- Email: [email protected]
Effect of epidermal growth factor on ulcerative mucositis in hamsters that receive cancer chemotherapy
Effect of epidermal growth factor on ulcerative mucositis in hamsters that receive cancer chemotherapy Stephen T. Sonis, DMD, DMSca~ b Joseph W. Costa, Jr, DMD,aj b Susan M. Evitts, BS,a Loryn E. Lindquist, BS,a and Margery Nicolson, PhD, c Boston, Mass., and Thousand Oaks, CaliJ: BRIGHAM
&
WOMEN’S
HOSPITAL,
HARVARD
SCHOOL
OF DENTAL
MEDICINE,
AND
AMGEN
CORPORATION
Ulcerative mucositis is a common, bothersome, and dose-limiting complication of cancer chemotherapy. It has been hypothesized that mucosal susceptibility to the degenerative effects of stomatotoxic drugs is related to the renewal rate of the buccal epithelium. This study was undertaken i:o evaluate the effect of epidermal growth factor, a molecule known to stimulate epidermal cell division, on the course, frequency, and healing of ulcerative mucositis in an animal model. Golden Syrian hamsters were subjected to a standard mucositis-induction protocol with 5-fluorouracil. Osmotic pumps were implanted into a space between the retractor muscle and the platysma cervicale muscle, and delivered epidermal growth factor or placebo at a constant rate for 7 or 14 days. Epidermal growth factor increased oral mucosal breakdown in the face of antineoplastic therapy. The course and extent of mucositis was influenced by the timing of epidermal growth factor pump placement relative to the initiation of stomatotoxic therapy. These results support the hypothesis that the epithelial basal cell rate is one of the key elements in determining mucosal sensitivity to cancer chemotherapy. (ORALSURGORALMEDORALPATHOL~~~~;~~:~~~-~~)
U
lcerative oral mucositis is one of the most common, bothersome, and dose-limiting complications of cancer chemotherapy. l-4 Because the oral mucosa undergoes frequent replication, it is especially sensitive to the direct cytotoxic effects of chemotherapy. Histologically, signs of oral mucosal atrophy become evident soon after tlherapy has begun. Inhibition of DNA replication of basal epithelial cells and cytolysis result in a thin, atrophic mucosa that is highly susceptible to spontaneous or traumatic ulceration. It has been hypothesized that the rate of basal epithelial cell proliferation affects the susceptibility of the oral mucosa to the stomatotoxic effect of chemotherapy.‘% 5, 6 Epidermal growth factor (EGF) is a 53-amino acid
Supported in part by the Brigham Surgical Group Foundation and the Amgen Corporation. aDivision of Dentistry, Brigham & Women’s Hospital, Boston. bHarvard School of Dental Medicine, Boston. CAmgen Corp., Thousand Oaks.
7/13/38980
polypeptide (mol. wt. 6000) first isolated from extracts of mouse submaxillary glands. The molecule has a number of activities including the induction of precocious eyelid opening and incisor eruption when injected into newborn mice.? This response is attributed to stimulation of epidermal growth and keratinization. In addition, EGF directly stimulates the proliferation of basal epithelial cells in skin organ culture systems.’ Other mitogenic effects of EGF have been noted in vitro with a variety of cell types. In vivo studies in neonatal mice by Steidler and Reade9 suggest that subcutaneous injections of EGF cause an increase in epithelial thickness and keratinization of skin and oral mucosa. Recent studies have shown that EGF promotes proliferation and differentiation of the gastrointestinal mucosa.lO, l l The mechanism of action EGF exerts after it binds to its receptor is currently under investigation. It is known that the EGF receptor is a glycoprotein with
749
750
Sonis et al.
ORALSURGQR.~LM~D
ORALPATHOL
December 1992
Fig. 1. AIzet osmotic pump filled with EGF (8.33 pg/pl) or placebo. Pump measures 3.0 cm in length and 0.7 cm in
diameter. a molecular weight of 175,000. On binding, EGF is able to stimulate an intrinsic tyrosine kinase activity that autophosphorylates the receptor.12 However, little is known about how the binding and autophosphorylation influence transduction of a mitogenic signal to the nucleus so as to alter gene regulation. Because EGF stimulates epithelial proliferation, we investigated its effect, when applied topically, on the frequency, course, and healing of ulcerative mucositis. We hypothesized that early exposure of the oral mucosa to EGF simultaneous to stomatotoxic cancer chemotherapy would enhance the development of ulcerative mucositis. We also hypothesized that its effect during healing might be favorable in reestablishing intact epithelium. MATERIAL AND METHODS Animals
Male Golden Syrian hamsters, 8 to were obtained from Charles River Wilmington, Mass. Animals were kept and provided with free access standard and water. Epidermal
growth
10 weeks old, Laboratories, five to a cage hamster food
factor
Human recombinant EGF was provided by Amgen Corp., Thousand Oaks, Calif., and loaded into 200 ~1Alzet osmotic pumps (Alza Corp., Palo Alto, Calif.). Pump placement
protocol
To ensure uniform topical EGF delivery, an implanted osmotic pump protocol was devised. After induction, animals were anesthesized with ethyl ether; sodium pentobarbital (Nembutal) (50 mg/kg) was administered by intraperitoneal injection. The left buccal pouch was everted and an incision made at the
ig. 2. Alzet osmotic pump sutured in place between retractor muscle and platysma cervicale muscle. Blunted cannula can be seen protruding through mucosa.
most superior aspect of the pouch in the mucobuccal fold. With blunt dissection a space was created between the retractor muscle and the platysma cervitale muscle. The pump, with a short contiguous cannula, was inserted and a purse-string
suture was
placed (Figs. 1 & 2). The cannula was allowed to protrude through the surgical site by about 3 mm. The end of the cannula was blunted to prevent mucosal irritation and dislodgement. Preliminary studies performed in our laboratory with dye-loaded pumps demonstrated their efficacy in delivering a uniform dose of material during a prolonged period. No infection or soft tissue anomalies were associated with pump placement. Pumps placed for 2 weeks delivered EGF at the rate of 0.5 pl/hr; l-week pumps delivered EGF at the rate of 1.0 pl/hr. Mucositis was induced in 60 hamsters by an established protocol.‘3 Briefly, hamsters received 5-fluerouracil(5-FU) (60 mgjkg) on days 0 and 5, with superficial irritation of the buccal pouch mucosa on day 7. Severe mucositis developed in all animals with this technique. Animals were randomly and equally divided into the following groups: Group I: On day 3 a ‘I-day pump containing EGF was implanted (8.33 pg/pl) Group 2: On day 7 a 7-day pump containing EGF was implanted (8.33 pg/hl) Group 3: On day 6 a 7-day pump containing EGF was implanted (8.33 pg/pl) Group 4: On day 1 a l&day pump containing EGF was implanted (8.33 pg/pl) Group 5: On day 7 a 7-day pump containing placebo was implanted (control for group 2) Group 6: On day 1 a 14-day pump containing placebo was implanted (control for group 4) Animals were observed and weighed daily and had
Effect of epidermal growth factor on mucositis
Volume Number
74 6
Table
I. Mean mucositis score and standard deviation by day”
Day 10
11
/ 8.39 1.25
11
8.21
12
1.65 9.39 0.56
13
NP NP
14 15 16 17 18 19 20
9.54 0.90 8.67 2.10 9.63 0.53 4.00 0.00 6.00 0.00 1.75 0.00 4.00 0.00
2
1
4.28 1.03 3.47 2.43 7.63 1.63 7.55 1.27 8.14 0.84 8.78 0.97 6.64 2.46 5.25 2.90 4.43 2.69 3.25 2.92 4.13 3.03
3 6.60 1.02
6.50
‘T
4
/
7.11 2.85 8.32
1.06
1.62
7.94 1.20 9.40 0.76 7.60 2.16 6.08 0.88 6.42 0.63 4.61 2.10 5.58 1.46 3.75 1.89 4.83 1.04
8.14 1.27 7.13 2.48 5.43 3.38 4.13 3.77 3.46 3.11 2.00 3.11 2.45 1.58 1.75 0.88 1.35 1.28
5
1
751
6
1.55t 0.52 2.55 2.08
6.78 1.79 8.03 2.09 6.94 1.74 6.19 2.39 5.71 1.76 1.68 0.62 0.75$ 0.75 0.64 0.67 1.71 0.89 1.89 1.38 2.00 1.61
NP
6.17 3.02 6.47 2.63 5.56t 3.02 4.56 3.34 3.25 1.89 3.38 3.34 2.06 1.91 2.75 2.60
NP, Photographs not available for these time points. *Mean mucositis scores for each group on each day. Top value in each row indicates mean mucositis score ranging from 0 to 10. Bottom value represents SD for that average score for each group on each day. Parametric statistical analysis was performed between groups 2 and 5 and groups 4 and 6 by unpaired t test. tStatistically significant (p < 0.01). fStatistically significant (p < 0.05).
free access to food and water. Beginning on day 10 left buccal pouches were photographed daily with a Yashica Dental Eye, camera (Kyocera Corp., Kyoto, Japan). At the conclusion of the experiment (day 20) the photographs were randomly numbered, mixed, and scored by four different observers, in blinded fashion, by comparing them with a standardized set of photographs with mucositis scores ranging from 0 (no mucositis) to 10 (severe mucositis). Data were collected and mean daily mucositis scores were calculated for each group (Table I). Parametric statistical analysis was performed between groups 2 and 5 and between groups 4 and 6 with the unpaired t test. Mean daily mucositis scores ( ? SE) were plotted for all groups to analyze trends in healing by comparing the experimental g;roups with the control. Daily weights were evalualed to assessthe overall health of the animals throughout the experiment. A survival curve was plotted to determine whether any significant differences in unplanned mortality existed between experimental groups and the control. RESULTS
The presence of EGF significantly modified the frequency and course of mucositis. Hamsters that re-
Table II. Relationship between pump placement and peak mucositis score Day pump 0 3 6 7
placed
Peak
mucositis
day
11 12-13 13 14-15
ceived EGF had more severe, prolonged mucositis than animals that received placebo. In addition, the timing of pump placement, that is, the initiation of EGF exposure, affected the course of mucositis. For example, hamsters in which 7-day EGF pumps were implanted 1 week after the commencement of chemotherapy (group 2) had more significant mucositis throughout the course of the experiment than animals in which 7-day placebo pumps were implanted (group 5), except for day 11 (Fig. 3). Similarly, the course of mucositis was worse in hamsters in which 14-day EGF pumps were implanted (group 4) compared with animals with 1Cday placebo pumps (group 6) (Fig. 4). Delivery of EGF by pumps placed on either day 3 or day 6 failed to alter favorably the course or severity
752
Sonis et al.
ORA~SURGQRALMED
ORALPATH~L
December
10
11
12
13
14
15 Day
16
17
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19
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20
Fig. 3. Comparison of mucositis scoresfor osmotic pumps containing EGF (0) or placebo (A) placed for 7 days. EGF-filled pumps (group 2) were inserted on day 7. Pumps loaded with placebo (group 5) were placed on same day. Pumps placed in both groups operated for 7 days.
0-j I 10
I 11
I 12
I 13
I 14
I 15 Day
I 16
I 17
I 18
I 19
I 20
Fig. 4. Comparison of mucositis scoresfor osmotic pumps containing EGF (0) or placebo (A) placed for 14 days. EGF-filled pumps (group 4) were inserted on day 1. Pumps loaded with placebo (group 6) were placed on same day. Pumps placed in both groups operated for 14 days. of mucositis (Figs. 5 and 6). The earlier the pumps were placed, the more rapid the onset of peak mucositis (Table II). In addition, placement of the pumps early in the experimental period resulted in a more rapid development of mucositis than the placement of pumps 1 week after the start of chemotherapy. Thus animals in groups 2 and 5 had the least severe mucositis as measured on day 10. As expected, a direct relationship existed between the severity of mucositis and weight loss (Fig. 7). Animals in group 6 had the least mucositis and demon-
strated little fluctuation in weight during the experimental period. In contrast, the prolonged mucositis noted in animals in group 1 resulted in a dramatic weight loss during the experiment. Overall, the onset of weight recovery was noted in all groups at about day 16 to 17. Survival appeared to be related more to the severity and onset of mucositis than to the presence or absence of EGF. Nonsurgical deaths were first of significance on day 10 and were noted in both EGF and placebo groups (Fig. 8). Hamsters that had
Effect of epidermal growth factor on mucositis
Volume 74 Number 6
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14
15 Day
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Fig. 5. Comparison of mucositis scoresfor osmotic pumps containing EGF placed for 7 days (0) or placebo pumps placed for 7 days (A) and 14 days (0). EGF-filled pumps (group 1) were inserted on day 3. Pumps loaded with placebo were inserted on day 7 (group 5) and day 1 (group 6). Pumps placed in group 1 and group 5 operated for 7 days. Pumps in group 6 operated for 14 days.
76El432l01
lb
1’1
I$
15
14I
15 I
16I
17I
18I
19I
20I
Day
Fig. 6. Comparison of mucositis scoresfor osmotic pumps containing EGF placed for 7 days (0) or placebo pumps placed for 7 days (A) and 14 days (Cl). EGF-filled pumps (group 3) were inserted on day 6. Pumps loaded with placebo were inserted on day 7 (group 5) and day 1 (group 6). Pumps placed in group 3 and group 5 operated for 7 days. Pumps in group 6 operated for 14 days. pumps placed on day 3 or day 6 demonstrated the most mortality. By day 20 no significant difference was noted in the survival of hamsters in the other groups. DISCUSSION
The results of the present study support the hypothesis that the renewal rate of the oral epithelium is a major factor in determining the susceptibility of
the mucosa to the stomatotoxic effects of cancer chemotherapy. EGF, a cytokine with a demonstrated ability to stimulate epithelial cell proliferation, significantly increased the severity of mucositis and prolonged its duration. Preliminary pharmacokinetic studies by our group using iodine 125labeled EGF have shown that it is absorbed well by the oral mucosa.14 Presumably, increased cell mitoses stimulated by EGF resulted in increased susceptibility of
754
Sonis et al.
ORAL
3uR~
ORAL
MEDQRA~
December
PATHOS.
1992
70 I
01
I
I
3
I
I
I
5 6 7
I
II
I
I
I
I
I
I
I
I
10 11 12 13 14 15 16 17 16 19 20
Day
Fig. 7. Daily mean weights of’ animals receiving ‘I-day EGF pumps inserted on day 3 (0; group 1), 7-day EGF pumps inserted on day 7 (A; group 2), 7-day EGF pumps inserted on day 6 (0; group 3), 1Cday EGF pumps inserted on day 1 (0; group 4), 7-day placebo pumps inserted on day 1 (+; group 5) and 14-day placebo pumps inserted on day 1 (0; group 6).
+i--+-
1I
oi
’
I
3
I
I
I
5 6 7
I
I
I
I
I
I
I
I
I
I
I
10 11 12 13 14 15 16 17 18 19 20 Day
Fig. 8. Survival curve of animals receiving 7-day EGF pumps inserted on day 3 (0; group l), 7-day EGF pumps inserted on day 7 (A; group 2), 7-day EGF pumps inserted on day (5; group 3), 14-day EGF pumps inserted on day 1 (0; group 4), I4-day EGF pumps inserted on day 7 (+; group 5), and 14-day placebo pumps inserted on day 1 (0; group 6). the oral epithelium to the direct stomatotoxicity of the 5-W. In addition, it seems likely that the presence of the healing margins of the pump placement site resulted in an enhanced site of EGF action and became a target for enhanced 5-W activity. These findings are in agreement with the clinical observation of increased rates of mucositis among young patients as compared with older persons.6 It is likely that this observation is due to the more rapid
mitotic rate of oral epithelial cells in children compared with adults. In addition, the number of EGF receptors is greater on epithelium from younger patients, thus increasing the likelihood of enhanced proliferation to endogenous stimulatory cytokines. The release of EGF from submaxillary glands7 may provide a ready stimulus for cell proliferation. The lack of enhanced resolution of ulcerative mucositis in response to EGF is somewhat of an enigma
Volume Number
74 6
and might be related to the timing of cytokine application relative to mucositis. Although differences in timing of pump placement affected peak mucositis, in all cases EGF was initiated before optimum mucosal breakdown. Had the application of EGF been delayed until after the stomatotoxic effects had resolved, probably after day 10, it is possible that it might have resulted in a favorable effect on ulcer resolution. These results also suggest that an effective approach to the prevenlion of mucositis might be therapy aimed at inhibiting or reducing the rate of mucosal proliferation. We have initiated studies to evaluate this tactic further.
EfSect of epidermal growth factor on mucositis
6. 7.
8. 9.
10.
11. 12.
REFERENCES 1. Sonis ST, Sonis AL, Lieberman A. Qral complications in patients receiving treatment for malignancies other than of the head and neck. J Am Dent Assoc 1978:97:468-72. 2. Lockhart PB, Sonis ST. Alterations in the oral mucosa caused by chemotherapeutic agents: a histologic study. J Dermatol Surg Oncol 1981;7:1019-25. 3. Dreizen S, McCredie K, Keating M, Bodey G. Oral infections associated with chemotherapy in adults with acute leukemia. Postgrad Med 1982;71:133-46. 4. Lockhart PB, Sonis ST. Relationships of oral complications to peripheral blood leukocyte and platelet counts in patients receiving cancer chemotherapy. ORAL SURG ORAL MED ORAL PATHOL 1979;48:21-8. 5. Guggenheimer J, Verbin R, Appel B, Schmutz J. Clinico-
13.
14.
755
pathologic effects of cancer chemotherapeutic agents on human buccal mucosa. ORAL SURG ORAL MED ORAL PATHOL 1977;44:58-63. Sonis A, Sonis ST. Oral complications of cancer chemotherapy in pediatric patients. J Pedod 1979;3:122-8. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J Biol Chem 1962;237:1555-62. Cohen S. The epidermal growth factor (EGF). Cancer 1983; 51:1787-91. Steidler NE, Reade PC. Histomorphological effects of epiderma1 growth factor on skin and oral mucosa in neonatal mice. Arch Oral Biol 1980;25:37-43. Weaver LT, Gonnella PA, Israel EJ, Walker WA. Uptake and transport of epidermal growth factor by the small intestinal epithelium of the fetal rat. Gastroenterology 1990;98:828-37. Weaver LT, Walker WA. Epidermal growth factor and the developing human gut. Gastroenterology 1988;94:845-8. Waterfield MD. Epidermal growth factor and related molecules. Lancet 1989;1:1243-6. Sonis ST, Tracey C, Shklar G, Jenson J, Florine D. An animal model for mucositis induced by cancer chemotherapy. ORAL SURG ORAL MED ORAL PATHOL 1990;69:437-43. Sonis ST, Costa JW, Evitts SM, Lindquist LE, Nicolson M. The pharmacokinetics of topically applied epidermal growth factor to ulcerated versus intact hamster oral mucosa. 1991. (Submitted for publication.)
Reprint requests: Stephen T. Sonis, DMD, DMSc Dental Division Brigham & Women’s Hospital 75 Francis St. Boston, MA 02115
&
WOMEN’S
HOSPITAL,
HARVARD
SCHOOL
OF DENTAL
MEDICINE,
AND
AMGEN
CORPORATION
Ulcerative mucositis is a common, bothersome, and dose-limiting complication of cancer chemotherapy. It has been hypothesized that mucosal susceptibility to the degenerative effects of stomatotoxic drugs is related to the renewal rate of the buccal epithelium. This study was undertaken i:o evaluate the effect of epidermal growth factor, a molecule known to stimulate epidermal cell division, on the course, frequency, and healing of ulcerative mucositis in an animal model. Golden Syrian hamsters were subjected to a standard mucositis-induction protocol with 5-fluorouracil. Osmotic pumps were implanted into a space between the retractor muscle and the platysma cervicale muscle, and delivered epidermal growth factor or placebo at a constant rate for 7 or 14 days. Epidermal growth factor increased oral mucosal breakdown in the face of antineoplastic therapy. The course and extent of mucositis was influenced by the timing of epidermal growth factor pump placement relative to the initiation of stomatotoxic therapy. These results support the hypothesis that the epithelial basal cell rate is one of the key elements in determining mucosal sensitivity to cancer chemotherapy. (ORALSURGORALMEDORALPATHOL~~~~;~~:~~~-~~)
U
lcerative oral mucositis is one of the most common, bothersome, and dose-limiting complications of cancer chemotherapy. l-4 Because the oral mucosa undergoes frequent replication, it is especially sensitive to the direct cytotoxic effects of chemotherapy. Histologically, signs of oral mucosal atrophy become evident soon after tlherapy has begun. Inhibition of DNA replication of basal epithelial cells and cytolysis result in a thin, atrophic mucosa that is highly susceptible to spontaneous or traumatic ulceration. It has been hypothesized that the rate of basal epithelial cell proliferation affects the susceptibility of the oral mucosa to the stomatotoxic effect of chemotherapy.‘% 5, 6 Epidermal growth factor (EGF) is a 53-amino acid
Supported in part by the Brigham Surgical Group Foundation and the Amgen Corporation. aDivision of Dentistry, Brigham & Women’s Hospital, Boston. bHarvard School of Dental Medicine, Boston. CAmgen Corp., Thousand Oaks.
7/13/38980
polypeptide (mol. wt. 6000) first isolated from extracts of mouse submaxillary glands. The molecule has a number of activities including the induction of precocious eyelid opening and incisor eruption when injected into newborn mice.? This response is attributed to stimulation of epidermal growth and keratinization. In addition, EGF directly stimulates the proliferation of basal epithelial cells in skin organ culture systems.’ Other mitogenic effects of EGF have been noted in vitro with a variety of cell types. In vivo studies in neonatal mice by Steidler and Reade9 suggest that subcutaneous injections of EGF cause an increase in epithelial thickness and keratinization of skin and oral mucosa. Recent studies have shown that EGF promotes proliferation and differentiation of the gastrointestinal mucosa.lO, l l The mechanism of action EGF exerts after it binds to its receptor is currently under investigation. It is known that the EGF receptor is a glycoprotein with
749
750
Sonis et al.
ORALSURGQR.~LM~D
ORALPATHOL
December 1992
Fig. 1. AIzet osmotic pump filled with EGF (8.33 pg/pl) or placebo. Pump measures 3.0 cm in length and 0.7 cm in
diameter. a molecular weight of 175,000. On binding, EGF is able to stimulate an intrinsic tyrosine kinase activity that autophosphorylates the receptor.12 However, little is known about how the binding and autophosphorylation influence transduction of a mitogenic signal to the nucleus so as to alter gene regulation. Because EGF stimulates epithelial proliferation, we investigated its effect, when applied topically, on the frequency, course, and healing of ulcerative mucositis. We hypothesized that early exposure of the oral mucosa to EGF simultaneous to stomatotoxic cancer chemotherapy would enhance the development of ulcerative mucositis. We also hypothesized that its effect during healing might be favorable in reestablishing intact epithelium. MATERIAL AND METHODS Animals
Male Golden Syrian hamsters, 8 to were obtained from Charles River Wilmington, Mass. Animals were kept and provided with free access standard and water. Epidermal
growth
10 weeks old, Laboratories, five to a cage hamster food
factor
Human recombinant EGF was provided by Amgen Corp., Thousand Oaks, Calif., and loaded into 200 ~1Alzet osmotic pumps (Alza Corp., Palo Alto, Calif.). Pump placement
protocol
To ensure uniform topical EGF delivery, an implanted osmotic pump protocol was devised. After induction, animals were anesthesized with ethyl ether; sodium pentobarbital (Nembutal) (50 mg/kg) was administered by intraperitoneal injection. The left buccal pouch was everted and an incision made at the
ig. 2. Alzet osmotic pump sutured in place between retractor muscle and platysma cervicale muscle. Blunted cannula can be seen protruding through mucosa.
most superior aspect of the pouch in the mucobuccal fold. With blunt dissection a space was created between the retractor muscle and the platysma cervitale muscle. The pump, with a short contiguous cannula, was inserted and a purse-string
suture was
placed (Figs. 1 & 2). The cannula was allowed to protrude through the surgical site by about 3 mm. The end of the cannula was blunted to prevent mucosal irritation and dislodgement. Preliminary studies performed in our laboratory with dye-loaded pumps demonstrated their efficacy in delivering a uniform dose of material during a prolonged period. No infection or soft tissue anomalies were associated with pump placement. Pumps placed for 2 weeks delivered EGF at the rate of 0.5 pl/hr; l-week pumps delivered EGF at the rate of 1.0 pl/hr. Mucositis was induced in 60 hamsters by an established protocol.‘3 Briefly, hamsters received 5-fluerouracil(5-FU) (60 mgjkg) on days 0 and 5, with superficial irritation of the buccal pouch mucosa on day 7. Severe mucositis developed in all animals with this technique. Animals were randomly and equally divided into the following groups: Group I: On day 3 a ‘I-day pump containing EGF was implanted (8.33 pg/pl) Group 2: On day 7 a 7-day pump containing EGF was implanted (8.33 pg/hl) Group 3: On day 6 a 7-day pump containing EGF was implanted (8.33 pg/pl) Group 4: On day 1 a l&day pump containing EGF was implanted (8.33 pg/pl) Group 5: On day 7 a 7-day pump containing placebo was implanted (control for group 2) Group 6: On day 1 a 14-day pump containing placebo was implanted (control for group 4) Animals were observed and weighed daily and had
Effect of epidermal growth factor on mucositis
Volume Number
74 6
Table
I. Mean mucositis score and standard deviation by day”
Day 10
11
/ 8.39 1.25
11
8.21
12
1.65 9.39 0.56
13
NP NP
14 15 16 17 18 19 20
9.54 0.90 8.67 2.10 9.63 0.53 4.00 0.00 6.00 0.00 1.75 0.00 4.00 0.00
2
1
4.28 1.03 3.47 2.43 7.63 1.63 7.55 1.27 8.14 0.84 8.78 0.97 6.64 2.46 5.25 2.90 4.43 2.69 3.25 2.92 4.13 3.03
3 6.60 1.02
6.50
‘T
4
/
7.11 2.85 8.32
1.06
1.62
7.94 1.20 9.40 0.76 7.60 2.16 6.08 0.88 6.42 0.63 4.61 2.10 5.58 1.46 3.75 1.89 4.83 1.04
8.14 1.27 7.13 2.48 5.43 3.38 4.13 3.77 3.46 3.11 2.00 3.11 2.45 1.58 1.75 0.88 1.35 1.28
5
1
751
6
1.55t 0.52 2.55 2.08
6.78 1.79 8.03 2.09 6.94 1.74 6.19 2.39 5.71 1.76 1.68 0.62 0.75$ 0.75 0.64 0.67 1.71 0.89 1.89 1.38 2.00 1.61
NP
6.17 3.02 6.47 2.63 5.56t 3.02 4.56 3.34 3.25 1.89 3.38 3.34 2.06 1.91 2.75 2.60
NP, Photographs not available for these time points. *Mean mucositis scores for each group on each day. Top value in each row indicates mean mucositis score ranging from 0 to 10. Bottom value represents SD for that average score for each group on each day. Parametric statistical analysis was performed between groups 2 and 5 and groups 4 and 6 by unpaired t test. tStatistically significant (p < 0.01). fStatistically significant (p < 0.05).
free access to food and water. Beginning on day 10 left buccal pouches were photographed daily with a Yashica Dental Eye, camera (Kyocera Corp., Kyoto, Japan). At the conclusion of the experiment (day 20) the photographs were randomly numbered, mixed, and scored by four different observers, in blinded fashion, by comparing them with a standardized set of photographs with mucositis scores ranging from 0 (no mucositis) to 10 (severe mucositis). Data were collected and mean daily mucositis scores were calculated for each group (Table I). Parametric statistical analysis was performed between groups 2 and 5 and between groups 4 and 6 with the unpaired t test. Mean daily mucositis scores ( ? SE) were plotted for all groups to analyze trends in healing by comparing the experimental g;roups with the control. Daily weights were evalualed to assessthe overall health of the animals throughout the experiment. A survival curve was plotted to determine whether any significant differences in unplanned mortality existed between experimental groups and the control. RESULTS
The presence of EGF significantly modified the frequency and course of mucositis. Hamsters that re-
Table II. Relationship between pump placement and peak mucositis score Day pump 0 3 6 7
placed
Peak
mucositis
day
11 12-13 13 14-15
ceived EGF had more severe, prolonged mucositis than animals that received placebo. In addition, the timing of pump placement, that is, the initiation of EGF exposure, affected the course of mucositis. For example, hamsters in which 7-day EGF pumps were implanted 1 week after the commencement of chemotherapy (group 2) had more significant mucositis throughout the course of the experiment than animals in which 7-day placebo pumps were implanted (group 5), except for day 11 (Fig. 3). Similarly, the course of mucositis was worse in hamsters in which 14-day EGF pumps were implanted (group 4) compared with animals with 1Cday placebo pumps (group 6) (Fig. 4). Delivery of EGF by pumps placed on either day 3 or day 6 failed to alter favorably the course or severity
752
Sonis et al.
ORA~SURGQRALMED
ORALPATH~L
December
10
11
12
13
14
15 Day
16
17
18
19
1992
20
Fig. 3. Comparison of mucositis scoresfor osmotic pumps containing EGF (0) or placebo (A) placed for 7 days. EGF-filled pumps (group 2) were inserted on day 7. Pumps loaded with placebo (group 5) were placed on same day. Pumps placed in both groups operated for 7 days.
0-j I 10
I 11
I 12
I 13
I 14
I 15 Day
I 16
I 17
I 18
I 19
I 20
Fig. 4. Comparison of mucositis scoresfor osmotic pumps containing EGF (0) or placebo (A) placed for 14 days. EGF-filled pumps (group 4) were inserted on day 1. Pumps loaded with placebo (group 6) were placed on same day. Pumps placed in both groups operated for 14 days. of mucositis (Figs. 5 and 6). The earlier the pumps were placed, the more rapid the onset of peak mucositis (Table II). In addition, placement of the pumps early in the experimental period resulted in a more rapid development of mucositis than the placement of pumps 1 week after the start of chemotherapy. Thus animals in groups 2 and 5 had the least severe mucositis as measured on day 10. As expected, a direct relationship existed between the severity of mucositis and weight loss (Fig. 7). Animals in group 6 had the least mucositis and demon-
strated little fluctuation in weight during the experimental period. In contrast, the prolonged mucositis noted in animals in group 1 resulted in a dramatic weight loss during the experiment. Overall, the onset of weight recovery was noted in all groups at about day 16 to 17. Survival appeared to be related more to the severity and onset of mucositis than to the presence or absence of EGF. Nonsurgical deaths were first of significance on day 10 and were noted in both EGF and placebo groups (Fig. 8). Hamsters that had
Effect of epidermal growth factor on mucositis
Volume 74 Number 6
O-1 11
12
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15 Day
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Fig. 5. Comparison of mucositis scoresfor osmotic pumps containing EGF placed for 7 days (0) or placebo pumps placed for 7 days (A) and 14 days (0). EGF-filled pumps (group 1) were inserted on day 3. Pumps loaded with placebo were inserted on day 7 (group 5) and day 1 (group 6). Pumps placed in group 1 and group 5 operated for 7 days. Pumps in group 6 operated for 14 days.
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Fig. 6. Comparison of mucositis scoresfor osmotic pumps containing EGF placed for 7 days (0) or placebo pumps placed for 7 days (A) and 14 days (Cl). EGF-filled pumps (group 3) were inserted on day 6. Pumps loaded with placebo were inserted on day 7 (group 5) and day 1 (group 6). Pumps placed in group 3 and group 5 operated for 7 days. Pumps in group 6 operated for 14 days. pumps placed on day 3 or day 6 demonstrated the most mortality. By day 20 no significant difference was noted in the survival of hamsters in the other groups. DISCUSSION
The results of the present study support the hypothesis that the renewal rate of the oral epithelium is a major factor in determining the susceptibility of
the mucosa to the stomatotoxic effects of cancer chemotherapy. EGF, a cytokine with a demonstrated ability to stimulate epithelial cell proliferation, significantly increased the severity of mucositis and prolonged its duration. Preliminary pharmacokinetic studies by our group using iodine 125labeled EGF have shown that it is absorbed well by the oral mucosa.14 Presumably, increased cell mitoses stimulated by EGF resulted in increased susceptibility of
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Fig. 7. Daily mean weights of’ animals receiving ‘I-day EGF pumps inserted on day 3 (0; group 1), 7-day EGF pumps inserted on day 7 (A; group 2), 7-day EGF pumps inserted on day 6 (0; group 3), 1Cday EGF pumps inserted on day 1 (0; group 4), 7-day placebo pumps inserted on day 1 (+; group 5) and 14-day placebo pumps inserted on day 1 (0; group 6).
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Fig. 8. Survival curve of animals receiving 7-day EGF pumps inserted on day 3 (0; group l), 7-day EGF pumps inserted on day 7 (A; group 2), 7-day EGF pumps inserted on day (5; group 3), 14-day EGF pumps inserted on day 1 (0; group 4), I4-day EGF pumps inserted on day 7 (+; group 5), and 14-day placebo pumps inserted on day 1 (0; group 6). the oral epithelium to the direct stomatotoxicity of the 5-W. In addition, it seems likely that the presence of the healing margins of the pump placement site resulted in an enhanced site of EGF action and became a target for enhanced 5-W activity. These findings are in agreement with the clinical observation of increased rates of mucositis among young patients as compared with older persons.6 It is likely that this observation is due to the more rapid
mitotic rate of oral epithelial cells in children compared with adults. In addition, the number of EGF receptors is greater on epithelium from younger patients, thus increasing the likelihood of enhanced proliferation to endogenous stimulatory cytokines. The release of EGF from submaxillary glands7 may provide a ready stimulus for cell proliferation. The lack of enhanced resolution of ulcerative mucositis in response to EGF is somewhat of an enigma
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and might be related to the timing of cytokine application relative to mucositis. Although differences in timing of pump placement affected peak mucositis, in all cases EGF was initiated before optimum mucosal breakdown. Had the application of EGF been delayed until after the stomatotoxic effects had resolved, probably after day 10, it is possible that it might have resulted in a favorable effect on ulcer resolution. These results also suggest that an effective approach to the prevenlion of mucositis might be therapy aimed at inhibiting or reducing the rate of mucosal proliferation. We have initiated studies to evaluate this tactic further.
EfSect of epidermal growth factor on mucositis
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REFERENCES 1. Sonis ST, Sonis AL, Lieberman A. Qral complications in patients receiving treatment for malignancies other than of the head and neck. J Am Dent Assoc 1978:97:468-72. 2. Lockhart PB, Sonis ST. Alterations in the oral mucosa caused by chemotherapeutic agents: a histologic study. J Dermatol Surg Oncol 1981;7:1019-25. 3. Dreizen S, McCredie K, Keating M, Bodey G. Oral infections associated with chemotherapy in adults with acute leukemia. Postgrad Med 1982;71:133-46. 4. Lockhart PB, Sonis ST. Relationships of oral complications to peripheral blood leukocyte and platelet counts in patients receiving cancer chemotherapy. ORAL SURG ORAL MED ORAL PATHOL 1979;48:21-8. 5. Guggenheimer J, Verbin R, Appel B, Schmutz J. Clinico-
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pathologic effects of cancer chemotherapeutic agents on human buccal mucosa. ORAL SURG ORAL MED ORAL PATHOL 1977;44:58-63. Sonis A, Sonis ST. Oral complications of cancer chemotherapy in pediatric patients. J Pedod 1979;3:122-8. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J Biol Chem 1962;237:1555-62. Cohen S. The epidermal growth factor (EGF). Cancer 1983; 51:1787-91. Steidler NE, Reade PC. Histomorphological effects of epiderma1 growth factor on skin and oral mucosa in neonatal mice. Arch Oral Biol 1980;25:37-43. Weaver LT, Gonnella PA, Israel EJ, Walker WA. Uptake and transport of epidermal growth factor by the small intestinal epithelium of the fetal rat. Gastroenterology 1990;98:828-37. Weaver LT, Walker WA. Epidermal growth factor and the developing human gut. Gastroenterology 1988;94:845-8. Waterfield MD. Epidermal growth factor and related molecules. Lancet 1989;1:1243-6. Sonis ST, Tracey C, Shklar G, Jenson J, Florine D. An animal model for mucositis induced by cancer chemotherapy. ORAL SURG ORAL MED ORAL PATHOL 1990;69:437-43. Sonis ST, Costa JW, Evitts SM, Lindquist LE, Nicolson M. The pharmacokinetics of topically applied epidermal growth factor to ulcerated versus intact hamster oral mucosa. 1991. (Submitted for publication.)
Reprint requests: Stephen T. Sonis, DMD, DMSc Dental Division Brigham & Women’s Hospital 75 Francis St. Boston, MA 02115