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Do Serum Levels of Eosinophil Granule-derived Protein Change in Patients Undergoing Pelvic Radiotherapy?
Clinical Oncology (2005) 17: 382–384 doi:10.1016/j.clon.2005.01.007
Short Report Do Serum Levels of Eosinophil Granule-derived Protein Change in Patients Undergoing Pelvic Radiotherapy? J. M. Bowen*, K. Newboldy, P. Blakey, G. Wildz, W. Egnerz, A. R. Normanx, H. J. N. Andreyev{ *Department of Oncology, East Gloucestershire NHS Trust, Cheltenham, UK; yDepartment of Radiotherapy, Royal Marsden Hospital, London and Sutton, UK; zDepartment of Immunology, Sheffield Teaching Hospitals NHS Trust, UK; xDepartment of Computing, Royal Marsden Hospital, London and Sutton, UK; {Imperial College Faculty of Medicine, Chelsea and Westminster Hospital, London, UK ABSTRACT: Aims: Eosinophils have an important role in the pathogenesis of inflammatory bowel disease, with faecal levels of the eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX) reflecting disease activity. Eosinophil crypt abscesses are a characteristic histological finding in acute gastrointestinal radiation-induced mucosal damage. This pilot study aimed to investigate changes in serum levels of ECP/EPX during pelvic radiotherapy. Materials and methods: Patients with no history of inflammatory bowel disease, starting a 5-week course of pelvic radiotherapy, had serum ECP/EPX levels measured before radiotherapy and during the fourth week of treatment. Bowel toxicity was graded at week 4 using the Common Toxicity Criteria Scale. Results: Fifteen patients who were to undergo adjuvant radiotherapy for gynaecological cancer were recruited. The mean serum levels of ECP and EPX before treatment were 17.3 mg/l (range 2.0–49.3 mg/l) and 37.3 mg/l (range 12.0–94.0 mg/l), respectively. The mean serum levels during week 4 of radiotherapy for ECP and EPX were 43.0 mg/l (range 2.4–164.0 mg/l) and 38.7 mg/l (range 9.0–79.0 mg/l), respectively. Serum ECP levels increased at week 4 compared with levels before radiotherapy (P Z 0.02). Acute bowel toxicity was seen in 12 patients (80%) at week 4: Grade 1 in 25% patients and Grade 2 in 75%. In this small study, no correlation was seen between acute bowel toxicity at week 4 and serum ECP or EPX levels. Conclusions: Serum ECP levels increase in response to pelvic irradiation. This may reflect the known involvement of eosinophils in the acute response to radiotherapy. Further study is required to determine when levels start to rise and their relationship to the degree of acute bowel toxicity. Bowen, J. M. et al. (2005). Clinical Oncology 17, 382–384 Ó 2005 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Key words: Acute toxicity, ECP, eosinophil, EPX, pelvic radiotherapy Received: 12 August 2004 Revised: 12 August 2004 Accepted: 20 January 2005
Introduction
Radical pelvic radiotherapy is used in the adjuvant setting and for primary treatment of pelvic malignancies. The presence of the small and large intestine in the pelvis is the major dose-limiting factor. Acute bowel toxicity is common, manifesting as diarrhoea, abdominal pain and haematochesia, with symptoms generally increasing in severity from the third week of treatment. More severe acute change may predispose to late toxicity. A reliable serum marker of significant early gastrointestinal damage would be helpful.
Author for correspondence: Dr Jervoise Nicholas Andreyev, MA, PhD, FRCP, Department of Medicine and Therapeutics, Imperial College Faculty of Medicine, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK. Tel: C44-208-746-8144; Fax: C44-208746-8887; E-mail: [email protected] 0936-6555/05/000000C03 $35.00/0
The pathological changes associated with acute gastrointestinal radiation-induced toxicity include atrophy of the surface epithelium, acute cryptitis and eosinophilic crypt abscesses [1]. Eosinophils have been found to play an important role in inflammation associated with inflammatory bowel disease [2]. Eosinophil activation results in the release of eosinophil granule proteins. These are highly cationic proteins with cytotoxic activity, and include eosinophil cationic protein (ECP) and eosinophil protein X (EPX). Faecal levels of these proteins correlate with disease severity in patients with inflammatory bowel disease, and serum levels have been shown to reflect disease activity in childhood bronchial asthma [3]. The aim of this pilot study was to investigate whether serum eosinophil granule protein levels increased during pelvic radiotherapy. If so, this would warrant further investigation to determine whether they are reliable markers of the severity of bowel damage.
Ó 2005 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.
383
SERUM ECP LEVELS IN PATIENTS UNDERGOING PELVIC RADIOTHERAPY
Methods Patients
All patients recruited to this study were planned to receive a radical course of adjuvant pelvic radiotherapy for gynaecological malignancy after surgical removal of all macroscopic tumour. Entry criteria included no history of inflammatory bowel disease, no previous pelvic radiotherapy and a normal bowel habit before radiotherapy as defined by a score of 0 using the Common Toxicity Criteria system version 2. Serum samples were taken before radiation therapy and during the fourth week of treatment. Acute bowel toxicity was assessed during week 4 using the Common Toxic Criteria scoring system. All patients provided written informed consent. The study was reviewed and approved by the Royal Marsden Hospital Research and Ethics Committee. Radiotherapy
Patients started external-beam radiotherapy at least 6 weeks after surgery. All had treatment to the pelvis only. Treatment was delivered with a four-field technique with anterior–posterior and lateral portals. A total of 6–10 MV photons were used to deliver a total dose of 45 Gy in 1.8 Gy daily fractions over 5 weeks.
surgery and were to receive a course of adjuvant pelvic radiotherapy. Paired serum levels of ECP were available on all 15 patients, and mean levels were significantly higher during week 4 of pelvic radiotherapy than before treatment: 43.0 mg/l (range 2.4–164.0 mg/l, 95% confidence intervals 16.6 to 69.4 to 25.7; standard deviation 47.6) vs 17.4 mg (2.0–49.3 mg/l, 95% confidence intervals 9.0–25.7, standard deviation 15.1), respectively (P Z 0.02). Paired serum EPX levels were not available for six patients due to a delay in sample processing. For the remaining nine patients, no significant change was observed in mean serum levels during radiotherapy, with mean pre-radiotherapy EPX levels 37.3 mg/l (12.0–94.0 mg/l, 95% confidence intervals 18.6–56.0, standard deviation 24.3) and mean week 4 levels 38.7 mg/l, 95% confidence intervals 20.3–57.0, standard deviation 23.9); P Z 1.0 (Table 1 and Fig. 1). Bowel toxicity data at week 4 were available for all patients. Three patients (20%) recorded no acute bowel symptoms at week 4, three patients (20%) recorded Grade 1 toxicity and nine patients (60%) Grade 2 toxicity. No correlation was seen between the severity of acute bowel symptoms and the change in levels of either ECP (r Z 0.05, P Z 0.86) or EPX (r Z 0.37; P Z 0.33) between pretreatment and week 4 values. The value of this result is questionable because this study was not powered to examine whether a correlation exists.
Serum Levels of Eosinophil Cationic Protein and Eosinophil Protein X
Blood samples were collected into plain tubes and allowed to coagulate at room temperature for 1 h. Serum was obtained by centrifuging the clotted samples at 1000 g for 10 min. The plasma was then stored at 4(C for up to 2 h before it was frozen, and stored for up to 2 months at 20(C before being transported on dry ice to the reference laboratory. ECP measurements were made using a Unicap 100 automated analyser (Pharmacia). EPX values were obtained using a double antibody radioimmuno-assay technique (Pharmacia). Statistics
Serum levels of ECP and EPX were compared before radiotherapy and at week 4 using the Wilcoxon Signed Rank test. Mean changes in serum levels, standard deviations and confidence intervals were assessed for both eosinophil granule proteins. Spearmans correlation coefficient was calculated with confidence intervals to look at the relationship between serum eosinophil granule protein levels and bowel toxicity scores. Statistical significance was achieved when P % 0.05. Results
Between June and August 2002, 15 patients, aged 52–72 years (median 69 years) were enrolled in this study. Thirteen patients had a diagnosis of endometrial cancer and two of cervical cancer. All had completed definitive
Discussion
Acute bowel toxicity occurs in around 70% patients undergoing pelvic radiotherapy. The pathological processes underlying this are complex, and include initial increases in crypt stem-cell division followed by gradual depletion during treatment and loss of bowel villi. Studies in patients undergoing a course of pelvic radiotherapy have shown mucosal abnormalities, with a characteristic development of eosinophilic crypt abscesses and marked increases in focal tissue eosinophilia, involving both the mucosa and submucosa occurring maximally during the first 2 weeks of treatment [4]. These participants, with no history of inflammatory bowel disease, showed a wide range of serum values for Table 1 – Serum ECP/EPX levels before radiotherapy and during week 4 of treatment Eosinophil cationic protein (mg/l) Before radiotherapy Patients numbers Mean Range P value
Eosinophil protein X(mg/l)
Week 4
Before radiotherapy
43 2.4–164
37.3 12–94
15 19 2–49.3 0.02
Week 4
9 38.7 9–792 1.0
384
CLINICAL ONCOLOGY
Serum ECP (micromol/l)
a 180 160 140 120 100 80 60 40 20 0
Pre-radiotherapy
Week 4
Serum EPX (micromol/l)
b 100
This pilot study showed an increase in serum ECP levels during pelvic radiotherapy, which may reflect the mucosal changes associated with radiation treatment. This was a small study, as reflected by the wide confidence intervals and, although no rise was seen in the serum levels of EPX, this result was based on paired serum samples for only nine patients, and should be interpreted with caution. It is not clear whether the serum levels of these highly cationic proteins reflect the release of these proteins in the bowel mucosa or from circulating eosinophils. Further studies might look for correlations between serum ECP/EPX levels and serum eosinophil levels. As eosinophilic crypt abscesses are seen maximally during the first 2 weeks of treatment, increased serum levels may be seen at an earlier stage in the course of radiotherapy.
90 80
Conclusion
70
In this study, no correlation was seen between serum levels of either ECP or EPX and bowel symptoms. This may relate to the small number of patients in this pilot study or reflect the poorly understood relationship between radiation-induced mucosal damage and clinical bowel toxicity. A larger study looking at serum ECP levels and bowel symptoms throughout radiotherapy is warranted.
60 50 40 30 20 10 0
Pre-radiotherapy
Week 4
Fig. 1 – (a) Eosinophil cationic protein values in 15 patients before pelvic radiotherapy and during week 4 of treatment; (b) eosinophil protein X levels in nine patients before pelvic radiotherapy and during the fourth week of treatment.
ECP and EPX before and during radiotherapy. This may reflect the influence of other causes of raised serum eosinophil granule proteins, which include coeliac disease, asthma, atopy or concurrent parasitic infection, and a similar variation has been seen in serum ECP/EPX levels in asthmatic children [3].
References 1 Hovdenak N, Fajardo LF, Hauer-Jensen M. Acute radiation proctitis: a sequential clinicopathologic study during pelvic radiotherapy. Int J Radiat Oncol Biol Phys 2000;48:1111–1117. 2 Saitoh O, Kojima K, Sugi K. Fecal eosinophil granule-derived proteins reflect disease activity in inflammatory bowel disease. Am J Gastroenterol 1999;94:3513–3520. 3 Kohller DYo, Halmerbauer G, Frischer T, Roithner B. Assessment of eosinophil granule proteins in various body fluids: is there a relation to clinical variables in childhood asthma? Clin Exp Allergy 1999;29: 786–793. 4 Gelfand MD, Tepper M, Katz LA. Acute irradiation proctitis in man: development of eosinophilic crypt abscesses. Gastroenterology 1968;54: 401–411.
Short Report Do Serum Levels of Eosinophil Granule-derived Protein Change in Patients Undergoing Pelvic Radiotherapy? J. M. Bowen*, K. Newboldy, P. Blakey, G. Wildz, W. Egnerz, A. R. Normanx, H. J. N. Andreyev{ *Department of Oncology, East Gloucestershire NHS Trust, Cheltenham, UK; yDepartment of Radiotherapy, Royal Marsden Hospital, London and Sutton, UK; zDepartment of Immunology, Sheffield Teaching Hospitals NHS Trust, UK; xDepartment of Computing, Royal Marsden Hospital, London and Sutton, UK; {Imperial College Faculty of Medicine, Chelsea and Westminster Hospital, London, UK ABSTRACT: Aims: Eosinophils have an important role in the pathogenesis of inflammatory bowel disease, with faecal levels of the eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX) reflecting disease activity. Eosinophil crypt abscesses are a characteristic histological finding in acute gastrointestinal radiation-induced mucosal damage. This pilot study aimed to investigate changes in serum levels of ECP/EPX during pelvic radiotherapy. Materials and methods: Patients with no history of inflammatory bowel disease, starting a 5-week course of pelvic radiotherapy, had serum ECP/EPX levels measured before radiotherapy and during the fourth week of treatment. Bowel toxicity was graded at week 4 using the Common Toxicity Criteria Scale. Results: Fifteen patients who were to undergo adjuvant radiotherapy for gynaecological cancer were recruited. The mean serum levels of ECP and EPX before treatment were 17.3 mg/l (range 2.0–49.3 mg/l) and 37.3 mg/l (range 12.0–94.0 mg/l), respectively. The mean serum levels during week 4 of radiotherapy for ECP and EPX were 43.0 mg/l (range 2.4–164.0 mg/l) and 38.7 mg/l (range 9.0–79.0 mg/l), respectively. Serum ECP levels increased at week 4 compared with levels before radiotherapy (P Z 0.02). Acute bowel toxicity was seen in 12 patients (80%) at week 4: Grade 1 in 25% patients and Grade 2 in 75%. In this small study, no correlation was seen between acute bowel toxicity at week 4 and serum ECP or EPX levels. Conclusions: Serum ECP levels increase in response to pelvic irradiation. This may reflect the known involvement of eosinophils in the acute response to radiotherapy. Further study is required to determine when levels start to rise and their relationship to the degree of acute bowel toxicity. Bowen, J. M. et al. (2005). Clinical Oncology 17, 382–384 Ó 2005 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Key words: Acute toxicity, ECP, eosinophil, EPX, pelvic radiotherapy Received: 12 August 2004 Revised: 12 August 2004 Accepted: 20 January 2005
Introduction
Radical pelvic radiotherapy is used in the adjuvant setting and for primary treatment of pelvic malignancies. The presence of the small and large intestine in the pelvis is the major dose-limiting factor. Acute bowel toxicity is common, manifesting as diarrhoea, abdominal pain and haematochesia, with symptoms generally increasing in severity from the third week of treatment. More severe acute change may predispose to late toxicity. A reliable serum marker of significant early gastrointestinal damage would be helpful.
Author for correspondence: Dr Jervoise Nicholas Andreyev, MA, PhD, FRCP, Department of Medicine and Therapeutics, Imperial College Faculty of Medicine, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK. Tel: C44-208-746-8144; Fax: C44-208746-8887; E-mail: [email protected] 0936-6555/05/000000C03 $35.00/0
The pathological changes associated with acute gastrointestinal radiation-induced toxicity include atrophy of the surface epithelium, acute cryptitis and eosinophilic crypt abscesses [1]. Eosinophils have been found to play an important role in inflammation associated with inflammatory bowel disease [2]. Eosinophil activation results in the release of eosinophil granule proteins. These are highly cationic proteins with cytotoxic activity, and include eosinophil cationic protein (ECP) and eosinophil protein X (EPX). Faecal levels of these proteins correlate with disease severity in patients with inflammatory bowel disease, and serum levels have been shown to reflect disease activity in childhood bronchial asthma [3]. The aim of this pilot study was to investigate whether serum eosinophil granule protein levels increased during pelvic radiotherapy. If so, this would warrant further investigation to determine whether they are reliable markers of the severity of bowel damage.
Ó 2005 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.
383
SERUM ECP LEVELS IN PATIENTS UNDERGOING PELVIC RADIOTHERAPY
Methods Patients
All patients recruited to this study were planned to receive a radical course of adjuvant pelvic radiotherapy for gynaecological malignancy after surgical removal of all macroscopic tumour. Entry criteria included no history of inflammatory bowel disease, no previous pelvic radiotherapy and a normal bowel habit before radiotherapy as defined by a score of 0 using the Common Toxicity Criteria system version 2. Serum samples were taken before radiation therapy and during the fourth week of treatment. Acute bowel toxicity was assessed during week 4 using the Common Toxic Criteria scoring system. All patients provided written informed consent. The study was reviewed and approved by the Royal Marsden Hospital Research and Ethics Committee. Radiotherapy
Patients started external-beam radiotherapy at least 6 weeks after surgery. All had treatment to the pelvis only. Treatment was delivered with a four-field technique with anterior–posterior and lateral portals. A total of 6–10 MV photons were used to deliver a total dose of 45 Gy in 1.8 Gy daily fractions over 5 weeks.
surgery and were to receive a course of adjuvant pelvic radiotherapy. Paired serum levels of ECP were available on all 15 patients, and mean levels were significantly higher during week 4 of pelvic radiotherapy than before treatment: 43.0 mg/l (range 2.4–164.0 mg/l, 95% confidence intervals 16.6 to 69.4 to 25.7; standard deviation 47.6) vs 17.4 mg (2.0–49.3 mg/l, 95% confidence intervals 9.0–25.7, standard deviation 15.1), respectively (P Z 0.02). Paired serum EPX levels were not available for six patients due to a delay in sample processing. For the remaining nine patients, no significant change was observed in mean serum levels during radiotherapy, with mean pre-radiotherapy EPX levels 37.3 mg/l (12.0–94.0 mg/l, 95% confidence intervals 18.6–56.0, standard deviation 24.3) and mean week 4 levels 38.7 mg/l, 95% confidence intervals 20.3–57.0, standard deviation 23.9); P Z 1.0 (Table 1 and Fig. 1). Bowel toxicity data at week 4 were available for all patients. Three patients (20%) recorded no acute bowel symptoms at week 4, three patients (20%) recorded Grade 1 toxicity and nine patients (60%) Grade 2 toxicity. No correlation was seen between the severity of acute bowel symptoms and the change in levels of either ECP (r Z 0.05, P Z 0.86) or EPX (r Z 0.37; P Z 0.33) between pretreatment and week 4 values. The value of this result is questionable because this study was not powered to examine whether a correlation exists.
Serum Levels of Eosinophil Cationic Protein and Eosinophil Protein X
Blood samples were collected into plain tubes and allowed to coagulate at room temperature for 1 h. Serum was obtained by centrifuging the clotted samples at 1000 g for 10 min. The plasma was then stored at 4(C for up to 2 h before it was frozen, and stored for up to 2 months at 20(C before being transported on dry ice to the reference laboratory. ECP measurements were made using a Unicap 100 automated analyser (Pharmacia). EPX values were obtained using a double antibody radioimmuno-assay technique (Pharmacia). Statistics
Serum levels of ECP and EPX were compared before radiotherapy and at week 4 using the Wilcoxon Signed Rank test. Mean changes in serum levels, standard deviations and confidence intervals were assessed for both eosinophil granule proteins. Spearmans correlation coefficient was calculated with confidence intervals to look at the relationship between serum eosinophil granule protein levels and bowel toxicity scores. Statistical significance was achieved when P % 0.05. Results
Between June and August 2002, 15 patients, aged 52–72 years (median 69 years) were enrolled in this study. Thirteen patients had a diagnosis of endometrial cancer and two of cervical cancer. All had completed definitive
Discussion
Acute bowel toxicity occurs in around 70% patients undergoing pelvic radiotherapy. The pathological processes underlying this are complex, and include initial increases in crypt stem-cell division followed by gradual depletion during treatment and loss of bowel villi. Studies in patients undergoing a course of pelvic radiotherapy have shown mucosal abnormalities, with a characteristic development of eosinophilic crypt abscesses and marked increases in focal tissue eosinophilia, involving both the mucosa and submucosa occurring maximally during the first 2 weeks of treatment [4]. These participants, with no history of inflammatory bowel disease, showed a wide range of serum values for Table 1 – Serum ECP/EPX levels before radiotherapy and during week 4 of treatment Eosinophil cationic protein (mg/l) Before radiotherapy Patients numbers Mean Range P value
Eosinophil protein X(mg/l)
Week 4
Before radiotherapy
43 2.4–164
37.3 12–94
15 19 2–49.3 0.02
Week 4
9 38.7 9–792 1.0
384
CLINICAL ONCOLOGY
Serum ECP (micromol/l)
a 180 160 140 120 100 80 60 40 20 0
Pre-radiotherapy
Week 4
Serum EPX (micromol/l)
b 100
This pilot study showed an increase in serum ECP levels during pelvic radiotherapy, which may reflect the mucosal changes associated with radiation treatment. This was a small study, as reflected by the wide confidence intervals and, although no rise was seen in the serum levels of EPX, this result was based on paired serum samples for only nine patients, and should be interpreted with caution. It is not clear whether the serum levels of these highly cationic proteins reflect the release of these proteins in the bowel mucosa or from circulating eosinophils. Further studies might look for correlations between serum ECP/EPX levels and serum eosinophil levels. As eosinophilic crypt abscesses are seen maximally during the first 2 weeks of treatment, increased serum levels may be seen at an earlier stage in the course of radiotherapy.
90 80
Conclusion
70
In this study, no correlation was seen between serum levels of either ECP or EPX and bowel symptoms. This may relate to the small number of patients in this pilot study or reflect the poorly understood relationship between radiation-induced mucosal damage and clinical bowel toxicity. A larger study looking at serum ECP levels and bowel symptoms throughout radiotherapy is warranted.
60 50 40 30 20 10 0
Pre-radiotherapy
Week 4
Fig. 1 – (a) Eosinophil cationic protein values in 15 patients before pelvic radiotherapy and during week 4 of treatment; (b) eosinophil protein X levels in nine patients before pelvic radiotherapy and during the fourth week of treatment.
ECP and EPX before and during radiotherapy. This may reflect the influence of other causes of raised serum eosinophil granule proteins, which include coeliac disease, asthma, atopy or concurrent parasitic infection, and a similar variation has been seen in serum ECP/EPX levels in asthmatic children [3].
References 1 Hovdenak N, Fajardo LF, Hauer-Jensen M. Acute radiation proctitis: a sequential clinicopathologic study during pelvic radiotherapy. Int J Radiat Oncol Biol Phys 2000;48:1111–1117. 2 Saitoh O, Kojima K, Sugi K. Fecal eosinophil granule-derived proteins reflect disease activity in inflammatory bowel disease. Am J Gastroenterol 1999;94:3513–3520. 3 Kohller DYo, Halmerbauer G, Frischer T, Roithner B. Assessment of eosinophil granule proteins in various body fluids: is there a relation to clinical variables in childhood asthma? Clin Exp Allergy 1999;29: 786–793. 4 Gelfand MD, Tepper M, Katz LA. Acute irradiation proctitis in man: development of eosinophilic crypt abscesses. Gastroenterology 1968;54: 401–411.