin that our patients
experience, like hypo pigmentation. If patients react with certain skin side effects the research
data will support better information to the patients that their normal defense mechanism in the
treated skin area can be affected and they need to be more careful in the sun then the normal
population. Increased knowledge on the skin’s structural changes and what processes are
affected over time will help healthcare personnel to give more appropriate advice about skin
care to the group of patients receiving this type of treatment. Other aspects of the puzzle of
knowledge in the understanding of melanocytes survival and communication is that in the
long run the research data can help the melanoma research further how to isolate mutated
melanocytes and treat these cells.
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References:
(1) Statens beredning för utvärdering av medicinsk metodik. SBU-rapport.
Strålbehandling vid cancer. En systematisk litteratur översikt. 2003 Nr 162
(2) Reitan A M , Schölberg T (red), Översättning Jones L. Onkologisk omvårdnad.
(2003). Stockholm: Liber AB.
(3) Ringborg U, Henriksson R, Friberg S. Onkologi. (1998). Stockholm: Liber AB.
(4) Denham J, Hauer-Jensen M. The radiotherapeutic injury – a complex ‘wound’.
Radiotherapy&Oncology. 2002;63:129-145.
(5) Sitton E. Early and late radiation -induced skin alterations. Part 1: Mechanisms of skin
changes. Oncology Nursing Forym 1992;19:801-807 (a).
(6) Rorsman H, Björnberg A, Vahlquist A. Dermatologi Venereologi. (2000). Lund:
Studentlitteratur.
(7) Nylén P m fl. Ultraviolett strålning och hälsa – ett kunskapsunderlag.
Arbetslivsinstitutet, Arbete och Hälsa vetenskaplig skriftserie. 2002 Nr 2002:5.
(8) Groenwald S, Hansen Frogge M, Goodman M & Henke Yarbro C. (1997). Cancer
Nursing Principles and Practice. Boston: Jones & Bartlett.
(9) Porock D. Factors influencing the severity of radiation skin and mucosal reactions:
development of a conceptual framework. Eur J Cancer Care. 2002;11:33-43.
(10)
Ponten F, Lindman H, Bostrom A, Berne B, Bergh J. Induction of p53
expression in skin by radiotherapy and UV radiation: a randomized study. J Natl
Cancer inst. 2001 Jan 17;93:2:128-33.
(11)
Joiner MC, Marples B, Lambin P, Short SC, Turesson I. Low-dose
hypersensitivity: current status and possible mechanisms. Int J Radiat Oncol Biol Phys
2001;49:2:379-89.
(12)
Turesson I, Bernefors R, Book M, Flogegård M, Hermansson I, Johansson KA,
Lindh A, Sigurdardottir S, Thunberg U, Nyman J. Normal tissue response to low
doses of radiotherapy assessed by molecular markers –a study of skin in patients
treated for prostate cancer. Acta Oncol 2001;40:8:941-951.
(13)
Nyman J, Turesson I. Basal cell density in human skin for various fractionation
schedules in radiotherapy. Radiother Oncol 1994;33:2:117-24.
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