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A comparative study of granzyme B expression in keratoacanthoma and squamous cell carcinoma
Journal of Dermatological Science (2006) 44, 109—112
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LETTER TO THE EDITOR A comparative study of granzyme B expression in keratoacanthoma and squamous cell carcinoma KEYWORDS Granzyme B; Keratoacanthoma; Squamous cell carcinoma
Keratoacanthoma (KA) is a cutaneous neoplasm characterized by initial intensive growth and usually spontaneous regression [1]. Squamous cell carcinoma (SCC) is a malignant proliferation of the epithelial cells of the skin and mucous membranes characterized by locally destructive growth and tendency to metastases [1]. The most important feature that separates these closely related entities is a tendency of KA to regress but causes and detailed mechanism of this regression are still not completely elucidated. Recent studies suggest that tumor regression depends mainly on immune response mediated by cytotoxic CD8+ T lymphocytes (CTLs) supported by CD4+ Tcells [2]. CTLs can kill tumor cells and mediate tumor regression in vivo through two distinct molecular mechanisms: (1) direct exocytosis of granules containing granzyme B and perforin; (2) binding of the CD95 receptor on target cells [2,3]. Granzyme B/ perforin pathway has been shown to efficiently kill tumor cells in vitro, induce apoptosis in multipledrug-resistant and death-receptor resistant cell lines [3] and could be involved in tumor regression process. One hundred and fifty skin specimens were obtained from 82 male and 68 female patients. Average age of the patients was 69.10 (11.39), 67.87 (11.64), 75.50 (10.20), 76.57 (9.74) and 65.86 years (14.84) for proliferative keratoacanthoma (pKA), regressing keratoacanthoma (rKA), well differentiated SCC (wdSCC), poorly differentiated SCC (pdSCC) and healthy controls, respectively. The specimens included 30 cases of
each: normal skin (NS), pKA, rKA, wdSCC and pdSCC. Tumors were predominately located on photoexposed skin (80% of pKA, rKA and wdSCC; 85% of pdSCC). SCCs were classified according to Broders’ grading into two categories, well (Broders’ grade I) and poorly (Broders’ grade III) differentiated type while KAs were classified according to previously used criteria to designate pKA and rKA [4]. Four micrometers thick sections of formalin fixed paraffin embedded tissue were stained with hematoxylin-eosin and two pathologists examined each slide independently. Specific antibodies were used to recognize CD8 (1:50, DAKO A/S, Glostrup, DK), CD3 (1:25, DAKO A/S, Glostrup, DK), CD4 (1:25, NOVOCASTRA, Newcastle upon Tyne, UK) and granzyme B (prediluted, NOVOCASTRA, Newcastle upon Tyne, UK). Immunostaining results for CD8, CD4, CD3 and granzyme B were quantified by expressing the number of positive cells/mm2 of the lesion. Expression of granzyme B was significantly increased in all skin tumors examined as compared to NS ( p < 0.0001). Median values and ranges of granzyme B, CD3, CD8 and CD4 expression are shown in Fig. 1. Granzyme B expression was significantly increased in KAs as compared to pdSCC and had the highest values in rKA. Localization of granzyme B immunoreactivity in examined tumors is shown in Fig. 2. Granzyme B was detected in a part of peritumoral and intra-tumoral lymphocytes as sparsely granular pattern. Majority of granzyme B positive cells was found at the interface between lymphocytic areas and tumor cells in the dermis and tumor stromal septa. In KAs, granzyme B positive T cells were scattered between malignant keratinocytes and weak positive staining was also observed in some of the malignant keratinocytes. Tumors were variably infiltrated with CD3+, CD8+ and CD4+ T cells with significantly higher density of the infiltrate comparing to NS ( p < 0.0001). We have detected significantly higher density of CD3+ and CD4+ T cells in KAs as compared to SCCs (Fig. 1). Majority of the tumor infiltrating cells in KAs and SCCs were CD8+. There was a significant increase of
0923-1811/$30.00 # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2006.07.003
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Letter to the Editor
Fig. 1 Expression of CD4, CD3, CD8 and granzyme B in keratoacanthoma and squamous cell carcinoma (statistical significance: *p < 0.05; **p < 0.001; ***p < 0.0001).
CD8+ T cells density in rKA as compared to pKA and SCCs (Fig. 1). The highest density of CD3+, CD8+ and CD4+ T cells was found in rKA with significant change in CD4/CD8 ratio (1.66 in pKA to 0.9 in rKA) due to increased density of CD8+ cells (density of CD4+ T cells did not change in KAs). While CD4/CD8 ratio in rKA and SCCs (0.9 in wdSCC; 0.94 in pdSCC) was similar, density of inflammatory infiltrate was much lower in SCCs. CD3+ T cells infiltrate was detected along tumor—host interface and dermis or tumor septa. Peri-tumoral infiltrate was composed of both CD8+ and CD4+ cells and intra-tumoral cells were mainly CD8+, as described previously [5]. Expression of granzyme B correlated positively with density of CD4+ T cells in pKA ( p = 0.037, r = 0.383) and pdSCC ( p = 0.026, r = 0.406). However, in wdSCC expression of granzyme B correlated with density of CD8+ T cells ( p < 0.0001, r = 853) and in rKA with both, CD8+ ( p < 0.0001, r = 726) and CD4+ ( p = 0.001, r = 0.566) T cells. These findings suggest discordant expression of granzyme B in human lymphocyte subsets and the possibility of granzyme B and/or other granzymes to be produced
by a different subset of T cells in immune response mounted against different tumors, what might influence the effectiveness of host immune response [6]. Our results also imply the presence of adequately stimulated CD8+ Tcells in rKA and indicate that both CD8+ and CD4+ T lymphocytes contribute to immunosurveillance against skin cancer as suggested recently [2]. We have detected a decrease in granzyme B expression in tumor and NS samples from sun exposed areas ( p < 0.05) suggesting inhibition of cytotoxic activity. Modulation of cytotoxic activity by UV-radiation could contribute to the suppression of anti-tumor immune response stimulating promotion and cancer progression. Presence of significantly lower density of CD3+, CD8+ and CD4+ T-cell infiltrate in SCCs (especially noted when comparing rKA and pdSCC), suggests insufficient host immune response mounted against SCCs. Besides the highest total inflammatory infiltrate in rKA, we have detected a significant increase in CD8+ T cells in rKA as compared to pKA accompanied by intense cytotoxic activity. These data confirm previously suggested significance of the
Letter to the Editor
111
Fig. 2 Granzyme B immunostaining in keratoacanthoma and squamous cell carcinoma. (A) Strong granzyme B expression in lymphocytes at the interface between lymphocytic areas and tumor cells in pKA. Numerous granzyme B positive cells could also be seen scattered between malignant keratinocytes in the basal layers (HE 100). (B) Intense granzyme B expression in lymphocytes surrounding rKA (HE 100). (C) Some granzyme B positive cells in the wdSCC stroma (HE 200). (D) Rare granzyme B positive cells in the stroma of pdSCC (HE 200).
quantity of inflammatory infiltrate and CD8+ cytotoxic T lymphocytes as well as their activation state in tumor regression [2,7,8]. As suggested previously, T cells infiltrating SCCs could be targeted for apoptosis resulting in decreased T cell density [8—10]. In rare cases of KAs but in majority of SCCs, especially pdSCCs, the number of CD4+ plus CD8+ cells did not add up to total CD3+ T cells. We suppose this is due to previously described presence of the unusual CD3+CD4 CD8 phenotype attributed to apoptosis of tumor infiltrating lymphocytes in strong FasL expressing tumors [8] since immunostaining indicated that these tumors were often infiltrated by Fas+ lymphocytes, many showing evidence of apoptosis [8—10]. Our data revealed significantly increased number of T cells expressing granzyme B in rKA comparing to SCCs, suggesting a possible role of granzyme B in KA regression. A weak cytoplasmic staining in some malignant keratinocytes, especially in KAs, suggests that these cells may have been exposed and damaged by cytotoxic proteins released from the neighboring T cells. Data from our study confirm previous findings that T lymphocytes infiltrating SCCs are functionally defective.
References [1] Baham A, Regauer S, Soyer HP, Beham-Schmid C. Keratoacanthoma: a clinically distinct variant of well differentiated squamous cell carcinoma. Adv Anat Pathol 1998;5:269—80. [2] Matsui S, Ahlers JD, Vortmeyer AO, Terabe M, Tsukui T, Carbone DP, et al. A model for CD8+ CTL tumor immunosurveillance and regulation of tumor escape by CD4+ cells through an effect on Quality of CTL. J Immunol 1999; 163:184—93. [3] Pardo J, Bosque A, Brehm R, Wallich R, Naval J, Mullbacher A, et al. Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis. J Cell Biol 2004;167:457—68. [4] Batinac T, Zamolo G, Coklo M, Hadzisejdic I, Stemberger C, Zauhar G. Expression of cell cycle and apoptosis regulatory proteins in keratoacanthoma and squamous cell carcinoma. Pathol Res Pract 2006;202:599—607. [5] Nakano O, Sato M, Naito Y, Suzuki K, Orikasa S, Aizawa M, et al. Proliferative activity of intratumoral CD8+ T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumour immunity. Cancer Res 2001;61:5132—6. [6] Grossman WJ, Verbsky JW, Tollefsen BL, Kemper C, Atkinson JP, Ley TJ. Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells. Blood 2004;104:2840—8. [7] Smith KJ, Hamza S, Skelton H. Topical imidazoquinoline therapy of cutaneous squamous cell carcinoma polarizes
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Letter to the Editor
lymphoid and monocyte/macrophage populations to a Th1 and M1 cytokine pattern. Clin Exp Dermatol 2004;29:505— 12. [8] Reichert TE, Strauss L, Wagner EM, Gooding W, Whiteside TL. Signaling abnormalities, apoptosis, and reduced proliferation of circulating and tumour-infiltrating lymphocytes in patients with oral carcinoma. Clin Cancer Res 2002;8:3137— 45. [9] Gastman BR, Atarashi Y, Reichert TE, Saito T, Balkir L, Rabinowich H, et al. Fas ligand is expressed on human squamous cell carcinomas of head and neck and it promotes apoptosis of T lymphocytes. Cancer Res 1999;59:5356—64. [10] Kuss I, Hathaway B, Ferris RL, Gooding W, Whiteside TL. Decreased absolute counts of T lymphocyte subsets and their relation to disease in squamous cell carcinoma of the head and neck. Clin Cancer Res 2004;10:3755—62.
Tanja Batinac Department of Dermatovenerology, University Hospital, Kresimirova 42, 51000 Rijeka, Croatia
Gordana Zamolo* Ita Hadzisejdic Department of Pathology, Faculty of Medicine, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia Gordana Zauhar Department of Physics, Faculty of Medicine, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia *Corresponding author. Tel.: +385 51 325 813; fax: +385 51 325 810 E-mail address: [email protected] (G. Zamolo) 26 April 2006
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR A comparative study of granzyme B expression in keratoacanthoma and squamous cell carcinoma KEYWORDS Granzyme B; Keratoacanthoma; Squamous cell carcinoma
Keratoacanthoma (KA) is a cutaneous neoplasm characterized by initial intensive growth and usually spontaneous regression [1]. Squamous cell carcinoma (SCC) is a malignant proliferation of the epithelial cells of the skin and mucous membranes characterized by locally destructive growth and tendency to metastases [1]. The most important feature that separates these closely related entities is a tendency of KA to regress but causes and detailed mechanism of this regression are still not completely elucidated. Recent studies suggest that tumor regression depends mainly on immune response mediated by cytotoxic CD8+ T lymphocytes (CTLs) supported by CD4+ Tcells [2]. CTLs can kill tumor cells and mediate tumor regression in vivo through two distinct molecular mechanisms: (1) direct exocytosis of granules containing granzyme B and perforin; (2) binding of the CD95 receptor on target cells [2,3]. Granzyme B/ perforin pathway has been shown to efficiently kill tumor cells in vitro, induce apoptosis in multipledrug-resistant and death-receptor resistant cell lines [3] and could be involved in tumor regression process. One hundred and fifty skin specimens were obtained from 82 male and 68 female patients. Average age of the patients was 69.10 (11.39), 67.87 (11.64), 75.50 (10.20), 76.57 (9.74) and 65.86 years (14.84) for proliferative keratoacanthoma (pKA), regressing keratoacanthoma (rKA), well differentiated SCC (wdSCC), poorly differentiated SCC (pdSCC) and healthy controls, respectively. The specimens included 30 cases of
each: normal skin (NS), pKA, rKA, wdSCC and pdSCC. Tumors were predominately located on photoexposed skin (80% of pKA, rKA and wdSCC; 85% of pdSCC). SCCs were classified according to Broders’ grading into two categories, well (Broders’ grade I) and poorly (Broders’ grade III) differentiated type while KAs were classified according to previously used criteria to designate pKA and rKA [4]. Four micrometers thick sections of formalin fixed paraffin embedded tissue were stained with hematoxylin-eosin and two pathologists examined each slide independently. Specific antibodies were used to recognize CD8 (1:50, DAKO A/S, Glostrup, DK), CD3 (1:25, DAKO A/S, Glostrup, DK), CD4 (1:25, NOVOCASTRA, Newcastle upon Tyne, UK) and granzyme B (prediluted, NOVOCASTRA, Newcastle upon Tyne, UK). Immunostaining results for CD8, CD4, CD3 and granzyme B were quantified by expressing the number of positive cells/mm2 of the lesion. Expression of granzyme B was significantly increased in all skin tumors examined as compared to NS ( p < 0.0001). Median values and ranges of granzyme B, CD3, CD8 and CD4 expression are shown in Fig. 1. Granzyme B expression was significantly increased in KAs as compared to pdSCC and had the highest values in rKA. Localization of granzyme B immunoreactivity in examined tumors is shown in Fig. 2. Granzyme B was detected in a part of peritumoral and intra-tumoral lymphocytes as sparsely granular pattern. Majority of granzyme B positive cells was found at the interface between lymphocytic areas and tumor cells in the dermis and tumor stromal septa. In KAs, granzyme B positive T cells were scattered between malignant keratinocytes and weak positive staining was also observed in some of the malignant keratinocytes. Tumors were variably infiltrated with CD3+, CD8+ and CD4+ T cells with significantly higher density of the infiltrate comparing to NS ( p < 0.0001). We have detected significantly higher density of CD3+ and CD4+ T cells in KAs as compared to SCCs (Fig. 1). Majority of the tumor infiltrating cells in KAs and SCCs were CD8+. There was a significant increase of
0923-1811/$30.00 # 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2006.07.003
110
Letter to the Editor
Fig. 1 Expression of CD4, CD3, CD8 and granzyme B in keratoacanthoma and squamous cell carcinoma (statistical significance: *p < 0.05; **p < 0.001; ***p < 0.0001).
CD8+ T cells density in rKA as compared to pKA and SCCs (Fig. 1). The highest density of CD3+, CD8+ and CD4+ T cells was found in rKA with significant change in CD4/CD8 ratio (1.66 in pKA to 0.9 in rKA) due to increased density of CD8+ cells (density of CD4+ T cells did not change in KAs). While CD4/CD8 ratio in rKA and SCCs (0.9 in wdSCC; 0.94 in pdSCC) was similar, density of inflammatory infiltrate was much lower in SCCs. CD3+ T cells infiltrate was detected along tumor—host interface and dermis or tumor septa. Peri-tumoral infiltrate was composed of both CD8+ and CD4+ cells and intra-tumoral cells were mainly CD8+, as described previously [5]. Expression of granzyme B correlated positively with density of CD4+ T cells in pKA ( p = 0.037, r = 0.383) and pdSCC ( p = 0.026, r = 0.406). However, in wdSCC expression of granzyme B correlated with density of CD8+ T cells ( p < 0.0001, r = 853) and in rKA with both, CD8+ ( p < 0.0001, r = 726) and CD4+ ( p = 0.001, r = 0.566) T cells. These findings suggest discordant expression of granzyme B in human lymphocyte subsets and the possibility of granzyme B and/or other granzymes to be produced
by a different subset of T cells in immune response mounted against different tumors, what might influence the effectiveness of host immune response [6]. Our results also imply the presence of adequately stimulated CD8+ Tcells in rKA and indicate that both CD8+ and CD4+ T lymphocytes contribute to immunosurveillance against skin cancer as suggested recently [2]. We have detected a decrease in granzyme B expression in tumor and NS samples from sun exposed areas ( p < 0.05) suggesting inhibition of cytotoxic activity. Modulation of cytotoxic activity by UV-radiation could contribute to the suppression of anti-tumor immune response stimulating promotion and cancer progression. Presence of significantly lower density of CD3+, CD8+ and CD4+ T-cell infiltrate in SCCs (especially noted when comparing rKA and pdSCC), suggests insufficient host immune response mounted against SCCs. Besides the highest total inflammatory infiltrate in rKA, we have detected a significant increase in CD8+ T cells in rKA as compared to pKA accompanied by intense cytotoxic activity. These data confirm previously suggested significance of the
Letter to the Editor
111
Fig. 2 Granzyme B immunostaining in keratoacanthoma and squamous cell carcinoma. (A) Strong granzyme B expression in lymphocytes at the interface between lymphocytic areas and tumor cells in pKA. Numerous granzyme B positive cells could also be seen scattered between malignant keratinocytes in the basal layers (HE 100). (B) Intense granzyme B expression in lymphocytes surrounding rKA (HE 100). (C) Some granzyme B positive cells in the wdSCC stroma (HE 200). (D) Rare granzyme B positive cells in the stroma of pdSCC (HE 200).
quantity of inflammatory infiltrate and CD8+ cytotoxic T lymphocytes as well as their activation state in tumor regression [2,7,8]. As suggested previously, T cells infiltrating SCCs could be targeted for apoptosis resulting in decreased T cell density [8—10]. In rare cases of KAs but in majority of SCCs, especially pdSCCs, the number of CD4+ plus CD8+ cells did not add up to total CD3+ T cells. We suppose this is due to previously described presence of the unusual CD3+CD4 CD8 phenotype attributed to apoptosis of tumor infiltrating lymphocytes in strong FasL expressing tumors [8] since immunostaining indicated that these tumors were often infiltrated by Fas+ lymphocytes, many showing evidence of apoptosis [8—10]. Our data revealed significantly increased number of T cells expressing granzyme B in rKA comparing to SCCs, suggesting a possible role of granzyme B in KA regression. A weak cytoplasmic staining in some malignant keratinocytes, especially in KAs, suggests that these cells may have been exposed and damaged by cytotoxic proteins released from the neighboring T cells. Data from our study confirm previous findings that T lymphocytes infiltrating SCCs are functionally defective.
References [1] Baham A, Regauer S, Soyer HP, Beham-Schmid C. Keratoacanthoma: a clinically distinct variant of well differentiated squamous cell carcinoma. Adv Anat Pathol 1998;5:269—80. [2] Matsui S, Ahlers JD, Vortmeyer AO, Terabe M, Tsukui T, Carbone DP, et al. A model for CD8+ CTL tumor immunosurveillance and regulation of tumor escape by CD4+ cells through an effect on Quality of CTL. J Immunol 1999; 163:184—93. [3] Pardo J, Bosque A, Brehm R, Wallich R, Naval J, Mullbacher A, et al. Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis. J Cell Biol 2004;167:457—68. [4] Batinac T, Zamolo G, Coklo M, Hadzisejdic I, Stemberger C, Zauhar G. Expression of cell cycle and apoptosis regulatory proteins in keratoacanthoma and squamous cell carcinoma. Pathol Res Pract 2006;202:599—607. [5] Nakano O, Sato M, Naito Y, Suzuki K, Orikasa S, Aizawa M, et al. Proliferative activity of intratumoral CD8+ T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumour immunity. Cancer Res 2001;61:5132—6. [6] Grossman WJ, Verbsky JW, Tollefsen BL, Kemper C, Atkinson JP, Ley TJ. Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells. Blood 2004;104:2840—8. [7] Smith KJ, Hamza S, Skelton H. Topical imidazoquinoline therapy of cutaneous squamous cell carcinoma polarizes
112
Letter to the Editor
lymphoid and monocyte/macrophage populations to a Th1 and M1 cytokine pattern. Clin Exp Dermatol 2004;29:505— 12. [8] Reichert TE, Strauss L, Wagner EM, Gooding W, Whiteside TL. Signaling abnormalities, apoptosis, and reduced proliferation of circulating and tumour-infiltrating lymphocytes in patients with oral carcinoma. Clin Cancer Res 2002;8:3137— 45. [9] Gastman BR, Atarashi Y, Reichert TE, Saito T, Balkir L, Rabinowich H, et al. Fas ligand is expressed on human squamous cell carcinomas of head and neck and it promotes apoptosis of T lymphocytes. Cancer Res 1999;59:5356—64. [10] Kuss I, Hathaway B, Ferris RL, Gooding W, Whiteside TL. Decreased absolute counts of T lymphocyte subsets and their relation to disease in squamous cell carcinoma of the head and neck. Clin Cancer Res 2004;10:3755—62.
Tanja Batinac Department of Dermatovenerology, University Hospital, Kresimirova 42, 51000 Rijeka, Croatia
Gordana Zamolo* Ita Hadzisejdic Department of Pathology, Faculty of Medicine, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia Gordana Zauhar Department of Physics, Faculty of Medicine, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia *Corresponding author. Tel.: +385 51 325 813; fax: +385 51 325 810 E-mail address: [email protected] (G. Zamolo) 26 April 2006