Radiotherapy and Oncology, 25 (1992) 49-55 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-8140/92/$05.00
49
RADION 01025
A pilot study of a method of estimating the number of functional eccrine sweat glands in irradiated human skin W. J a m e s Morris, Stanley Dische and G o d f r e y M o t t Regional Centre/or Radiotherapy and Oncology, Mount Vernon Hospital, Northwood, Middlesex, UK (Received 12 September 1991, revision received 18 March 1992, accepted 30 March 1992)
Key words: Eccrine glands; Sweating; Radiotherapy; Clinical radiobiology; Normal tissue; Skin appendages
Summary Following stimulation with pilocarpine, the secretion from eccrine sweat glands produces characteristic imprints in hardening silicone polymers applied to the skin. This permits an accurate determination of the numerical density of functional eccrine glands in irradiated skin which can be compared to non-irradiated skin. A description of this inexpensive, noninvasive, and quantitative technique is presented as well as preliminary results determined in six normal subjects and 28 irradiated patients. Eleven patients, with atrophy and telangiectasia after radiotherapy to the skin to a high dose, were found to have no functional eccrine glands by this technique. A range of results from normal numbers of eccrine glands through partial and, rarely, complete loss was observed in patients given lower doses and in whom the skin was visually normal. When the irradiated side outside the boost area in 16 breast cancer patients who received postoperative radiotherapy was compared to an equivalent area on the untreated, contralateral side, 11 showed a greater than 50% reduction in the density of functional eccrine glands. The method appears to be a sensitive, quantitative assay for a permanent change in skin and so ought to facilitate meaningful comparison of different regimens of radiotherapy. Further studies are required to determine the dose-response relationship, latency and progression of the observed changes.
Introduction Studies of the late changes in skin following therapeutic irradiation have included observations of fibrosis, pigmentation, atrophy, and telangiectasia. Telangiectasia, in particular has provided useful dose-response relationships when arbitrary scales have been applied to its severity [2,12]. Irradiated skin often feels drier than normal, and it is a common clinical observation that sweating is reduced in irradiated axillae. Despite this, no systematic study has been published on changes in sweat gland function in irradiated human skin. Eccrine sweat glands are unbranched, tubular, coiled excretory structures which develop by epithelial invagination during the fourth month of fetal life [2]. The glands lie in the dermis adjacent to the deep cutaneous
capillary plexus at a depth of 0.7 to 2 ram, except over the posterior trunk and the glabrous skin of the palms and soles where they lie as deep as 4 mm [4]. A single duct arises from each gland and penetrates the epidermis ending in a 5 to 10 ~tm aperture through which a watery secretion is expelled aiding thermoregulation [2,4,10]. This function is principally regulated by cholinergic innervation from the autonomic nervous system. Eccrine glands are among the most abundant skin appendages; their numerical density is genetically determined and varies between individuals and within individuals according to anatomical location. Average values per cm 2 are 64 on the trunk, 108 on the forearm, 181 on the forehead and 600 to 700 on the palms and soles [ 10]. Methods have been developed to measure eccrine function following thermal stress, direct nerve stimula-
Address for correspondence: Dr. W. J. Morris, British Columbia Cancer Agency, Vancouver Clinic, 600 West 10th Avenue, Vancouver, B.C. V5Z 4E6, Canada.
50 tion, and the local application of cholinergic stimulants [2]. These fall essentially into two approaches. The first attempts to quantitate physiological parameters such as the volume and rate of secretion by collecting sweat on pads, measuring changes in the electrical resistance of skin, or recording rates of evaporation over known areas of skin [2,6]. The second approach determines the numerical density of individual sweat droplets using special indicator solutions or imprints produced in silicone elastomer moulds [7,9]. This paper concerns the latter. Silicone elastomer moulds are considered to provide the most sensitive method of determining the numerical density of actively secreting eccrine glands [ 9]. With this technique, a low viscosity suspension of a silicone monomer, containing the required catalyst, is applied to the skin and allowed to harden producing a high resolution, permanent negative replica of the skin surface. During the hardening process, the hydrophobic silicone suspension withdraws from any sweat droplets emerging from the eccrine pores causing the formation of characteristic imprints in the surface of the cured silicone polymer. These imprints can easily be identified and counted using low power microscopy. The method has been successfully applied to the study of autonomic neuropathy in diabetic patients and has been shown to have excellent repeatability [ 5,7 ]. Such imprints do not occur in silicone moulds of unstimulated skin. Moreover, the number of imprints visible in silicone moulds taken from the digit pads of mice following stimulation with subcutaneous pilocarpine has been shown to correspond to the number of eccrine glands seen in serial sections of histologic preparations of the digit pads [8]. This paper reports the adaptation of this technique to determine the numerical density of functional eccrine glands in human skin following therapeutic irradiation and presents the results of a pilot study. Material and methods
Eccrine gland stimulation The method of pilocarpine iontophoresis used to stimulate the eccrine glands was adapted from the method of Gibson and Cooke [3,11]. The skin in the area of interest was cleansed with isopropyl alcohol and dried with gauze. A 3 × 5 cm sheet of blotting paper was soaked in 4 % w/v pilocarpine nitrate (Schering-Plough ophthalmic solution containing 0.20% benzalkonium preservative in normal saline) and applied to the test area of skin. Two 2 x 3 cm gauze pads soaked in sterile normal saline served as iontophoresis electrodes. One was placed over the blotting paper and the other on a
separate area of nearby skin. The electrodes were then connected to a constant current D.C. apparatus such that the positive electrode was over the pilocarpine. A current of 0.04 mA per cm 2 was applied for 5 min. In an initial testing and development phase, pilocarpine iontophoresis was performed on 19 subjects; following this the technique was performed on the 28 irradiated patients and the six non-irradiated subjects reported here. A mild tingling or itching sensation was reported by most subjects, however, none of the 53 consecutive subjects found it more than mildly uncomfortable and in all cases the procedure was completed without interruption. The sensation ends within a few minutes of interrupting the current. A slight to moderate local erythema, lasting for 1 to 2 h, was common following pilocarpine iontophoresis. The apparatus used to provide the constant current was designed, built and safety tested by one of the authors (G.M.). It is powered by a 9 V dry cell battery, measures 16.5 x 8.0 x 4.0 cm, and cost £250 to construct.
Silicone elastomer mould preparation A diluted silicone elastomer was prepared by mixing 5 parts of silicone monomer suspension (MED-6382, McGhan Nusil Corp., 1150 Mark Ave., Carpenteria, Ca., 903013) with 4 parts (w/w) of the same manufacture's naphtha-based thinner. MED-6382 catalyst is added just prior to use with a syringe fitted with a 19gauge needle at the rate of one drop per gram of diluted suspension. Following the 5-min period of iontophoresis, the catalyst was thoroughly mixed, the skin dried, and the suspension applied to the test skin as a thin layer (0.1-0.5 mm)with a wooden spatula. After 10 min or less the polymer had hardened sufficiently to be removed, usually as an intact sheet of translucent white material. A further 24 h is required for complete curing, after which it forms a robust, non-adhering material with excellent geometric stability. During the hardening process there is a noticeable odour of organic solvents, principally naphtha, and therefore the procedure is best performed in a well ventilated room.
Estimation of the density of eccrine pore imprints During the hardening process, the hydrophobic silicone suspension withdraws from the droplets of sweat secreted by functional eccrine glands forming hemispherical imprints and often minute holes. The largest ones, about 0.2 mm in diameter, can be seen with the naked eye, but more can be identified with a low power mi-
51 croscope. The fully cured silicone mould was trimmed and placed in a standard glass-sided 35 mm transparency mount permitting microscopic examination and compact, dust-free storage. Pilocarpine iontophoresis was applied to 15 c m 2 in each test region of skin and the central 8 c m 2 of this stimulated region was used in estimating the numerical density of eccrine pore imprints using direct counting under low power microscopy. Except in four cases (patient nos. 2, 5, 8 and 10) the numerical density was calculated from the mean of 35 low power fields (11.9 mm 2 per LPF). Using this counting method the entire 8 cm 2 was sampled and more than 50~o of the surface area was counted. It is important to count a large area since the distribution of eccrine pores is not uniform over small (mm) scales. Gases escaping from solution in the silicone suspension during the hardening process can leave minute bubbles in the silicone elastomer mould, particularly if the material is applied in a thick layer. These can be readily distinguished from eccrine pore imprints since the latter are always open to the skin surface side of the silicone mould while the gas bubbles never are. These bubbles can largely be prevented by de-gassing
the diluted silicone monomer suspension in a vacuum for 1 h and then allowing it to stand for 24 h prior to use.
Patients and treatment techniques
The first objective was to see if there were a reduced number of functional eccrine glands in irradiated skin. Consent was obtained from 12 patients who received radiotherapy to a variety of anatomic sites with a range of doses, schedules, photon/electron energies and times since completion of treatment (Table I). The second objective was to determine if a quantitative reduction in the density of functional eccrine glands could be demonstrated in irradiated skin in which little or no visual change was apparent. Chosen were patients with unilateral breast cancer treated either by mastectomy and chest wall irradiation (8 patients) or lumpectomy followed by breast irradiation (8 patients). In both cases the contralateral side was used for comparison. All patients had been treated using a tangent pair technique with megavoltage equipment and bolus material (equivalent to 5 mm of tissue) coveting at least a 10 cm circle over the original tumour
TABLE I Numerical density of eccrine pore imprints in silicone elastomer m o u l d s taken of skin following pilocarpine iontophoresis in 12 patients. Patient no.
1 2 3 4 5 6 7 8 9 10 ll 12
Diagnoses
H & N SC Sem. BCC H & N SC BCC NHL H & N SC BCC Sem. Sere. Thyr. NHL Endo.
Tumour dose/fractions
66 Gy/33 35 Gy/20 35 Gy/5 40.5 Gy/27 b 35 G y / 5 30 G y / 1 2 63 G y / 3 0 60 G y / 3 0 45 Gy/25 36 G y / 1 2 64 G y / 3 2 40 G y / 2 0 50.6 Gy/22 c
Energy
5 MV 8 MV 100 kVp 8 MV 250 kVp 8 MV 5 MV 6 MeV ~ 8 MV 250 kVp Cobalt 8 MV 8 MV
Site of skin examined
Neck Abdomen Forehead Neck Nose Neck Neck Pinna Abdomen Groin Neck Neck Pelvis
Time since XRT
1 mth 4 mths 10 m t h s 1 yr 2 yrs 1 m t h 5 yrs 7 yrs 8 yrs 9 yrs I m t h 9 yrs 1 m t h 11 yrs 11 yrs 7 m t h s 12 yrs 11 m t h s
Bolus
No No No No No No N/K Yes No No Yes No No
Obvious skin changes
Yes No Yes No Yes No Yes No No Yes Yes No No
Eccrine pore imprints ( n u m b e r cm 2) Untreated area ~
Treated area
80.4 100 49.5 41.9 5.98 40.2 23.0 17.9 39.5 66.3 25.6
< 1 73.0 < 1 70.3 < 1 8.61 < 1 < 1 10.5 < 1 < 1 56.9 35.2
For patients w h o h a d unilateral treatment, an equivalent area on the contrateral side was tested for comparison. b Three fractions per day ( C H A R T ) : This patient h a d a s q u a m o u s cell c a r c i n o m a of the mobile tongue treated with combined external b e a m to the primary t u m o u r and uninvolved neck n o d e s followed by interstitial brachytherapy to the turnout. c Four-field plan. The values given were calculated from the m e a n s o f 35 low power microscope fields (11.9 m m 2 per L P F ) which sampled the central 8 cm 2 o f the original 15 c m : silicone m o u l d except in the cases of patients 2, 5, 8 and 10 where a smaller area was s a m p l e d a n d fewer L P F counted. H & N SC = h e a d and neck s q u a m o u s cell carcinoma; Sem. = s e m i n o m a ; B C C = b a s a l cell carcinoma; N H L = n o n - h o d g k i n ' s l y m p h o m a ; Thyr. = thyroid carcinoma; Endo. = endometrial adenocarcinoma).
52
Fig. 1. Photomicrographs of silicone elastomer moulds of human skin. (a) Volar forearm of normal subject prior to pilocarpine iontophoresis. (b) Same subject immediately following pilocarpine iontophoresis. (c) Forehead 10 months post-radiotherapy for basal cell carcinoma (35 Gy in 5 daily fractions, field edge runs vertically through the middle of the photomicrograph). (d) Breast patient no. 24, non-irradiated UIQ right breast. (e) Breast patient no. 24, irradiated UIQ left breast. (f) Breast patient no. 24, boost region of left breast. Note: the scale bars in all panels equal 1 ram.
site. Fourteen of these patients received a minimum tumour dose of 40 Gy in 15 fractions treating once daily Monday to Friday, while the remaining two patients received 50 Gy in 25 fractions. Five patients received boost doses to the tumour bed using electrons; in these
cases test areas were selected both within and outside the boost region. The average time between completion of treatment and testing was 66 months (range 2 to 204 months). Two patients had adjuvant C M F chemotherapy.
53 The actual dose of radiation received at the depth of the eccrine glands in these breast cancer patients could not be determined accurately. It is estimated that the dose to the eccrine glands ranged between 28 and 40 Gy in the non-boost regions of the 14 patients who received 15 treatments over 3 weeks, 35-50 Gy in the two patients who received 25 treatments over 5 weeks, and up to 50 Gy within the boost area of the five patients who received a 10 Gy electron boost following the standard 15 fraction course. Patients treated by these techniques usually showed acute erythema and some dry desquamation; rarely did moist desquamation occur. In follow up, telangiectasia and atrophy are normally observed in boost areas, but little if any visible change was present elsewhere on the skin. In order to provide an estimate of the variation in the density to be encountered when comparing one side of the chest with the other, silicone elastomer moulds of skin from both breasts were made of six non-irradiated subjects following pilocarpine iontophoresis. Results
Photomicrographs (Fig. la,b) show silicone elastomer moulds taken from the same region of volar forearm skin from a normal subject before and after 5 min of pilocarpine iontophoresis. It can be seen that no eccrine pore imprints are present in the mould taken from the unstimulated skin but many are visible after cholinergic stimulation. A photomicrograph of a silicone elastomer mould taken following stimulation with pilocarpine in a patient successfully treated for a basal cell carcinoma on the forehead with a 100 kVp X-ray apparatus (35 Gy in 5 daily fractions, patient 3 Table I) is shown in Fig. lc.
The heavily irradiated skin on the left side of the photomicrograph has no eccrine pore imprints while that outside the treatment field shows imprints. Silicone moulds taken from the skin of one of the breast cancer patients (no. 24, Table III) 20 months after radiotherapy are shown in photomicrographs (Fig. ld,e,f). A test area from the upper inner quadrant of the untreated right breast is shown in Fig. ld and contrasts with an equivalent test area from the upper inner quadrant of the treated left breast (Fig. le). An area of the upper outer quadrant of the left breast which was in the boost region and has marked telangiectasia, is shown in (Fig. lf). Compared to the untreated breast it is apparent that there is a reduction in the number of eccrine pore imprints visible following pilocarpine stimulation in the treated breast and none are visible within the boost region. Table I gives the results of 13 irradiated sites on the initial 12 patients tested with the technique. No eccrine pore imprints could be observed in six sites where there were marked post-radiation changes and where the skin had received a dose of radiation similar to that of the tumour, either because of the energy of the irradiation or because of the use of bolus material. One other site (patient 8) had also received a high dose in the skin, but had a normal appearance and no eccrine pore imprints were detected. The remaining six had normal appearing skin; only one showed a reduction in eccrine pore imprints of more than 50~o. The estimated dose in these areas of skin at the depth of the eccrine glands ranged from 20 to 35 Gy given in 12 to 27 fractions. Such doses and doses per fraction are likely to give a low index of permanent radiation change. The results from testing six normal subjects to evaluate the range in right-left asymmetry in the density of
TABLE II Numerical density of eccrine pore imprints in silicone elastomer moulds of skin in six normal subjects (values given were calculated from the means of 35 LPF). Subject no.
Eccrine pore imprints (number per cm 2) Upper inner quadrant left breast
Upper inner quadrant right breast
Mean
% Difference*
13 14 15 16 17 18
34.9 37.8 91.0 62.4 67.9 37.8
46.1 22.0 92.1 46.4 54.3 33.7
40.5 29.9 91.6 54.4 61.1 35.8
27.6 52.8 1.20 29.4 22.3 11.5
Mean
55.3
49.1
52.2
24.1 (S.D. 17.6)
*Percent difference is defined as the larger - the smaller/mean x 100.
54 TABLE
III
N u m e r i c a l d e n s i t y o f e c c r i n e p o r e i m p r i n t s in s i l i c o n e e l a s t o m e r m o u l d s t a k e n o f s k i n in 16 p a t i e n t s w h o r e c e i v e d p o s t o p e r a t i v e r a d i o t h e r a p y f o r c a r c i n o m a o f t h e b r e a s t . S u r g i c a l t r e a t m e n t w a s e i t h e r b y m a s t e c t o m y ( M ) o r l u m p e c t o m y (L). Patient
Months
Surgical
Tumour
no.
since XRT
treatment
dose/ fractions
E c c r i n e p o r e i m p r i n t s ( n u m b e r p e r c m 2) Untreateda
Treateda
side
side
~o C h a n g ~
area
19 u
2
M
50 G y / 2 5
4
M
40 Gy/15
21 b
5
L
40 Gy/15 + B
22
10
L
50 G y / 2 5
27.5
30.9
+ 11.9
-
23
13
L
40 Gy/15
43.3
31.3
- 27.7
-
24
20
L
40 Gy/15 + B
67.0
10.0
- 85.1
< 1
- 100
25
47
L
40 Gy/15 + B
19.6
< 1
- 100
26
48
L
40 Gy/15 + B
78.7
1.90
- 97.6
< 1
- 100
27
60
M
40 Gy/15
23.2
1.91
- 88.7
-
-
28
92
M
40 Gy/15
7.89
+ 24.2
-
-
29
93
L
40 Gy/15
96.4
- 88.6
-
-
30
98
M
40 Gy/15
11.7
31
105
L
40 Gy/15 + B
30.4
32
127
M
40 Gy/15
21.0
33
135
M
40 Gy/15
34
204
M
40 Gy/15
a p< 0.02. b Received aduvant CMF
< 1 34.7
5.98
5.50 38.3 34.0
5.98
Boost
20
Mean
40.5
~o C h a n g e
- 85.2
-
< 1
Nil
-
< 1
- 100
< 1
11.0 < 1 12.0 9.57 < 1 33.5 9.75
- 100
- 100 - 60.5 - 54.4 - 100 - 13.1
< 1
< 1 -
- 100 -
- 100 -
-
-
-
-
- 7 1 . 3 ~o
chemotherapy.
eccrine pores are given in Table II. On the basis of this information, it was felt that a real lowering in the number of eccrine glands would be accepted when there was a reduction in the density of eccrine pore imprints of 50~o or more when comparison was made between the irradiated and the non-irradiated side in the breast cancer patients. The results from 16 breast cancer patients are presented in Table III. Eleven of the patients had a reduction in eccrine pore density that exceeded 50~o in the treated breast compared to the contralateral side. Of the remaining five patients, two had a reduction of less than 5 0 ~ , two had higher counts in the treated side and one had no visible eccrine pore imprints in silicone elastomer moulds taken from the treated or the untreated breast. Five patients had boost doses to a region of the breast in which obvious radiation changes were present, and no eccfine pore imprints were visible in silicone moulds taken of skin from these areas. Discussion The paper reports the use of an inexpensive, noninvasive and quantitative technique to determine the density of functional eccrine glands in human skin as a mani-
festation of changes after therapeutic irradiation. The number of eccrine pore imprints present in silicone elastomer moulds following pilocarpine iontophoresis in skin showing changes typical of late radiation change is reduced, usually to zero. In a group of 16 breast cancer patients with irradiated skin that appeared visually normal, 11 showed a reduction in eccrine pore imprints of more than 50~o when compared with the contralateral non-irradiated side. This difference is greater than that found between the fight and left breasts in six normal subjects. There was no obvious relationship between the type of surgical treatment or the time elapsed since radiotherapy and the results obtained. In one subject (no. 20, Table III) the technique failed to demonstrate the presence of eccrine pore imprints in a silicone elastomer mould from non-irradiated skin; the reason for this is not clear, but it is unlikely to represent an actual absence of eccrine glands. The observation of complete loss of functional eccrine glands in skin showing late changes due to radiotherapy can be contrasted with the normal number found in skin where the dose at 0.7-2 mm ranged from 20 to 35 Gy given at less than 1.5 Gy per fraction. A useful dose-response relationship is suggested by the data derived from the breast cancer patients treated to intermediate dose levels (between 28 and 50 Gy mostly
55 at 1.9-2.7 Gy per fraction) where, in 6 of 16 cases, there was a reduction, but not a complete loss, in the number of functional eccrine glands. The pilot study suggests that the technique should provide useful quantitative data on normal tissue effects following radiotherapy. A precise comparison may then be possible of permanent changes in human skin after use of altered fractionation regimens such as CHART with that after conventional daily treatment. In collaboration with colleagues at the Gray Laboratory the method is being employed in fractionation studies in the mouse foot. Clinical studies designed to determine the dose-response curve, latency and pro-
gression for the loss of eccrine glands in human skin are being conducted.
Acknowledgements The authors wish to thank Mr. D. Gavin, Mr. M. Nathan, and Mr. R. Sibley of the Mount Vernon Hospital Maxillo-Facial Laboratory for help and advice regarding silicone elastomers. W. J. M. held a fellowship of the British Columbia Cancer Agency during the performance of this work. S. D. is a life fellow of the Cancer Research Campaign. The support of these organizations is gratefully acknowledged.
References 1 Bentzen, S. M. and Overgaard, M. Relationship between early and later normal-tissue injury after postmastectomy radiotherapy. Radiother. Oncol. 20: 159-165, 1991. 2 Champion, R.H. Disorders of sweat glands. In: Textbook of Dermatology, 4th edn., Vol. Ill, pp. 1881-1887. Editors: A. Rook, D. S. Wilkinson, F. J. G. Ebling, R. H. Champion and J. L. Burton, Blackwell Scientific, Oxford, 1986. 3 Gibson, L. E. and Cooke, R.E. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 39: 545-549, 1959. 4 Gray's Anatomy, 37th edn, pp. 70-94. Editors: P. L. Williams, R. Warwick, M. Dyson, and L. H. Bannister. Churchill Livingstone, Edinburgh, 1989. 5 Harris, D. R., Polk, B. F. and Willis I. Evaluating sweat gland activity with imprint techniques. J. Invest. Dermatol. 58: 78-84, 1972. 6 Kennedy, W. R., Sakuta, M., Sutherland, D. and Goetz, F. The sweating deficiency in diabetes mellitus: methods of quantitation and clinical correlation. Neurology 34: 758-763, 1984.
7 Kennedy, W.R., Dakuta, M., Sutherland, D. and Goetz, F. Quantitation of the sweating deficiency in diabetes mellitus. Ann. Neurol. 15: 482-488, 1984. 8 Kennedy, W. R., Sakuta, M. and Quick, D . C . Rodent eccrine sweat glands: a case of multiple efferent innervation. Neuroscience 11: 741-749, 1984. 9 Ryder, R . E . J . , Marshall, R., Johnson, K., Ryder, A.P., Owens, D. R. and Hayes, T.M. Acetylcholine sweatspot test for autonomic denervation. Lancet I: 1303-1305, 1988. 10 Sato, K., Kang, W. H., Saga, K. and Sato, K.T. Biology of sweat glands and their disorders, I: Normal sweat gland function. J. Am. Acad. Dermatol. 20: 537-563, 1989. 11 Sloan, J. B. and Solanti, K. Iontophoresis in dermatology: a review. J. Am. Acad. Dermatol. 15: 671-684, 1986. 12 Turesson, I. Characteristics of dose-response relationships for late radiation effects: an analysis of skin telangiectasia and head and neck morbidity. Radiother. Oncol. 20: 149-158, 1991.
49
RADION 01025
A pilot study of a method of estimating the number of functional eccrine sweat glands in irradiated human skin W. J a m e s Morris, Stanley Dische and G o d f r e y M o t t Regional Centre/or Radiotherapy and Oncology, Mount Vernon Hospital, Northwood, Middlesex, UK (Received 12 September 1991, revision received 18 March 1992, accepted 30 March 1992)
Key words: Eccrine glands; Sweating; Radiotherapy; Clinical radiobiology; Normal tissue; Skin appendages
Summary Following stimulation with pilocarpine, the secretion from eccrine sweat glands produces characteristic imprints in hardening silicone polymers applied to the skin. This permits an accurate determination of the numerical density of functional eccrine glands in irradiated skin which can be compared to non-irradiated skin. A description of this inexpensive, noninvasive, and quantitative technique is presented as well as preliminary results determined in six normal subjects and 28 irradiated patients. Eleven patients, with atrophy and telangiectasia after radiotherapy to the skin to a high dose, were found to have no functional eccrine glands by this technique. A range of results from normal numbers of eccrine glands through partial and, rarely, complete loss was observed in patients given lower doses and in whom the skin was visually normal. When the irradiated side outside the boost area in 16 breast cancer patients who received postoperative radiotherapy was compared to an equivalent area on the untreated, contralateral side, 11 showed a greater than 50% reduction in the density of functional eccrine glands. The method appears to be a sensitive, quantitative assay for a permanent change in skin and so ought to facilitate meaningful comparison of different regimens of radiotherapy. Further studies are required to determine the dose-response relationship, latency and progression of the observed changes.
Introduction Studies of the late changes in skin following therapeutic irradiation have included observations of fibrosis, pigmentation, atrophy, and telangiectasia. Telangiectasia, in particular has provided useful dose-response relationships when arbitrary scales have been applied to its severity [2,12]. Irradiated skin often feels drier than normal, and it is a common clinical observation that sweating is reduced in irradiated axillae. Despite this, no systematic study has been published on changes in sweat gland function in irradiated human skin. Eccrine sweat glands are unbranched, tubular, coiled excretory structures which develop by epithelial invagination during the fourth month of fetal life [2]. The glands lie in the dermis adjacent to the deep cutaneous
capillary plexus at a depth of 0.7 to 2 ram, except over the posterior trunk and the glabrous skin of the palms and soles where they lie as deep as 4 mm [4]. A single duct arises from each gland and penetrates the epidermis ending in a 5 to 10 ~tm aperture through which a watery secretion is expelled aiding thermoregulation [2,4,10]. This function is principally regulated by cholinergic innervation from the autonomic nervous system. Eccrine glands are among the most abundant skin appendages; their numerical density is genetically determined and varies between individuals and within individuals according to anatomical location. Average values per cm 2 are 64 on the trunk, 108 on the forearm, 181 on the forehead and 600 to 700 on the palms and soles [ 10]. Methods have been developed to measure eccrine function following thermal stress, direct nerve stimula-
Address for correspondence: Dr. W. J. Morris, British Columbia Cancer Agency, Vancouver Clinic, 600 West 10th Avenue, Vancouver, B.C. V5Z 4E6, Canada.
50 tion, and the local application of cholinergic stimulants [2]. These fall essentially into two approaches. The first attempts to quantitate physiological parameters such as the volume and rate of secretion by collecting sweat on pads, measuring changes in the electrical resistance of skin, or recording rates of evaporation over known areas of skin [2,6]. The second approach determines the numerical density of individual sweat droplets using special indicator solutions or imprints produced in silicone elastomer moulds [7,9]. This paper concerns the latter. Silicone elastomer moulds are considered to provide the most sensitive method of determining the numerical density of actively secreting eccrine glands [ 9]. With this technique, a low viscosity suspension of a silicone monomer, containing the required catalyst, is applied to the skin and allowed to harden producing a high resolution, permanent negative replica of the skin surface. During the hardening process, the hydrophobic silicone suspension withdraws from any sweat droplets emerging from the eccrine pores causing the formation of characteristic imprints in the surface of the cured silicone polymer. These imprints can easily be identified and counted using low power microscopy. The method has been successfully applied to the study of autonomic neuropathy in diabetic patients and has been shown to have excellent repeatability [ 5,7 ]. Such imprints do not occur in silicone moulds of unstimulated skin. Moreover, the number of imprints visible in silicone moulds taken from the digit pads of mice following stimulation with subcutaneous pilocarpine has been shown to correspond to the number of eccrine glands seen in serial sections of histologic preparations of the digit pads [8]. This paper reports the adaptation of this technique to determine the numerical density of functional eccrine glands in human skin following therapeutic irradiation and presents the results of a pilot study. Material and methods
Eccrine gland stimulation The method of pilocarpine iontophoresis used to stimulate the eccrine glands was adapted from the method of Gibson and Cooke [3,11]. The skin in the area of interest was cleansed with isopropyl alcohol and dried with gauze. A 3 × 5 cm sheet of blotting paper was soaked in 4 % w/v pilocarpine nitrate (Schering-Plough ophthalmic solution containing 0.20% benzalkonium preservative in normal saline) and applied to the test area of skin. Two 2 x 3 cm gauze pads soaked in sterile normal saline served as iontophoresis electrodes. One was placed over the blotting paper and the other on a
separate area of nearby skin. The electrodes were then connected to a constant current D.C. apparatus such that the positive electrode was over the pilocarpine. A current of 0.04 mA per cm 2 was applied for 5 min. In an initial testing and development phase, pilocarpine iontophoresis was performed on 19 subjects; following this the technique was performed on the 28 irradiated patients and the six non-irradiated subjects reported here. A mild tingling or itching sensation was reported by most subjects, however, none of the 53 consecutive subjects found it more than mildly uncomfortable and in all cases the procedure was completed without interruption. The sensation ends within a few minutes of interrupting the current. A slight to moderate local erythema, lasting for 1 to 2 h, was common following pilocarpine iontophoresis. The apparatus used to provide the constant current was designed, built and safety tested by one of the authors (G.M.). It is powered by a 9 V dry cell battery, measures 16.5 x 8.0 x 4.0 cm, and cost £250 to construct.
Silicone elastomer mould preparation A diluted silicone elastomer was prepared by mixing 5 parts of silicone monomer suspension (MED-6382, McGhan Nusil Corp., 1150 Mark Ave., Carpenteria, Ca., 903013) with 4 parts (w/w) of the same manufacture's naphtha-based thinner. MED-6382 catalyst is added just prior to use with a syringe fitted with a 19gauge needle at the rate of one drop per gram of diluted suspension. Following the 5-min period of iontophoresis, the catalyst was thoroughly mixed, the skin dried, and the suspension applied to the test skin as a thin layer (0.1-0.5 mm)with a wooden spatula. After 10 min or less the polymer had hardened sufficiently to be removed, usually as an intact sheet of translucent white material. A further 24 h is required for complete curing, after which it forms a robust, non-adhering material with excellent geometric stability. During the hardening process there is a noticeable odour of organic solvents, principally naphtha, and therefore the procedure is best performed in a well ventilated room.
Estimation of the density of eccrine pore imprints During the hardening process, the hydrophobic silicone suspension withdraws from the droplets of sweat secreted by functional eccrine glands forming hemispherical imprints and often minute holes. The largest ones, about 0.2 mm in diameter, can be seen with the naked eye, but more can be identified with a low power mi-
51 croscope. The fully cured silicone mould was trimmed and placed in a standard glass-sided 35 mm transparency mount permitting microscopic examination and compact, dust-free storage. Pilocarpine iontophoresis was applied to 15 c m 2 in each test region of skin and the central 8 c m 2 of this stimulated region was used in estimating the numerical density of eccrine pore imprints using direct counting under low power microscopy. Except in four cases (patient nos. 2, 5, 8 and 10) the numerical density was calculated from the mean of 35 low power fields (11.9 mm 2 per LPF). Using this counting method the entire 8 cm 2 was sampled and more than 50~o of the surface area was counted. It is important to count a large area since the distribution of eccrine pores is not uniform over small (mm) scales. Gases escaping from solution in the silicone suspension during the hardening process can leave minute bubbles in the silicone elastomer mould, particularly if the material is applied in a thick layer. These can be readily distinguished from eccrine pore imprints since the latter are always open to the skin surface side of the silicone mould while the gas bubbles never are. These bubbles can largely be prevented by de-gassing
the diluted silicone monomer suspension in a vacuum for 1 h and then allowing it to stand for 24 h prior to use.
Patients and treatment techniques
The first objective was to see if there were a reduced number of functional eccrine glands in irradiated skin. Consent was obtained from 12 patients who received radiotherapy to a variety of anatomic sites with a range of doses, schedules, photon/electron energies and times since completion of treatment (Table I). The second objective was to determine if a quantitative reduction in the density of functional eccrine glands could be demonstrated in irradiated skin in which little or no visual change was apparent. Chosen were patients with unilateral breast cancer treated either by mastectomy and chest wall irradiation (8 patients) or lumpectomy followed by breast irradiation (8 patients). In both cases the contralateral side was used for comparison. All patients had been treated using a tangent pair technique with megavoltage equipment and bolus material (equivalent to 5 mm of tissue) coveting at least a 10 cm circle over the original tumour
TABLE I Numerical density of eccrine pore imprints in silicone elastomer m o u l d s taken of skin following pilocarpine iontophoresis in 12 patients. Patient no.
1 2 3 4 5 6 7 8 9 10 ll 12
Diagnoses
H & N SC Sem. BCC H & N SC BCC NHL H & N SC BCC Sem. Sere. Thyr. NHL Endo.
Tumour dose/fractions
66 Gy/33 35 Gy/20 35 Gy/5 40.5 Gy/27 b 35 G y / 5 30 G y / 1 2 63 G y / 3 0 60 G y / 3 0 45 Gy/25 36 G y / 1 2 64 G y / 3 2 40 G y / 2 0 50.6 Gy/22 c
Energy
5 MV 8 MV 100 kVp 8 MV 250 kVp 8 MV 5 MV 6 MeV ~ 8 MV 250 kVp Cobalt 8 MV 8 MV
Site of skin examined
Neck Abdomen Forehead Neck Nose Neck Neck Pinna Abdomen Groin Neck Neck Pelvis
Time since XRT
1 mth 4 mths 10 m t h s 1 yr 2 yrs 1 m t h 5 yrs 7 yrs 8 yrs 9 yrs I m t h 9 yrs 1 m t h 11 yrs 11 yrs 7 m t h s 12 yrs 11 m t h s
Bolus
No No No No No No N/K Yes No No Yes No No
Obvious skin changes
Yes No Yes No Yes No Yes No No Yes Yes No No
Eccrine pore imprints ( n u m b e r cm 2) Untreated area ~
Treated area
80.4 100 49.5 41.9 5.98 40.2 23.0 17.9 39.5 66.3 25.6
< 1 73.0 < 1 70.3 < 1 8.61 < 1 < 1 10.5 < 1 < 1 56.9 35.2
For patients w h o h a d unilateral treatment, an equivalent area on the contrateral side was tested for comparison. b Three fractions per day ( C H A R T ) : This patient h a d a s q u a m o u s cell c a r c i n o m a of the mobile tongue treated with combined external b e a m to the primary t u m o u r and uninvolved neck n o d e s followed by interstitial brachytherapy to the turnout. c Four-field plan. The values given were calculated from the m e a n s o f 35 low power microscope fields (11.9 m m 2 per L P F ) which sampled the central 8 cm 2 o f the original 15 c m : silicone m o u l d except in the cases of patients 2, 5, 8 and 10 where a smaller area was s a m p l e d a n d fewer L P F counted. H & N SC = h e a d and neck s q u a m o u s cell carcinoma; Sem. = s e m i n o m a ; B C C = b a s a l cell carcinoma; N H L = n o n - h o d g k i n ' s l y m p h o m a ; Thyr. = thyroid carcinoma; Endo. = endometrial adenocarcinoma).
52
Fig. 1. Photomicrographs of silicone elastomer moulds of human skin. (a) Volar forearm of normal subject prior to pilocarpine iontophoresis. (b) Same subject immediately following pilocarpine iontophoresis. (c) Forehead 10 months post-radiotherapy for basal cell carcinoma (35 Gy in 5 daily fractions, field edge runs vertically through the middle of the photomicrograph). (d) Breast patient no. 24, non-irradiated UIQ right breast. (e) Breast patient no. 24, irradiated UIQ left breast. (f) Breast patient no. 24, boost region of left breast. Note: the scale bars in all panels equal 1 ram.
site. Fourteen of these patients received a minimum tumour dose of 40 Gy in 15 fractions treating once daily Monday to Friday, while the remaining two patients received 50 Gy in 25 fractions. Five patients received boost doses to the tumour bed using electrons; in these
cases test areas were selected both within and outside the boost region. The average time between completion of treatment and testing was 66 months (range 2 to 204 months). Two patients had adjuvant C M F chemotherapy.
53 The actual dose of radiation received at the depth of the eccrine glands in these breast cancer patients could not be determined accurately. It is estimated that the dose to the eccrine glands ranged between 28 and 40 Gy in the non-boost regions of the 14 patients who received 15 treatments over 3 weeks, 35-50 Gy in the two patients who received 25 treatments over 5 weeks, and up to 50 Gy within the boost area of the five patients who received a 10 Gy electron boost following the standard 15 fraction course. Patients treated by these techniques usually showed acute erythema and some dry desquamation; rarely did moist desquamation occur. In follow up, telangiectasia and atrophy are normally observed in boost areas, but little if any visible change was present elsewhere on the skin. In order to provide an estimate of the variation in the density to be encountered when comparing one side of the chest with the other, silicone elastomer moulds of skin from both breasts were made of six non-irradiated subjects following pilocarpine iontophoresis. Results
Photomicrographs (Fig. la,b) show silicone elastomer moulds taken from the same region of volar forearm skin from a normal subject before and after 5 min of pilocarpine iontophoresis. It can be seen that no eccrine pore imprints are present in the mould taken from the unstimulated skin but many are visible after cholinergic stimulation. A photomicrograph of a silicone elastomer mould taken following stimulation with pilocarpine in a patient successfully treated for a basal cell carcinoma on the forehead with a 100 kVp X-ray apparatus (35 Gy in 5 daily fractions, patient 3 Table I) is shown in Fig. lc.
The heavily irradiated skin on the left side of the photomicrograph has no eccrine pore imprints while that outside the treatment field shows imprints. Silicone moulds taken from the skin of one of the breast cancer patients (no. 24, Table III) 20 months after radiotherapy are shown in photomicrographs (Fig. ld,e,f). A test area from the upper inner quadrant of the untreated right breast is shown in Fig. ld and contrasts with an equivalent test area from the upper inner quadrant of the treated left breast (Fig. le). An area of the upper outer quadrant of the left breast which was in the boost region and has marked telangiectasia, is shown in (Fig. lf). Compared to the untreated breast it is apparent that there is a reduction in the number of eccrine pore imprints visible following pilocarpine stimulation in the treated breast and none are visible within the boost region. Table I gives the results of 13 irradiated sites on the initial 12 patients tested with the technique. No eccrine pore imprints could be observed in six sites where there were marked post-radiation changes and where the skin had received a dose of radiation similar to that of the tumour, either because of the energy of the irradiation or because of the use of bolus material. One other site (patient 8) had also received a high dose in the skin, but had a normal appearance and no eccrine pore imprints were detected. The remaining six had normal appearing skin; only one showed a reduction in eccrine pore imprints of more than 50~o. The estimated dose in these areas of skin at the depth of the eccrine glands ranged from 20 to 35 Gy given in 12 to 27 fractions. Such doses and doses per fraction are likely to give a low index of permanent radiation change. The results from testing six normal subjects to evaluate the range in right-left asymmetry in the density of
TABLE II Numerical density of eccrine pore imprints in silicone elastomer moulds of skin in six normal subjects (values given were calculated from the means of 35 LPF). Subject no.
Eccrine pore imprints (number per cm 2) Upper inner quadrant left breast
Upper inner quadrant right breast
Mean
% Difference*
13 14 15 16 17 18
34.9 37.8 91.0 62.4 67.9 37.8
46.1 22.0 92.1 46.4 54.3 33.7
40.5 29.9 91.6 54.4 61.1 35.8
27.6 52.8 1.20 29.4 22.3 11.5
Mean
55.3
49.1
52.2
24.1 (S.D. 17.6)
*Percent difference is defined as the larger - the smaller/mean x 100.
54 TABLE
III
N u m e r i c a l d e n s i t y o f e c c r i n e p o r e i m p r i n t s in s i l i c o n e e l a s t o m e r m o u l d s t a k e n o f s k i n in 16 p a t i e n t s w h o r e c e i v e d p o s t o p e r a t i v e r a d i o t h e r a p y f o r c a r c i n o m a o f t h e b r e a s t . S u r g i c a l t r e a t m e n t w a s e i t h e r b y m a s t e c t o m y ( M ) o r l u m p e c t o m y (L). Patient
Months
Surgical
Tumour
no.
since XRT
treatment
dose/ fractions
E c c r i n e p o r e i m p r i n t s ( n u m b e r p e r c m 2) Untreateda
Treateda
side
side
~o C h a n g ~
area
19 u
2
M
50 G y / 2 5
4
M
40 Gy/15
21 b
5
L
40 Gy/15 + B
22
10
L
50 G y / 2 5
27.5
30.9
+ 11.9
-
23
13
L
40 Gy/15
43.3
31.3
- 27.7
-
24
20
L
40 Gy/15 + B
67.0
10.0
- 85.1
< 1
- 100
25
47
L
40 Gy/15 + B
19.6
< 1
- 100
26
48
L
40 Gy/15 + B
78.7
1.90
- 97.6
< 1
- 100
27
60
M
40 Gy/15
23.2
1.91
- 88.7
-
-
28
92
M
40 Gy/15
7.89
+ 24.2
-
-
29
93
L
40 Gy/15
96.4
- 88.6
-
-
30
98
M
40 Gy/15
11.7
31
105
L
40 Gy/15 + B
30.4
32
127
M
40 Gy/15
21.0
33
135
M
40 Gy/15
34
204
M
40 Gy/15
a p< 0.02. b Received aduvant CMF
< 1 34.7
5.98
5.50 38.3 34.0
5.98
Boost
20
Mean
40.5
~o C h a n g e
- 85.2
-
< 1
Nil
-
< 1
- 100
< 1
11.0 < 1 12.0 9.57 < 1 33.5 9.75
- 100
- 100 - 60.5 - 54.4 - 100 - 13.1
< 1
< 1 -
- 100 -
- 100 -
-
-
-
-
- 7 1 . 3 ~o
chemotherapy.
eccrine pores are given in Table II. On the basis of this information, it was felt that a real lowering in the number of eccrine glands would be accepted when there was a reduction in the density of eccrine pore imprints of 50~o or more when comparison was made between the irradiated and the non-irradiated side in the breast cancer patients. The results from 16 breast cancer patients are presented in Table III. Eleven of the patients had a reduction in eccrine pore density that exceeded 50~o in the treated breast compared to the contralateral side. Of the remaining five patients, two had a reduction of less than 5 0 ~ , two had higher counts in the treated side and one had no visible eccrine pore imprints in silicone elastomer moulds taken from the treated or the untreated breast. Five patients had boost doses to a region of the breast in which obvious radiation changes were present, and no eccfine pore imprints were visible in silicone moulds taken of skin from these areas. Discussion The paper reports the use of an inexpensive, noninvasive and quantitative technique to determine the density of functional eccrine glands in human skin as a mani-
festation of changes after therapeutic irradiation. The number of eccrine pore imprints present in silicone elastomer moulds following pilocarpine iontophoresis in skin showing changes typical of late radiation change is reduced, usually to zero. In a group of 16 breast cancer patients with irradiated skin that appeared visually normal, 11 showed a reduction in eccrine pore imprints of more than 50~o when compared with the contralateral non-irradiated side. This difference is greater than that found between the fight and left breasts in six normal subjects. There was no obvious relationship between the type of surgical treatment or the time elapsed since radiotherapy and the results obtained. In one subject (no. 20, Table III) the technique failed to demonstrate the presence of eccrine pore imprints in a silicone elastomer mould from non-irradiated skin; the reason for this is not clear, but it is unlikely to represent an actual absence of eccrine glands. The observation of complete loss of functional eccrine glands in skin showing late changes due to radiotherapy can be contrasted with the normal number found in skin where the dose at 0.7-2 mm ranged from 20 to 35 Gy given at less than 1.5 Gy per fraction. A useful dose-response relationship is suggested by the data derived from the breast cancer patients treated to intermediate dose levels (between 28 and 50 Gy mostly
55 at 1.9-2.7 Gy per fraction) where, in 6 of 16 cases, there was a reduction, but not a complete loss, in the number of functional eccrine glands. The pilot study suggests that the technique should provide useful quantitative data on normal tissue effects following radiotherapy. A precise comparison may then be possible of permanent changes in human skin after use of altered fractionation regimens such as CHART with that after conventional daily treatment. In collaboration with colleagues at the Gray Laboratory the method is being employed in fractionation studies in the mouse foot. Clinical studies designed to determine the dose-response curve, latency and pro-
gression for the loss of eccrine glands in human skin are being conducted.
Acknowledgements The authors wish to thank Mr. D. Gavin, Mr. M. Nathan, and Mr. R. Sibley of the Mount Vernon Hospital Maxillo-Facial Laboratory for help and advice regarding silicone elastomers. W. J. M. held a fellowship of the British Columbia Cancer Agency during the performance of this work. S. D. is a life fellow of the Cancer Research Campaign. The support of these organizations is gratefully acknowledged.
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