Journal of Ciilaneoiix Piilholagy 1975: 2: 284-293
Topical 5-Fluorouracil Treatment of Superficial Basal Cell Epithelioma. A Light and Electron Microscopic Study STUVEN J. HODGE', G . RANDOLPH SCIIRODI', LAFAYEITE G. ROBERT G . FREEMAN^
' Department of Dermatology and Pathology, University of Louisville School of Medicine, Louisville, Kentucky; and " Departments of Dermatology and Pathology, The University of Texas Southwestern Medical School, Dallas, Texas, U. S. A. A 27-year-old patient developed superficial basal cell epitheliomas approximately 20 years after taking Fowler's solution. One of the lesions was successfully treated with topical 2 % 5-fluorouracil solution under occlusion. Sequential biopsies of the lesion before, during and after therapy were examined by light and electron microscopy, and the changes at various stages are described. Changes occurred only in tumor cells and adjacent epidermis, and only after occlusion of 5-FU. After one week of occlusive therapy, focal discontinuities in the basal lamina and intercellular spaces were wider with reduction and condensation of tonofilaments. Mitochondrial degeneration was seen along with irregularities in nucleoli. These changes were most prominent after two weeks of occlusive therapy, and many degenerating keratinocytes were seen detached from other cells. One month after cessation of therapy, the entire area was excised, and no evidence of tumor was seen. (Received for pubticalion August 31, 1975)
It has long been recognized that inorganic arsenic ingcstion may induce skin cancer, either squamous cell or basal cell in type. The basal cell epitheliomas so induced are most often multiple superlicial lesions and appear predominately on the trunk (Neubaurer 1947). They often progress very slowly and may or may not be accompanied by the other pigmentary and keratotic alterations induced by arsenic (Sommers & McManus 1953). Histologieal features usually include an atrophic epidermis overlying multifocal small nests of basal cell tumor arising along the dermo-epidermal junction (Demis et al. 1974). In the past decade the clinical response of actinic keratosis and superficial skin can-
cers to topical 5-fluorouracil (5-FU) has been thoroughly documented and the efficacy of this mode ot therapy has been established for properly selected cases (Dillaha et al. 1963, Williams & Klein 1970, Simmonds 1972). Basal cell epitheliomas do not show a predictable response to topical 5-FU because of varying thickness of lesions. Some may disappear completely after superficial erosion, but there is a high incidence of recurrence. Superficial erythematous basal cell epitheliomas would be expected to respond more completely than nodular ones to such treatment (Jansen 1974). An ullrastructural study of a superficial basal eell epithelioma during therapy with
5-FLUOROURACIL AND BASAL CELL EPITHELIOMA
topical 5-FU demonstrates an cffccl on the tumor. The purpose of this paper is to present observations regarding the light and electron microscopic findings of a superficial basal cell epitheliomu dining and after therapy with topical 5-FU, and lo compare these findings with published ultrastructural studies of basal cell epithelioma (Zelickson 1962, Ishibashi et al. 1971, Reidbord et al. 1971). Material and Methods
PalienI A 27-year-old dermatology resident (one of the authors, S. J. H.) had two pink slightly elevated plaques on the right upper abdominal quadrant for three years. They enlarged slowly and were asytnptoinatic except on rare oeeasions wben erosion was accompanied by burning and pruritus. Examination revealed two erythematous macular lesions, 1.0 and 0.5 cm in diameter respectively, located in the anterior axillary line of the right upper abdominal quadrant (Fig. 1), with a distinct thread-like, serpiginous, pearly border. Epidermal atrophy
n Fig. /. Superficial basal cell epithelioma. Slightly clevatCLl erythematous plaque on abdomen, 1 cm in diameter.
285
with fine scaling was noted. The smallest lesion was excised and bistologic examination revealed changes characteristic of a superficial basal cell epithelioma. Tbe patient had been treated for childhood asthma, receiving an unknown quantity of Fowler's solution over a one year period at age seven. No other lesions were noted. No rib or jaw lesions were foutid on roentgenograms and tbere were no palmar pits to suggest basal cell nevus syndrome. All laboratory tests were within normal limits, including a quantitative determination for arsenie on a 24 h urine specimen. Two years later, tbe patient developed several small hyperkeratotie papules over the plantar surfaces of both feet. The lesions varied from 2 to 5 mm in diameter and were consistent with arsenical keratoses. Treatment After an initial 2 mm punch biopsy specimen confirmed tbe diagnosis of superfieial basal cell epithelioma on the remaining large lesion, it was treated with 2 % 5-FU solution (Efudex®), applied thinly twiee daily. Wben no clinical or histologic response was observed after two weeks of such treatment, the solution was applied under ocelusion with Saran Wrap and bandage thereafter for four additional weeks. Biopsies
Sequential biopsies were taken from different areas of the same lesion at intervals of two weeks, tbree weeks and four weeks of occkisive therapy. Treatment was stopped after four weeks of oeelusive therapy. The area was completely excised one month after cessation of therapy and serial sections were made of the skin ellipse. Control biopsy specimens were taken from adjacent abdominal skin at the same time intervals and examined with both light and electron microscopy.
286
HODGE ET AL.
Light Microscopy Each biopsy specimen was divided in two, with one-half fixed in 10 % formalin, embedded in paraffin and stained with hematoxylin and eosin for light microscopy. Electron Microscopy The other part was fixed in phosphatebuffered 3 % glutaraldehyde for electron microscopy. After post-fixation in 1.33% osmium tetroxide solution buffered with scollidine, the tissue was dehydrated in graded alcohols and embedded in Maraglas. Thick 1 me sections were stained with toluidine blue. Ultrathin sections were cut with a Porter-Blum ultramierotone, triplestained with Reynold's lead citrate and saturated uranly acetate (Reynolds 1963) and examined with a Siemen's Elmiskop 1.
Results
Clijiical No changes were observed in the lesion
during two weeks of topical therapy with 5-FU without occlusion. Initiation of occlusion resulted in gradually increasing erythema with pruritus until uleeration occurred after two weeks of occlusive therapy. This was followed by gradual disappearance of the uleeration with resolution of the lesion over a three week period. One month after cessation of therapy, at the time of exeision, there was epidermal atrophy with mild hyperpigmentation and foeal scarring from the punch biopsy sites. Light Microscopy Before treatment, hematoxylin-eosin stained sections revealed small nests of basal tumor cells arising along the dermo-epidermal junetion with some nests in the superficial dermis. There was no dermal elastosis or actinic degeneration of collagen. There was prominent fibroblastic proliferation of the stroma surrounding the tumor masses as described by Milestone & Helwig (1973) (Fig. 2).
Fig. 2. Superficial basal cell cpitlielioina. Small nests of basal ttjmor cells arising along dermoepidermal junction with small nest in dermis. H & E, X 100.
5-FLUOROURACIL AND BASAL CELL EPITHELIOMA All changes descrihed during therapy were limited to the tumor cells and adjacent keratinocytes. Significant changes were not evident on biopsy until one week after occlusion was initiated (three weeks therapy). Changes seen at this time included widened intercellular spaces, most marked a t the basal layer, and focal acantholysis. Mitotic figures were slightly increased and greater nuclear hyperchromatism and plcomorphism were noted in the basal layer, appearing limited to tumor cells. Nucleoli were more prominent and a mild perivascular infiltration of lymphocytes was noted in the upper dermis. The stroma appeared unchanged. After two weeks under occlusion (four •vveeks therapy), the greatest changes were noted, predominantly in the basal tumor cells and stroma of the upper dermis. Cellular atypia was evident in the tumor cells of the basal layer and upper dermis. There was greater nuclear pleomorphism and hyperchromatism, with increased mitotic fi-
287
gures, some bizarre, and very enlarged nucleoli. Death of tumor cells, as manifest by nuclear pyknosis and karyorrhcxis, was a prominent feature, ln addition, acantholysis was increased with vesicle formation in some areas of the tumor. An intense infiltration of histiocytes and lymphocytes in the dermis was most marked at the dermo-epidermal junction and around the clumps of tumor cells with migration of inflammatory cells into the epidermis. The inflammatory cell infiltration was limited to the papillary and upper rcticular dermis. Marked fibroblastic proliferation with many plump immature fibroblasts was concentrated in the papillary dermis near the dermo-epidermal junction and around nests of tumor cells. There was marked vascular dilatation with extravasation of crythrocytes in the upper dermis with some epidermal transmigration. One month after cessation of therapy the excised skin showed mild hyperkeratosis with a thin epidermis showing no atypia.
Fig. 3. Lesion one month after therapy. A thin epidermis with scar formation in the dermis is seen without evidence of tumor. H & E, X 100.
288
HODGE ET AL.
There was no residual tumor in cither the epidermis or dermis. Many plump fibroblasts were seen in the mid-dermis with areas of immature collagen, and many erythrocytcs consistent with the stage of wound healing at the time of biopsy. Mild vascular dilatation was noted in the upper dermis with a mild perivascular infiltrate of lymphocytes and histiocytes (Fig. 3). Electron Microscopy Before therapy the tumor cells exhibited characteristics previously described (Zelick-
son 1962, Ishibashi et al. 1971, Reidbord et al. 1971) (Fig. 4). The cells were similar in size and shape with round granular nuclei containing single small dark nucleoli, and sharply limited by a double membrane. Some peripheral aggregation of chromatin was seen. The cytoplasm was granular with sparse, short, fine tonofilaments, sometimes arranged around the nucleus. Melanin granules were prominent and mitochondria were few in number with normal appearance. Endoplasmic reticulum was sparse and golgi structures were normal. Several
Fig. 4. Superficial basal cell epithelioma. Epidermal keratinocytes are similar in size and shape with sparse granular cytoplasm (C). Basal lamina (bottom, arrows) is intact. (N) represents nucleus. (1) represents Intercellular space. X 5,000.
5-FLUOROURAClL AND BASAL CELL EPITHELIOMA electron dense bodies were present as noted b y Zelickson (1962) and interpreted to presutnably be lysosomes. Intercellular spaces were slightly widened focally with occasional convolution and folding. Desmosomes were sparse but normal in configuration with normal tonofilament attachment. An occasional cell as described by Reidbord et al. (1971) was seen. This contained dark granular cytoplastn and an irregular nucleus with irregular cell margins. There were many desmosomes and tonofibrils. The tumor was separated in all areas from t h e dermis by a definite, unbroken basal lamina that had an utiusual smudged, thickened appearance (Fig. 5). Hemi-desmosomes o n the cell side of the basal lamina appeared slightly increased in number. Most appeared
289
normal, but there was occasional focal stnudging. The dermis showed numerous collagen bundles of normal appearance and fibroblasts with a rare lymphocyte or histiocyte. The sequential ultrastructural changes paralleled those seen by light microscopy, the changes occurring only in tumor cells and adjacent epidermis. The changes noted after one week under occlusion were similar and included for the first time changes in the basal lamina. Focal discotitinuities were seen occasionally and the intact basal lamina no longer had a smudged appearance (Fig. 6). The hemi-desmosomes and anchoring tonofilaments appeared normal. Intercellular spaces were wider with accentuation of villous-like projections of the plasma membrane. Tonofilament abnormalities included
^. 5. Before therapy. Basal lamina (arrows), surrounding tumor nest in dermis is thickened with no discontinuity. (D) represents dermis, (C) represents cytoplasm of tumor cells. (H) represents hemidcsmosomes. X 45,000.
290
HODGE ET AL.
Fig. 6. Three weeks therapy. Focal gap (on right (G) in otherwise normal basal lamina (arrows). (C) represents collagen, (P) represents melanin pigment. X 36,000. reduction in number, and grouping into short thick fascicles with either a perinuclear or random arrangement. The desmosomes and their attachments appeared normal. Cytoplasmic vascuoles appeared which were interpreted as probable degenerated mitochondria or possibly lipid droplets of low electron density (Lapis & Bendeczky 1966). Mitochondrial degeneration was seen focally as evidenced by detachment, vesiculation and fragmentation of christae (Fig. 7). Nucleoli were larger and more electrondense and irregtilar. After two weeks under occlusion (he basal lamina defects persisted, but were similar to those whicli appeared after one week of occlusion. Intercellular spaces were extremely wide both in areas of tumor and higher in the epidermis. Many degenerating keratinocytcs were seen detached from other
cells (Fig. 8). Desmosomal alterations were marked, characterized by homogenization, decrease in number, and some were retracted to one plasma membrane, unattached to the other. Tonolilaments were condensed and sparse and plasma membranes became focally indistinct. Cytoplasmic organelle degeneration was widespread with severe mitochondrial damage. Nucleoli became larger, more electron-dense, and bizarre configurations were prominent. Dermal collagen and elastin were unaltered throughout therapy. One month after cessation of therapy, only slight widening of the intercellular spaces and mild tonofilament condensation was seen. The dermis showed heavy proliferation of fibroblasis wi(h many erythrocytes. A few lymphocytes and histiocytes were scattered throughout. A great amount
5-FLUOROURACIL AND BASAL CELL EPITHELTOMA
291
M
\f/
>^
Fig. 7. Three weeks llicrapy. Degenerated mitochondria (M) demonstrate iletaclimeiit and fragmentation of christae (C). X 50,000.
of immature collagen was seen along with some areas of collagen degeneration and vascular proliferation. These findings were similar to that seen in early sear formation. A control biopsy of clinically normal skin near the lesion, treated with 5-FU, showed only a mild infiltration of lymphocytes and histiocytes perivascularly in the upper dermis with no epidermal change. These changes were similar throughout therapy. Discussion
The patient was not aware of the fact that he had received the arsenic-containing Fowler's sokition as a child until the initial diagnosis of superficial basal cell epithelioma resulted in a careful search for the etiology of the lesions. The plantar keratoses two years later helped confirm an arsenical
etiology. Careful physical and x-ray examination ruled out the basal cell nevus syndrome. The lack of actinic degeneration of collagen and elastin, and the non-sunexposed abdominal location tended to preclude an actinic etiology. Eveti though in general, the response of basal cell epitheliotna to 5-FU is unpredictable, a good response was expected because of the superficial nature of the lesion. Tt was interesting to note that tbe lesion did not respond significantly clinically, histologically or even ultrastrueturally to topical 2 % 5-FU until occlusion was initiated. Whether this response was a result of increased peneti'alion of 5-FU, or a combination of penetration and primary irritation is not clear. ]n studies by Sloll and Klein (Klein et al. 1965a,b, Klein ct al. 1966, Stoll et al. 1967) local administration of 5-FU
292
HODGE ET AL.
Fig. 8. Four weeks therapy. Degenerating keratinocyte (k) in widened intercellular space (I). X 10,000.
had marked effects upon the natural course of single and multiple basal cell epitheliomas. They did not find a statistically significant difference in the effects of several concentrations of 5-FU cream upon tumor regression (Klein et al. 1965a). However, they demonstrated that the incidence of tumor regression was significantly higher with occlusion than with non-occlusion of the sites of application of 5-FU cream (Stoll & Klein 1969). The fibroblastic activity and early scar formation noted in the excised skin one month after cessation of therapy were probably due to the result of the multiple punch biopsies which were taken from the original 1 cm lesion. Collagen was not affected in a study of the effects of 5-FU on actinic keratoses (Hodge et al. 1974). Since these areas of scar could have masked occult
tumor cells, the entire area of the original lesion was excised and serially sectioned to rule out this possibility. The question of how much the punch biopsies with resulting scar contributed to resolution of the tumor remains unanswered. The ultrastructural findings parallel those observed at the light microscopic level and are similar to the findings seen with topical 5-FU therapy of actinic keratoses (Hodge et al. 1974). Change in tumor cells were not significant until three weeks of therapy, and were most marked at four weeks therapy (two weeks of oeclusion). Because of lack of tissue, another biopsy was not performed until the entire lesion was excised one month after cessation of therapy. Surprisingly, little is known about the mechanism of action of 5-FU in human carcinoma, but many agree that the specific
5-FLUOROURAClL AND BASAL CELL EPITHELIOMA
inhibition of DNA synthesis is the most probable mechanism lor the growth-inhibiting effect of 5-FU. Cutaneous application selectively inhibits growth of abnormal tissue. These abnormal cells in our study appeared to undergo necrosis, being replaeed by normally regenerating keratinocytes. Dillaha et al. (L963) diseussed the possibility of an affinity of 5-FU for neoplastic cells because of their increased mitotic activity. Our purely morphologic description allows us only to speculate as to the reason for the speeificity, and further correlation of morphologic and biochemical findings appears necessary.
References Demis, J. J., Crounse, R. G., Dobson, R. L. & McGuire, J. (1974) Clinical Dcrinatology, Vol. 4, Unit 28:18, pp.6. Hagcrstown Md., NY & London: Harper & Row. Dillaha, C , Jansen, T., Honeycutt, M. & Bradford, C. (1963) Selective cytotoxic effect of topical 5-fluorouracil. Archives of Dermatology 88, 247. Hodge, S. J., Schrodt, G. R. & Owen, L. G. (1974) Topical 5-fliiorouracil treatment of actinic keratoses: A light and electron microscopic study. Journal of Cutaneous Pathologv I, 238-248. Ishibashi, A., Kasuga, T. & Tsuchlya, E. (1971) Electron microscopic study of hasal cell carcinoma. Journal of Investigative Derniatologv 56, 298. Jansen, T. G. (1974) Questions and answers. Journal of American Medical Association 288, 209. Klein, E., Stoll, H. L. Jr., Milgrom, H. Case, R. W., Traenkle, H. L., Graham, S., Laor, T. & Helm, F. (1965a) Tumors of the skin IV. Double blind study on effects of local administration of anti-tumor agents in hasal cell carcinoma. Journal of Investigative Dermatology 44, 351. Klein, E., Stoll, H.L. Jr., Milgrom, H., Tiaenkle, H. L., Case, R. W., Laor, T., Helm, F. & Nadel, R. W. (1965b) Tumors of the skin V. Local administralion of anti-tumor agents to multiple superficial basal cell carcinomas. Jonriuil of Investigative Dermatology 45, 489.
293
Klein, E., Stoll, H. L. Jr., Milgrom, H., Traenkle, H. L., Graham, S. & Helm, F. (1966) Tumours of the skin VL Study on effects of local administration of 5-fluorouracil in basal cell carcinoma. Journal of Investigative Dermatology 47, 22. Lapis, K. & Bendeczky, I. (1966) Antimetabolite - induced changes in the fine structure of tumor cells. Acta Biologica Hungaria 17, 199. Milestone, E. B. & Helwig, E. B. (1973) Basal cell carcinoma in children. Archives of Dermatology 108, 523. Neubaurer, O. (1947) Arsenical cancer: A review. British Journal of Cancer 192. 251. Reidbord, H. E., Wechsler, H.L. & Fisher, E. R. (1971) Uitrastructural study of basal cell carcinoma and its variants with comments on histogenesis. Archives of Dernuitology 104, 132. Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208. Simmonds, W. L. (1972) Topical management of actinic keratosis with 5-fluorouracil: Results of a 6-year follow-up study. Cutis 10, Sommers, S. C. & McManus, R. G. (1953) Multiple arsenical cancers of skin and internal organs. Cancer 6, 347. Stoll, H. L. Jr., Klein, E. & Case, R. W. (1967) Tumors of the skin VJJJ. Effects of varying the concentration of locally administered 5fluorouracil on basal cell carcinoma. Journal of Investigative Dermatology 49, 219. Stoll, H. L. Jr. & Klein, E. (1969) Tumors of the skin XL Effect of occlusion dressing on the local administration of 5-fIuorouraciI to superficial basal cell carcinoma. Journal of Investigative Dermatology 52, 9. Williams, A. & Klein, E. (1970) Experiences with local chemotherapy and immunotherapy in premalignant and malignant skin lesions. Cancer 25, 450. Zelickson, A. S. (1962) An electron microscope study of the basal cell epithelioma. Journal of Investigative Dermatology 39, 183. Address: Steven J. Hodge, M.D. Dcpt. of Dermatology Louisville General Hospital 323 East Chestnut Street Louisville, Kentucky, U.S.A.
Topical 5-Fluorouracil Treatment of Superficial Basal Cell Epithelioma. A Light and Electron Microscopic Study STUVEN J. HODGE', G . RANDOLPH SCIIRODI', LAFAYEITE G. ROBERT G . FREEMAN^
' Department of Dermatology and Pathology, University of Louisville School of Medicine, Louisville, Kentucky; and " Departments of Dermatology and Pathology, The University of Texas Southwestern Medical School, Dallas, Texas, U. S. A. A 27-year-old patient developed superficial basal cell epitheliomas approximately 20 years after taking Fowler's solution. One of the lesions was successfully treated with topical 2 % 5-fluorouracil solution under occlusion. Sequential biopsies of the lesion before, during and after therapy were examined by light and electron microscopy, and the changes at various stages are described. Changes occurred only in tumor cells and adjacent epidermis, and only after occlusion of 5-FU. After one week of occlusive therapy, focal discontinuities in the basal lamina and intercellular spaces were wider with reduction and condensation of tonofilaments. Mitochondrial degeneration was seen along with irregularities in nucleoli. These changes were most prominent after two weeks of occlusive therapy, and many degenerating keratinocytes were seen detached from other cells. One month after cessation of therapy, the entire area was excised, and no evidence of tumor was seen. (Received for pubticalion August 31, 1975)
It has long been recognized that inorganic arsenic ingcstion may induce skin cancer, either squamous cell or basal cell in type. The basal cell epitheliomas so induced are most often multiple superlicial lesions and appear predominately on the trunk (Neubaurer 1947). They often progress very slowly and may or may not be accompanied by the other pigmentary and keratotic alterations induced by arsenic (Sommers & McManus 1953). Histologieal features usually include an atrophic epidermis overlying multifocal small nests of basal cell tumor arising along the dermo-epidermal junction (Demis et al. 1974). In the past decade the clinical response of actinic keratosis and superficial skin can-
cers to topical 5-fluorouracil (5-FU) has been thoroughly documented and the efficacy of this mode ot therapy has been established for properly selected cases (Dillaha et al. 1963, Williams & Klein 1970, Simmonds 1972). Basal cell epitheliomas do not show a predictable response to topical 5-FU because of varying thickness of lesions. Some may disappear completely after superficial erosion, but there is a high incidence of recurrence. Superficial erythematous basal cell epitheliomas would be expected to respond more completely than nodular ones to such treatment (Jansen 1974). An ullrastructural study of a superficial basal eell epithelioma during therapy with
5-FLUOROURACIL AND BASAL CELL EPITHELIOMA
topical 5-FU demonstrates an cffccl on the tumor. The purpose of this paper is to present observations regarding the light and electron microscopic findings of a superficial basal cell epitheliomu dining and after therapy with topical 5-FU, and lo compare these findings with published ultrastructural studies of basal cell epithelioma (Zelickson 1962, Ishibashi et al. 1971, Reidbord et al. 1971). Material and Methods
PalienI A 27-year-old dermatology resident (one of the authors, S. J. H.) had two pink slightly elevated plaques on the right upper abdominal quadrant for three years. They enlarged slowly and were asytnptoinatic except on rare oeeasions wben erosion was accompanied by burning and pruritus. Examination revealed two erythematous macular lesions, 1.0 and 0.5 cm in diameter respectively, located in the anterior axillary line of the right upper abdominal quadrant (Fig. 1), with a distinct thread-like, serpiginous, pearly border. Epidermal atrophy
n Fig. /. Superficial basal cell epithelioma. Slightly clevatCLl erythematous plaque on abdomen, 1 cm in diameter.
285
with fine scaling was noted. The smallest lesion was excised and bistologic examination revealed changes characteristic of a superficial basal cell epithelioma. Tbe patient had been treated for childhood asthma, receiving an unknown quantity of Fowler's solution over a one year period at age seven. No other lesions were noted. No rib or jaw lesions were foutid on roentgenograms and tbere were no palmar pits to suggest basal cell nevus syndrome. All laboratory tests were within normal limits, including a quantitative determination for arsenie on a 24 h urine specimen. Two years later, tbe patient developed several small hyperkeratotie papules over the plantar surfaces of both feet. The lesions varied from 2 to 5 mm in diameter and were consistent with arsenical keratoses. Treatment After an initial 2 mm punch biopsy specimen confirmed tbe diagnosis of superfieial basal cell epithelioma on the remaining large lesion, it was treated with 2 % 5-FU solution (Efudex®), applied thinly twiee daily. Wben no clinical or histologic response was observed after two weeks of such treatment, the solution was applied under ocelusion with Saran Wrap and bandage thereafter for four additional weeks. Biopsies
Sequential biopsies were taken from different areas of the same lesion at intervals of two weeks, tbree weeks and four weeks of occkisive therapy. Treatment was stopped after four weeks of oeelusive therapy. The area was completely excised one month after cessation of therapy and serial sections were made of the skin ellipse. Control biopsy specimens were taken from adjacent abdominal skin at the same time intervals and examined with both light and electron microscopy.
286
HODGE ET AL.
Light Microscopy Each biopsy specimen was divided in two, with one-half fixed in 10 % formalin, embedded in paraffin and stained with hematoxylin and eosin for light microscopy. Electron Microscopy The other part was fixed in phosphatebuffered 3 % glutaraldehyde for electron microscopy. After post-fixation in 1.33% osmium tetroxide solution buffered with scollidine, the tissue was dehydrated in graded alcohols and embedded in Maraglas. Thick 1 me sections were stained with toluidine blue. Ultrathin sections were cut with a Porter-Blum ultramierotone, triplestained with Reynold's lead citrate and saturated uranly acetate (Reynolds 1963) and examined with a Siemen's Elmiskop 1.
Results
Clijiical No changes were observed in the lesion
during two weeks of topical therapy with 5-FU without occlusion. Initiation of occlusion resulted in gradually increasing erythema with pruritus until uleeration occurred after two weeks of occlusive therapy. This was followed by gradual disappearance of the uleeration with resolution of the lesion over a three week period. One month after cessation of therapy, at the time of exeision, there was epidermal atrophy with mild hyperpigmentation and foeal scarring from the punch biopsy sites. Light Microscopy Before treatment, hematoxylin-eosin stained sections revealed small nests of basal tumor cells arising along the dermo-epidermal junetion with some nests in the superficial dermis. There was no dermal elastosis or actinic degeneration of collagen. There was prominent fibroblastic proliferation of the stroma surrounding the tumor masses as described by Milestone & Helwig (1973) (Fig. 2).
Fig. 2. Superficial basal cell cpitlielioina. Small nests of basal ttjmor cells arising along dermoepidermal junction with small nest in dermis. H & E, X 100.
5-FLUOROURACIL AND BASAL CELL EPITHELIOMA All changes descrihed during therapy were limited to the tumor cells and adjacent keratinocytes. Significant changes were not evident on biopsy until one week after occlusion was initiated (three weeks therapy). Changes seen at this time included widened intercellular spaces, most marked a t the basal layer, and focal acantholysis. Mitotic figures were slightly increased and greater nuclear hyperchromatism and plcomorphism were noted in the basal layer, appearing limited to tumor cells. Nucleoli were more prominent and a mild perivascular infiltration of lymphocytes was noted in the upper dermis. The stroma appeared unchanged. After two weeks under occlusion (four •vveeks therapy), the greatest changes were noted, predominantly in the basal tumor cells and stroma of the upper dermis. Cellular atypia was evident in the tumor cells of the basal layer and upper dermis. There was greater nuclear pleomorphism and hyperchromatism, with increased mitotic fi-
287
gures, some bizarre, and very enlarged nucleoli. Death of tumor cells, as manifest by nuclear pyknosis and karyorrhcxis, was a prominent feature, ln addition, acantholysis was increased with vesicle formation in some areas of the tumor. An intense infiltration of histiocytes and lymphocytes in the dermis was most marked at the dermo-epidermal junction and around the clumps of tumor cells with migration of inflammatory cells into the epidermis. The inflammatory cell infiltration was limited to the papillary and upper rcticular dermis. Marked fibroblastic proliferation with many plump immature fibroblasts was concentrated in the papillary dermis near the dermo-epidermal junction and around nests of tumor cells. There was marked vascular dilatation with extravasation of crythrocytes in the upper dermis with some epidermal transmigration. One month after cessation of therapy the excised skin showed mild hyperkeratosis with a thin epidermis showing no atypia.
Fig. 3. Lesion one month after therapy. A thin epidermis with scar formation in the dermis is seen without evidence of tumor. H & E, X 100.
288
HODGE ET AL.
There was no residual tumor in cither the epidermis or dermis. Many plump fibroblasts were seen in the mid-dermis with areas of immature collagen, and many erythrocytcs consistent with the stage of wound healing at the time of biopsy. Mild vascular dilatation was noted in the upper dermis with a mild perivascular infiltrate of lymphocytes and histiocytes (Fig. 3). Electron Microscopy Before therapy the tumor cells exhibited characteristics previously described (Zelick-
son 1962, Ishibashi et al. 1971, Reidbord et al. 1971) (Fig. 4). The cells were similar in size and shape with round granular nuclei containing single small dark nucleoli, and sharply limited by a double membrane. Some peripheral aggregation of chromatin was seen. The cytoplasm was granular with sparse, short, fine tonofilaments, sometimes arranged around the nucleus. Melanin granules were prominent and mitochondria were few in number with normal appearance. Endoplasmic reticulum was sparse and golgi structures were normal. Several
Fig. 4. Superficial basal cell epithelioma. Epidermal keratinocytes are similar in size and shape with sparse granular cytoplasm (C). Basal lamina (bottom, arrows) is intact. (N) represents nucleus. (1) represents Intercellular space. X 5,000.
5-FLUOROURAClL AND BASAL CELL EPITHELIOMA electron dense bodies were present as noted b y Zelickson (1962) and interpreted to presutnably be lysosomes. Intercellular spaces were slightly widened focally with occasional convolution and folding. Desmosomes were sparse but normal in configuration with normal tonofilament attachment. An occasional cell as described by Reidbord et al. (1971) was seen. This contained dark granular cytoplastn and an irregular nucleus with irregular cell margins. There were many desmosomes and tonofibrils. The tumor was separated in all areas from t h e dermis by a definite, unbroken basal lamina that had an utiusual smudged, thickened appearance (Fig. 5). Hemi-desmosomes o n the cell side of the basal lamina appeared slightly increased in number. Most appeared
289
normal, but there was occasional focal stnudging. The dermis showed numerous collagen bundles of normal appearance and fibroblasts with a rare lymphocyte or histiocyte. The sequential ultrastructural changes paralleled those seen by light microscopy, the changes occurring only in tumor cells and adjacent epidermis. The changes noted after one week under occlusion were similar and included for the first time changes in the basal lamina. Focal discotitinuities were seen occasionally and the intact basal lamina no longer had a smudged appearance (Fig. 6). The hemi-desmosomes and anchoring tonofilaments appeared normal. Intercellular spaces were wider with accentuation of villous-like projections of the plasma membrane. Tonofilament abnormalities included
^. 5. Before therapy. Basal lamina (arrows), surrounding tumor nest in dermis is thickened with no discontinuity. (D) represents dermis, (C) represents cytoplasm of tumor cells. (H) represents hemidcsmosomes. X 45,000.
290
HODGE ET AL.
Fig. 6. Three weeks therapy. Focal gap (on right (G) in otherwise normal basal lamina (arrows). (C) represents collagen, (P) represents melanin pigment. X 36,000. reduction in number, and grouping into short thick fascicles with either a perinuclear or random arrangement. The desmosomes and their attachments appeared normal. Cytoplasmic vascuoles appeared which were interpreted as probable degenerated mitochondria or possibly lipid droplets of low electron density (Lapis & Bendeczky 1966). Mitochondrial degeneration was seen focally as evidenced by detachment, vesiculation and fragmentation of christae (Fig. 7). Nucleoli were larger and more electrondense and irregtilar. After two weeks under occlusion (he basal lamina defects persisted, but were similar to those whicli appeared after one week of occlusion. Intercellular spaces were extremely wide both in areas of tumor and higher in the epidermis. Many degenerating keratinocytcs were seen detached from other
cells (Fig. 8). Desmosomal alterations were marked, characterized by homogenization, decrease in number, and some were retracted to one plasma membrane, unattached to the other. Tonolilaments were condensed and sparse and plasma membranes became focally indistinct. Cytoplasmic organelle degeneration was widespread with severe mitochondrial damage. Nucleoli became larger, more electron-dense, and bizarre configurations were prominent. Dermal collagen and elastin were unaltered throughout therapy. One month after cessation of therapy, only slight widening of the intercellular spaces and mild tonofilament condensation was seen. The dermis showed heavy proliferation of fibroblasis wi(h many erythrocytes. A few lymphocytes and histiocytes were scattered throughout. A great amount
5-FLUOROURACIL AND BASAL CELL EPITHELTOMA
291
M
\f/
>^
Fig. 7. Three weeks llicrapy. Degenerated mitochondria (M) demonstrate iletaclimeiit and fragmentation of christae (C). X 50,000.
of immature collagen was seen along with some areas of collagen degeneration and vascular proliferation. These findings were similar to that seen in early sear formation. A control biopsy of clinically normal skin near the lesion, treated with 5-FU, showed only a mild infiltration of lymphocytes and histiocytes perivascularly in the upper dermis with no epidermal change. These changes were similar throughout therapy. Discussion
The patient was not aware of the fact that he had received the arsenic-containing Fowler's sokition as a child until the initial diagnosis of superficial basal cell epithelioma resulted in a careful search for the etiology of the lesions. The plantar keratoses two years later helped confirm an arsenical
etiology. Careful physical and x-ray examination ruled out the basal cell nevus syndrome. The lack of actinic degeneration of collagen and elastin, and the non-sunexposed abdominal location tended to preclude an actinic etiology. Eveti though in general, the response of basal cell epitheliotna to 5-FU is unpredictable, a good response was expected because of the superficial nature of the lesion. Tt was interesting to note that tbe lesion did not respond significantly clinically, histologically or even ultrastrueturally to topical 2 % 5-FU until occlusion was initiated. Whether this response was a result of increased peneti'alion of 5-FU, or a combination of penetration and primary irritation is not clear. ]n studies by Sloll and Klein (Klein et al. 1965a,b, Klein ct al. 1966, Stoll et al. 1967) local administration of 5-FU
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HODGE ET AL.
Fig. 8. Four weeks therapy. Degenerating keratinocyte (k) in widened intercellular space (I). X 10,000.
had marked effects upon the natural course of single and multiple basal cell epitheliomas. They did not find a statistically significant difference in the effects of several concentrations of 5-FU cream upon tumor regression (Klein et al. 1965a). However, they demonstrated that the incidence of tumor regression was significantly higher with occlusion than with non-occlusion of the sites of application of 5-FU cream (Stoll & Klein 1969). The fibroblastic activity and early scar formation noted in the excised skin one month after cessation of therapy were probably due to the result of the multiple punch biopsies which were taken from the original 1 cm lesion. Collagen was not affected in a study of the effects of 5-FU on actinic keratoses (Hodge et al. 1974). Since these areas of scar could have masked occult
tumor cells, the entire area of the original lesion was excised and serially sectioned to rule out this possibility. The question of how much the punch biopsies with resulting scar contributed to resolution of the tumor remains unanswered. The ultrastructural findings parallel those observed at the light microscopic level and are similar to the findings seen with topical 5-FU therapy of actinic keratoses (Hodge et al. 1974). Change in tumor cells were not significant until three weeks of therapy, and were most marked at four weeks therapy (two weeks of oeclusion). Because of lack of tissue, another biopsy was not performed until the entire lesion was excised one month after cessation of therapy. Surprisingly, little is known about the mechanism of action of 5-FU in human carcinoma, but many agree that the specific
5-FLUOROURAClL AND BASAL CELL EPITHELIOMA
inhibition of DNA synthesis is the most probable mechanism lor the growth-inhibiting effect of 5-FU. Cutaneous application selectively inhibits growth of abnormal tissue. These abnormal cells in our study appeared to undergo necrosis, being replaeed by normally regenerating keratinocytes. Dillaha et al. (L963) diseussed the possibility of an affinity of 5-FU for neoplastic cells because of their increased mitotic activity. Our purely morphologic description allows us only to speculate as to the reason for the speeificity, and further correlation of morphologic and biochemical findings appears necessary.
References Demis, J. J., Crounse, R. G., Dobson, R. L. & McGuire, J. (1974) Clinical Dcrinatology, Vol. 4, Unit 28:18, pp.6. Hagcrstown Md., NY & London: Harper & Row. Dillaha, C , Jansen, T., Honeycutt, M. & Bradford, C. (1963) Selective cytotoxic effect of topical 5-fluorouracil. Archives of Dermatology 88, 247. Hodge, S. J., Schrodt, G. R. & Owen, L. G. (1974) Topical 5-fliiorouracil treatment of actinic keratoses: A light and electron microscopic study. Journal of Cutaneous Pathologv I, 238-248. Ishibashi, A., Kasuga, T. & Tsuchlya, E. (1971) Electron microscopic study of hasal cell carcinoma. Journal of Investigative Derniatologv 56, 298. Jansen, T. G. (1974) Questions and answers. Journal of American Medical Association 288, 209. Klein, E., Stoll, H. L. Jr., Milgrom, H. Case, R. W., Traenkle, H. L., Graham, S., Laor, T. & Helm, F. (1965a) Tumors of the skin IV. Double blind study on effects of local administration of anti-tumor agents in hasal cell carcinoma. Journal of Investigative Dermatology 44, 351. Klein, E., Stoll, H.L. Jr., Milgrom, H., Tiaenkle, H. L., Case, R. W., Laor, T., Helm, F. & Nadel, R. W. (1965b) Tumors of the skin V. Local administralion of anti-tumor agents to multiple superficial basal cell carcinomas. Jonriuil of Investigative Dermatology 45, 489.
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Klein, E., Stoll, H. L. Jr., Milgrom, H., Traenkle, H. L., Graham, S. & Helm, F. (1966) Tumours of the skin VL Study on effects of local administration of 5-fluorouracil in basal cell carcinoma. Journal of Investigative Dermatology 47, 22. Lapis, K. & Bendeczky, I. (1966) Antimetabolite - induced changes in the fine structure of tumor cells. Acta Biologica Hungaria 17, 199. Milestone, E. B. & Helwig, E. B. (1973) Basal cell carcinoma in children. Archives of Dermatology 108, 523. Neubaurer, O. (1947) Arsenical cancer: A review. British Journal of Cancer 192. 251. Reidbord, H. E., Wechsler, H.L. & Fisher, E. R. (1971) Uitrastructural study of basal cell carcinoma and its variants with comments on histogenesis. Archives of Dernuitology 104, 132. Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208. Simmonds, W. L. (1972) Topical management of actinic keratosis with 5-fluorouracil: Results of a 6-year follow-up study. Cutis 10, Sommers, S. C. & McManus, R. G. (1953) Multiple arsenical cancers of skin and internal organs. Cancer 6, 347. Stoll, H. L. Jr., Klein, E. & Case, R. W. (1967) Tumors of the skin VJJJ. Effects of varying the concentration of locally administered 5fluorouracil on basal cell carcinoma. Journal of Investigative Dermatology 49, 219. Stoll, H. L. Jr. & Klein, E. (1969) Tumors of the skin XL Effect of occlusion dressing on the local administration of 5-fIuorouraciI to superficial basal cell carcinoma. Journal of Investigative Dermatology 52, 9. Williams, A. & Klein, E. (1970) Experiences with local chemotherapy and immunotherapy in premalignant and malignant skin lesions. Cancer 25, 450. Zelickson, A. S. (1962) An electron microscope study of the basal cell epithelioma. Journal of Investigative Dermatology 39, 183. Address: Steven J. Hodge, M.D. Dcpt. of Dermatology Louisville General Hospital 323 East Chestnut Street Louisville, Kentucky, U.S.A.
