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Use of a murine monoclonal antibody which binds to malignant keratinocytes to detect tumor cells in microscopically controlled surgery
Use of a murine monoclonal antibody which binds to malignant keratinocytes to detect tumor cells in microscopically controlled surgery Allan R. Oseroff, M.D., Ph.D., Robert Roth, M.D., Susan Lipman, M.D., and Vera B. Morhenn, M.D. Stanford, CA A new murine monoclonal antibody, termed VM-1, stains basal cell carcinoma ceils and at least some squamous cell carcinoma cells. This antibody was used to visualize the tumor margins during microscopically controlled surgery using immunofluorescence (IF) and immunoperoxidase (IP) technics. Our experiments demonstrate that VM-1 is helpful in detecting small islands of tumor cells in the tissue at the margins of skin cancers. Thus, tissue staining with this antibody should improve the accuracy of microscopically controlled excision, and hopefully will decrease the rate of tumor recurrence after surgery. (J AM ACADDERMATOL8:616-619, 1983.)
Mohs' chemosurgery (MCS) and the fresh tissue modification described in 1974 are used extensively to treat squamous cell carcinomas (SCCs) and certain types of basal cell carcinomas (BCCs) of the skin, which are characterized by strands and nests of tumor cells which are not visible on the skin surface, t-a Conventional forms of surgery and radiation often fail to eradicate these "silent" extensions of tumor, so that relatively high recurrence rates result. In MCS, the clinically visible tumor is removed, the next layer of tissue is excised, immediately frozen, cut on the cryostat, stained with hematoxylin and eosin, and checked forresidual tumor cells with the light microscope .4 MCS is the treatment of choice in recurrent From the Department of Dermatology and the Division of Immunology, Stanford University Medical Center. Accepted for publication Sept. 22, 1982. Reprint requests to: Dr. Vera B. Morhenn, Department of Dermatology, Stanford University Medical Center, Stanford, CA 94305.
616
BCCs and SCCs. With this form of therapy, the cure rate for recurrent BCCs is about 98% whereas the cure rate with other forms of therapy (e.g., radiation, conventional surgical excision) ranges from 50% to 85%. 4 For SCCs, MCS results in a 5-year cure rate of 94% in the group of Patients who have not received previous treatment. 5 However, in recurrent SCCs, the 5-year cure rate is only 76.3%.5 Size and location of the lesion also influence the 5-year cure rates. Thus, BCCs and SCCs of the nose which are 3 cm or greater in size have a 5-year cure rate of 84.3% and 56%, respectively. 5 BCCs and SCCs of the ear canal have an 88.9% and a 30% 5-year success rate, respectively. ~ Unsuccessful treatment of BCCs causes increased morbidity, repeated treatments with the possibility of mutilation, and economic losses. Recurrences of SCCs may lead to metastasis, which in Mohs' series of SCCs led to death in ninety-seven patients (4.3%).5 Residual and undetected tumor cells are thought to be the main reason for recurrence after MCS. 4
Volume 8 Number 5 May, 1983
Murine monoclonal antibody to detect tumor cells
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Fig. 1. A, BCC of the skin stained with hematoxylin and eosin, showing invasion of tumor cells (---->)in the dermis. (Magnification, x 100.) B, Serial section of the same tumor stained with VM-1 and the technic of IP. Tumor cells (-->) are heavily labeled with black stain characteristic of peroxidase. (Magnification, x 250.) C, BCC cells (--->)stained with VM-1 and the technic of IF. (Magnification, ×250.) When single cells or small islands of tumor cells remain in the tissue, they are sometimes difficult to distinguish on frozen section with conventional staining methods, since they may be confused with inflammatory, nerve, or vascular cells. Thus, a staining technic which clearly defines tumor cells should decrease the rate of recurrence and mortality. Furthermore, a definitive staining technic should lower the need for extensive experience in reading frozen sections, and for taking extra tissue at the margins if the presence or absence of tumor cells is in doubt on the frozen sections of already removed skin. Monoclonal antibodies, prepared by the hybridoma technic of K6hler and Milstein, 6 allow the generation o f large quantities of reagents with defined specificity and avidity. In dermatology, monoclonal antibodies against thymus-derived lymphocytes can be used for therapy of mycosis fungoides. 7'8 A monoclonal antibody which binds to basal cell and SCC cells would be helpful to detect small islands of tumor cells. We report the
use of an antibasal cell antibody, VM-1, as a diagnostic aid in MCS of cutaneous carcinomas. MATERIALS AND METHODS
Monoclonal antibody preparation The mouse monoclonal antibody used for these experiments is called VM-1 (or W-13) and was produced by standard technics with the use of epidermal cells from psoriatic plaques as the immunogen?~'~This antibody is an IgG of the Yt subtype and stains the basal cell layer on frozen sections of normal human skin.*
Preparation of frozen sections and immunofluorescence (IF) staining To prepare frozen sections of skin, tissue obtained at surgery is snap-frozen on a chuck placed on dry ice using OCT compound (Lab-Tek Division, Miles Laboratories Inc., Naperville, IL). Sections are cut with a microtome. The binding of antibodies to epidermal cells in situ is determined by IF using rabbit antimouse fiuorescein isothiocyanate-conjugated Ig (R/M-FITC). This re*Morhenn VB: In preparation.
618
Oseroff et al
Fig. 2. SCC of the skin stained with VM-1 and the technic of IP. (Magnification, x250.) agent has been adsorbed on dispersed human skin cells. Frozen sections of skin are incubated with monoclonal antibody in humidified glass petri dishes for 15 minutes at room temperature, washed with phosphate-buffered saline (PBS), labeled with R/M-FITC for 15 minutes, and washed with PBS. 1° Sections are covered with fluorescent antibody mounting fluid (Difco Laboratories Inc., Detroit, MI) and examined with a Zeiss fluorescence microscope.
Immunoperoxidase(IP) labeling Frozen sections of human skin are labeled, using the IP staining technic, n Briefly, the tissue is fixed with acetone, incubated with VM-1, washed with PBS, incubated with biotin-conjugated G/M-IgG (Tago, Inc., Burlingame, CA), washed with PBS, labeled with avidin-conjugated horseradish peroxidase, washed with PBS and H20, incubated with diaminobenzidine (DAB) solution, and rinsed with PBS and H~O. All incubations are at room temperature. The sections are processed in 0.5% CuSO4 solution, counterstained with Giemsa, cleared, and mounted. RESULTS AND DISCUSSION Skin obtained at the time of MCS for skin tumors was frozen and cut. Consecutive sections were stained with either hematoxylin and eosin (Fig. 1,A) or VM-1, using both IP (Fig. 1,B) or IF (Fig. 1, C) technics. In tissue from seven of seven different patients with BCC, including a morphea form tumor, and two patients with SCC (Fig. 2), VM-1 distinctly stained the tumor cells. Staining was uniform in all of the BCC and in one of the SCC specimens. In the other SCC, antibody binding was most dense at the periphery of large
Journal of the American Academy of Dermatology
Fig. 3. BCC stained with VM-1 and IP, showing small nests of tumor cells (~) in the dermis. (Magnification, x 250.) tumor islands, and less dense in the centers, where the cells appeared more squamoid. In smaller tumor nests the staining was quite uniform. Small islands of tumor were easily detectable (Fig. 3) in all specimens by both IP and IF technics. Control staining with a murine myeloma antibody of the same 71 subtype as VM-1 did not delineate the tumor cells. Using the technic of IF we observed fluorescence of at least some of the BCC cells in the control slides, which were unstained with VM-1. Thus, at least some infiltrating BCC cells are autofluorescent. In our hands, this autofluorescence has not been a problem since it augments the staining due to VM-1. Invading tumor cells are easily distinguished from hyperplastic downgrowths of rete ridges because, in the latter areas, the cells of the stratum spinosum and above are not labeled, whereas the tumor cells label more uniformly. Furthermore, the tumor cells label more intensely than do normal cells. In addition to the carcinoma cells, the antibody also stained cells of the external root sheath of hair follicles within the dermis (not shown). While it usually was possible to differentiate between islands of tumor cells and hair follicles, especially if the hair shaft was visible, this does represent a limitation of staining with VM-1. Particularly in the scalp, which has a high recurrence rate for skin cancers, differentiation of BCC cells from the cells of hair follicles may prove difficult. H o w -
Volume 8 Number 5 May, 1983
Murine monoclonal antibody to detect tumor cells
ever, in t h o s e cases in which minor cells infiltrate into subcutaneous tissue in areas devoid of hair follicles, labeling with VM-1 can be very helpful for the detection of tumor cells. B e c a u s e it requires fewer steps, IF staining takes a p p r o x i m a t e l y one-half the time needed for the IP t e c h n i c . It is also relatively easy to identify small g r o u p s of tumor cells, even under low p o w e r . I n the rare instances in which skin tumor ceils i n v a d e tissue c o m p o s e d of cells containing e n d o g e n o u s peroxidase (e.g., the parotid gland), IF is the stain of choice since the D A B used in IP will r e a c t with intracellular peroxidase and may lead to false-positive results. Disadvantages of IF are the n e e d for a fluorescence microscope and the b l e a c h i n g o f the stain with prolonged observation (this m a y be reduced by using a para-phenylenediamine mounting buffer. ~z W h i l e the IP method requires more time, it produces permanent, readily quantifiable staining w h i c h c a n be examined with an ordinary microscope in an illuminated room. By directly conj u g a t i n g VM- 1 with fluorescein or biotin, the time r e q u i r e d for IF or IP staining can be reduced to about 20 and 40 minutes, respectively, making both technics practical for diagnosis of residual t u m o r during M o h s ' surgery. R e c e n t experiments in our laboratory have s h o w n that VM-1 inhibits the growth of normal h u m a n keratinocytes in culture. 9,* At present, we are determining whether VM-1 also inhibits the g r o w t h o f keratinocytes in primary cultures obtained f r o m BCC. If VM-1 is toxic to malignant k e r a t i n o c y t e s , this antibody might be used to treat *Morhenn VB: In preparation.
619
skin malignancies by injecting it intralesionally. Alternatively, it may be possible to covalently bind cytotoxic drugs or radioactive isotopes to the antibody and thereby deliver these agents to cells which are to be destroyed. REFERENCES
1. Mohs FE: Chemosurgery:A microscopicallycontrolled method of cancer excision. Arch Surg 42:279-295, 1941. 2. TromovitchTA, Stegman SJ: Microscopicallycontrolled excision of skin tumors: Chemosurgery (Mohs)--fresh tissue technique. Arch Dermatol 110:231-232, 1974. 3. Tromovitch TA, Stegman SJ: Microscopic controlled excision of cutaneous tumors. Cancer41:653-658, 1978. 4. Stegman SJ, Tromovitch TA: Modern chemosurgery-microscopically controlled excision. West J Med 132: 7-12, 1980. 5. Mohs FE: Chemosurgery, microscopically controlled surgery for skin cancer. Springfield,IL, 1978, Charles C Thomas, Publisher, pp. 155-159. 6. KShler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 265:495-497, 1975. 7. Miller RA, Levy R: Response of cutaneous T cell lymphoma to therapy with hybridomamonoclonal antibody. Lancet 2:226-230, 1981. 8. Oseroff AR, Miller RA, Stratte PJ, Levy R: Monoclonal anti-T cell antibody in the therapy of mycosis fungoides. Clin Res 30:601, 1982. (Abst.) 9. Oseroff AF, Fleischmann H, Pfendt EA, Morhenn VB: A monoclonal antibodyagainst human basal cells affects the growth of epidermal cells in vitro. Clin Res 30:601, 1982. (Abst.) 10. Harrist JJ, Mihm MC Jr: Cutaneous immunopathology. Hum Pathol 10:625-653, 1979. 11. Warnke R, Levy R: Detection of T and B cell antigens with hybridoma antibodies: A biotin-avidin-horseradish peroxidase method. J HistochemCytochem28:771-776, 1980. 12. Huff JC, Weston WL, Wanda KD: Enhancement of specific immunofluorescentfindings with use of a paraphenylenediamine mounting buffer. J Invest Derrnatol 78:449-450, 1982.
Mohs' chemosurgery (MCS) and the fresh tissue modification described in 1974 are used extensively to treat squamous cell carcinomas (SCCs) and certain types of basal cell carcinomas (BCCs) of the skin, which are characterized by strands and nests of tumor cells which are not visible on the skin surface, t-a Conventional forms of surgery and radiation often fail to eradicate these "silent" extensions of tumor, so that relatively high recurrence rates result. In MCS, the clinically visible tumor is removed, the next layer of tissue is excised, immediately frozen, cut on the cryostat, stained with hematoxylin and eosin, and checked forresidual tumor cells with the light microscope .4 MCS is the treatment of choice in recurrent From the Department of Dermatology and the Division of Immunology, Stanford University Medical Center. Accepted for publication Sept. 22, 1982. Reprint requests to: Dr. Vera B. Morhenn, Department of Dermatology, Stanford University Medical Center, Stanford, CA 94305.
616
BCCs and SCCs. With this form of therapy, the cure rate for recurrent BCCs is about 98% whereas the cure rate with other forms of therapy (e.g., radiation, conventional surgical excision) ranges from 50% to 85%. 4 For SCCs, MCS results in a 5-year cure rate of 94% in the group of Patients who have not received previous treatment. 5 However, in recurrent SCCs, the 5-year cure rate is only 76.3%.5 Size and location of the lesion also influence the 5-year cure rates. Thus, BCCs and SCCs of the nose which are 3 cm or greater in size have a 5-year cure rate of 84.3% and 56%, respectively. 5 BCCs and SCCs of the ear canal have an 88.9% and a 30% 5-year success rate, respectively. ~ Unsuccessful treatment of BCCs causes increased morbidity, repeated treatments with the possibility of mutilation, and economic losses. Recurrences of SCCs may lead to metastasis, which in Mohs' series of SCCs led to death in ninety-seven patients (4.3%).5 Residual and undetected tumor cells are thought to be the main reason for recurrence after MCS. 4
Volume 8 Number 5 May, 1983
Murine monoclonal antibody to detect tumor cells
617
Fig. 1. A, BCC of the skin stained with hematoxylin and eosin, showing invasion of tumor cells (---->)in the dermis. (Magnification, x 100.) B, Serial section of the same tumor stained with VM-1 and the technic of IP. Tumor cells (-->) are heavily labeled with black stain characteristic of peroxidase. (Magnification, x 250.) C, BCC cells (--->)stained with VM-1 and the technic of IF. (Magnification, ×250.) When single cells or small islands of tumor cells remain in the tissue, they are sometimes difficult to distinguish on frozen section with conventional staining methods, since they may be confused with inflammatory, nerve, or vascular cells. Thus, a staining technic which clearly defines tumor cells should decrease the rate of recurrence and mortality. Furthermore, a definitive staining technic should lower the need for extensive experience in reading frozen sections, and for taking extra tissue at the margins if the presence or absence of tumor cells is in doubt on the frozen sections of already removed skin. Monoclonal antibodies, prepared by the hybridoma technic of K6hler and Milstein, 6 allow the generation o f large quantities of reagents with defined specificity and avidity. In dermatology, monoclonal antibodies against thymus-derived lymphocytes can be used for therapy of mycosis fungoides. 7'8 A monoclonal antibody which binds to basal cell and SCC cells would be helpful to detect small islands of tumor cells. We report the
use of an antibasal cell antibody, VM-1, as a diagnostic aid in MCS of cutaneous carcinomas. MATERIALS AND METHODS
Monoclonal antibody preparation The mouse monoclonal antibody used for these experiments is called VM-1 (or W-13) and was produced by standard technics with the use of epidermal cells from psoriatic plaques as the immunogen?~'~This antibody is an IgG of the Yt subtype and stains the basal cell layer on frozen sections of normal human skin.*
Preparation of frozen sections and immunofluorescence (IF) staining To prepare frozen sections of skin, tissue obtained at surgery is snap-frozen on a chuck placed on dry ice using OCT compound (Lab-Tek Division, Miles Laboratories Inc., Naperville, IL). Sections are cut with a microtome. The binding of antibodies to epidermal cells in situ is determined by IF using rabbit antimouse fiuorescein isothiocyanate-conjugated Ig (R/M-FITC). This re*Morhenn VB: In preparation.
618
Oseroff et al
Fig. 2. SCC of the skin stained with VM-1 and the technic of IP. (Magnification, x250.) agent has been adsorbed on dispersed human skin cells. Frozen sections of skin are incubated with monoclonal antibody in humidified glass petri dishes for 15 minutes at room temperature, washed with phosphate-buffered saline (PBS), labeled with R/M-FITC for 15 minutes, and washed with PBS. 1° Sections are covered with fluorescent antibody mounting fluid (Difco Laboratories Inc., Detroit, MI) and examined with a Zeiss fluorescence microscope.
Immunoperoxidase(IP) labeling Frozen sections of human skin are labeled, using the IP staining technic, n Briefly, the tissue is fixed with acetone, incubated with VM-1, washed with PBS, incubated with biotin-conjugated G/M-IgG (Tago, Inc., Burlingame, CA), washed with PBS, labeled with avidin-conjugated horseradish peroxidase, washed with PBS and H20, incubated with diaminobenzidine (DAB) solution, and rinsed with PBS and H~O. All incubations are at room temperature. The sections are processed in 0.5% CuSO4 solution, counterstained with Giemsa, cleared, and mounted. RESULTS AND DISCUSSION Skin obtained at the time of MCS for skin tumors was frozen and cut. Consecutive sections were stained with either hematoxylin and eosin (Fig. 1,A) or VM-1, using both IP (Fig. 1,B) or IF (Fig. 1, C) technics. In tissue from seven of seven different patients with BCC, including a morphea form tumor, and two patients with SCC (Fig. 2), VM-1 distinctly stained the tumor cells. Staining was uniform in all of the BCC and in one of the SCC specimens. In the other SCC, antibody binding was most dense at the periphery of large
Journal of the American Academy of Dermatology
Fig. 3. BCC stained with VM-1 and IP, showing small nests of tumor cells (~) in the dermis. (Magnification, x 250.) tumor islands, and less dense in the centers, where the cells appeared more squamoid. In smaller tumor nests the staining was quite uniform. Small islands of tumor were easily detectable (Fig. 3) in all specimens by both IP and IF technics. Control staining with a murine myeloma antibody of the same 71 subtype as VM-1 did not delineate the tumor cells. Using the technic of IF we observed fluorescence of at least some of the BCC cells in the control slides, which were unstained with VM-1. Thus, at least some infiltrating BCC cells are autofluorescent. In our hands, this autofluorescence has not been a problem since it augments the staining due to VM-1. Invading tumor cells are easily distinguished from hyperplastic downgrowths of rete ridges because, in the latter areas, the cells of the stratum spinosum and above are not labeled, whereas the tumor cells label more uniformly. Furthermore, the tumor cells label more intensely than do normal cells. In addition to the carcinoma cells, the antibody also stained cells of the external root sheath of hair follicles within the dermis (not shown). While it usually was possible to differentiate between islands of tumor cells and hair follicles, especially if the hair shaft was visible, this does represent a limitation of staining with VM-1. Particularly in the scalp, which has a high recurrence rate for skin cancers, differentiation of BCC cells from the cells of hair follicles may prove difficult. H o w -
Volume 8 Number 5 May, 1983
Murine monoclonal antibody to detect tumor cells
ever, in t h o s e cases in which minor cells infiltrate into subcutaneous tissue in areas devoid of hair follicles, labeling with VM-1 can be very helpful for the detection of tumor cells. B e c a u s e it requires fewer steps, IF staining takes a p p r o x i m a t e l y one-half the time needed for the IP t e c h n i c . It is also relatively easy to identify small g r o u p s of tumor cells, even under low p o w e r . I n the rare instances in which skin tumor ceils i n v a d e tissue c o m p o s e d of cells containing e n d o g e n o u s peroxidase (e.g., the parotid gland), IF is the stain of choice since the D A B used in IP will r e a c t with intracellular peroxidase and may lead to false-positive results. Disadvantages of IF are the n e e d for a fluorescence microscope and the b l e a c h i n g o f the stain with prolonged observation (this m a y be reduced by using a para-phenylenediamine mounting buffer. ~z W h i l e the IP method requires more time, it produces permanent, readily quantifiable staining w h i c h c a n be examined with an ordinary microscope in an illuminated room. By directly conj u g a t i n g VM- 1 with fluorescein or biotin, the time r e q u i r e d for IF or IP staining can be reduced to about 20 and 40 minutes, respectively, making both technics practical for diagnosis of residual t u m o r during M o h s ' surgery. R e c e n t experiments in our laboratory have s h o w n that VM-1 inhibits the growth of normal h u m a n keratinocytes in culture. 9,* At present, we are determining whether VM-1 also inhibits the g r o w t h o f keratinocytes in primary cultures obtained f r o m BCC. If VM-1 is toxic to malignant k e r a t i n o c y t e s , this antibody might be used to treat *Morhenn VB: In preparation.
619
skin malignancies by injecting it intralesionally. Alternatively, it may be possible to covalently bind cytotoxic drugs or radioactive isotopes to the antibody and thereby deliver these agents to cells which are to be destroyed. REFERENCES
1. Mohs FE: Chemosurgery:A microscopicallycontrolled method of cancer excision. Arch Surg 42:279-295, 1941. 2. TromovitchTA, Stegman SJ: Microscopicallycontrolled excision of skin tumors: Chemosurgery (Mohs)--fresh tissue technique. Arch Dermatol 110:231-232, 1974. 3. Tromovitch TA, Stegman SJ: Microscopic controlled excision of cutaneous tumors. Cancer41:653-658, 1978. 4. Stegman SJ, Tromovitch TA: Modern chemosurgery-microscopically controlled excision. West J Med 132: 7-12, 1980. 5. Mohs FE: Chemosurgery, microscopically controlled surgery for skin cancer. Springfield,IL, 1978, Charles C Thomas, Publisher, pp. 155-159. 6. KShler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 265:495-497, 1975. 7. Miller RA, Levy R: Response of cutaneous T cell lymphoma to therapy with hybridomamonoclonal antibody. Lancet 2:226-230, 1981. 8. Oseroff AR, Miller RA, Stratte PJ, Levy R: Monoclonal anti-T cell antibody in the therapy of mycosis fungoides. Clin Res 30:601, 1982. (Abst.) 9. Oseroff AF, Fleischmann H, Pfendt EA, Morhenn VB: A monoclonal antibodyagainst human basal cells affects the growth of epidermal cells in vitro. Clin Res 30:601, 1982. (Abst.) 10. Harrist JJ, Mihm MC Jr: Cutaneous immunopathology. Hum Pathol 10:625-653, 1979. 11. Warnke R, Levy R: Detection of T and B cell antigens with hybridoma antibodies: A biotin-avidin-horseradish peroxidase method. J HistochemCytochem28:771-776, 1980. 12. Huff JC, Weston WL, Wanda KD: Enhancement of specific immunofluorescentfindings with use of a paraphenylenediamine mounting buffer. J Invest Derrnatol 78:449-450, 1982.