- Email: [email protected]
Histological expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in human primary melanoma
Pathology (December 2004) 36(6), pp. 561–565
ANATOMICAL PATHOLOGY
Histological expression of tumour necrosis factor-related apoptosisinducing ligand (TRAIL) in human primary melanoma LUC P. BRON*, RICHARD A. SCOLYER{{§, JOHN F. THOMPSON{§,
AND
PETER HERSEY{§}
*Sydney Head and Neck Cancer Institute, {Department of Anatomical Pathology, {Sydney Melanoma Unit and §the Melanoma and Skin Cancer Research Institute, Royal Prince Alfred Hospital, Sydney, ,Discipline of Surgery, Faculty of Medicine, University of Sydney, Sydney and }Oncology and Immunology Unit, Newcastle Mater Hospital, Newcastle, NSW, Australia
Summary Aims: Tumour necrosis factor-related apoptosis ligand (TRAIL) appears to selectively induce apoptosis in a wide range of cultured malignant cells, including melanoma. This study was designed to attempt to clarify the role of TRAIL in the biology of human melanoma. Methods: Tissue sections cut from formalin-fixed, paraffin-embedded tissue blocks of 45 primary cutaneous melanomas were tested for expression of TRAIL using immunohistochemistry. The intensity, pattern of staining and percentage of positively stained tumour cells were evaluated in each melanoma. Breslow thickness, ulcerative state, dermal mitotic rate and the presence of tumour infiltrating lymphocytes were measured/determined in each case. Median follow up for the cohort of patients was 10 months (range 1–18). Survival analysis was conducted using the Kaplan–Meier method. The level of expression of TRAIL was compared with the various histological determinants using the two-tailed Fisher’s exact test and the x2-test. Results: Overall and disease-free survival were 72 and 48%, respectively, and did not correlate with TRAIL expression. Among the pathological prognostic determinants, only mitotic rate showed a statistically significant correlation with TRAIL expression using the x2-test (P~0.04). Conclusion: We conclude that TRAIL expression in melanoma defines a more aggressive/proliferative phenotype, either through selection of apoptotic resistant cells or by secondary induction of other factors enhancing proliferation of more malignant cells. Analysis of a larger group of patients with longer follow-up is required to determine whether TRAIL expression correlates with survival of patients. Key words: Tumour necrosis factor-related apoptosis-inducing ligand, tumor necrosis factor-related apoptosis-inducing ligand, TRAIL, cutaneous melanoma, apoptosis, pathology. Received 12 January, revised and accepted 26 May 2004
INTRODUCTION Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that, like TNF-a and Fas ligand, is a type II membrane protein that can induce apoptotic cell death in a variety of cell types.1–3
TRAIL appears to be particularly important because it can induce apoptosis in a wide range of cultured malignant cells but not in normal tissues,4–9 with the possible exception of some human liver cells.10 The potential importance of TRAIL as an anticancer agent has been supported by studies in animal models showing selective toxicity to transplanted human tumours but not to normal tissues.11–13 Induction of apoptosis by TRAIL is believed to be mediated by interaction with two death receptors on cells referred to as TRAIL-R1 and TRAIL-R2 (see Wiley et al.3 for alternate nomenclature). Normal cells are postulated to be protected from TRAIL-induced apoptosis by their expression of TRAIL-R3 and TRAIL-R4, which lack cytoplasmic death domains and act to sequester TRAIL (decoy receptors) or to mediate anti-apoptotic signals.4–14 We have shown previously that receptors for TRAIL were expressed on cultured melanoma and were able to induce varying degrees of apoptosis in approximately twothirds of the melanoma cell lines tested.7–9 Sensitivity of melanoma cells to TRAIL-induced apoptosis showed an overall correlation with the level of death receptors, particularly TRAIL-R2 expression, but did not correlate with the level of expression of the decoy receptors, TRAIL-R3 and TRAIL-R4. In previous studies we15 and others16 have shown that TRAIL is expressed on activated lymphocytes and is upregulated on CD4- and CD8-positive T cells,15,16 NK cells,17 dendritic cells and monocytes18,19 by IFN-a, IL-2, IL-15 and IFN-c.20,21 TRAIL-induced apoptosis, therefore, appears to represent a second major cytotoxic mechanism used by lymphocytes in addition to or in place of the perforin granzyme system. Fas ligand (FasL) is another member of the TNF family that is expressed on lymphocytes and, like TRAIL, can induce apoptosis of certain cultured cancer cell lines that express receptors for Fas (CD95). A number of studies have shown that FasL is also expressed on tumour cells,22–24 including human melanoma.25 The latter authors also reported that the expression of FasL on melanoma cells could induce apoptosis of effector T lymphocytes and suggested that FasL could contribute to immune privilege of tumours. Similarly, human colon cancer cells were reported to express FasL and were capable of inducing apoptosis in Jurkat T cells but not colon carcinoma cells.26 Studies by others supported this concept of immune privilege.
ISSN 0031-3025 printed/ISSN 1465–3931 # 2004 Royal College of Pathologists of Australasia DOI: 10.1080/00313020400011268
562
Pathology (2004), 36(6), December
BRON et al.
TABLE 1 Clinical and pathological characteristics of the melanoma patients and their tumours Characteristics
No. of patients
Percentage
45
68 (23–83)*
29 16
64 36 3.5 (0.1–16)* 60 40
Age Gender Male Female Breslow thickness w1 mm v1 mm Ulcerative state Present Absent Dermal tumour mitotic rate (per mm2)
27 18 9 36 45
20 80 2 (0–22)*
Sydney. Five-mm thick sections were cut from a representative tissue block of each tumour. Sections were deparaffinised in xylene and then rehydrated through graded decreasing concentrations of alcohol. They were then incubated with rabbit antihuman TRAIL antibody (IgG isotype; Santa Cruz, USA) at appropriate concentration in PBS. Binding sites of the primary antibodies were visualised using the Vectastain ABC Kit (Vector Laboratories, USA) according to the manufacturer’s instructions. Finally, the sections were faintly counterstained with H&E and mounted with aqueous media. Negative controls were performed by omission of the primary antibody. Once stained, slides of melanoma were analysed by one pathologist (RAS), who had no prior knowledge of the clinical data. Percentage of positively stained cells (0–100%), intensity and pattern of staining, on a scale of 0–3 was estimated and recorded. The presence of tumour infiltrating lymphocytes was also evaluated in each case.
*Median (range). Statistical analysis
The similarity between the functional role of FasL and TRAIL raises the question as to whether TRAIL expression on tumours may also cause the death of immune effector cells. This appeared to be so in that murine adenocarcinoma cells engineered to express TRAIL were more resistant to immune killing both in vitro and in vivo.27 In view of these findings, we examined TRAIL expression in tissue sections taken from a series of primary melanomas to determine if TRAIL expression was correlated with important prognostic determinants in primary melanoma.
Statistical analysis was performed with JMP Statistics Made Visual software (SAS Institute Inc., USA). Correlation between TRAIL expression and other histological features of the melanomas were performed using the two-tailed Fisher’s exact test or the x2-test, as appropriate. The correlation of TRAIL expression to disease-free survival and overall survival was calculated by the Kaplan–Meier estimate. Multivariate analyses were performed using the Cox stepwise-regression analysis to determine the independent relationship between TRAIL expression and pathological prognostic determinants of primary melanoma.
RESULTS PATIENTS AND METHODS Patients Histological sections were cut from formalin-fixed, paraffin-embedded tissue blocks of primary cutaneous melanomas from 45 patients treated at the Sydney Melanoma Unit over the period from December 2000 to March 2002. Demographic details of the patients and histological features of their primary melanomas are summarised in Table 1. Depending on their Breslow thickness, primary melanomas were separated into thin (v1 mm) and thick (w1 mm) melanomas. All patients had regular follow up at the Sydney Melanoma Unit. Median follow-up was 10 months (range 1–18). Immunohistochemistry Archival paraffin blocks of the melanomas were retrieved from the Department of Anatomical Pathology, Royal Prince Alfred Hospital,
Fig. 1
TRAIL expression in primary melanomas Forty-five melanomas were examined for TRAIL expression. Patient demographics and histological characteristics are described in Table 1. Overall, 33 cases (74%) showed some TRAIL expression of varying intensity and 12 tumours (26%) were completely negative. Among the positive cases, two tumours (4%) showed focal expression of TRAIL (v25% of cells), 15 (33%) showed expression in 26–50% of cells, seven (16%) showed expression in 51–75% of tumour cells and nine (20%) had a high expression of TRAIL (w75% of cells). Examples of TRAIL expression in sections from primary melanoma are shown in Fig. 1. In our series of melanomas, there were 14 nodular melanomas, 10 superficial spreading melanomas, six Hutchinson’s melanotic freckles, three desmoplastic
Example of negative, mild and strong TRAIL expression in melanoma (TRAIL immunohistochemical stains, original magnification, 6400).
TRAIL EXPRESSION IN MELANOMA
TABLE 2
563
Summary of TRAIL expression among 45 cases of melanoma and its correlation with histopathological features of the primary tumour
TRAIL expression (% cells) Pathological feature of primary melanoma
0–25%
Breslow thickness v1 mm 6 w1 mm 8 Tumour infiltrating lymphocytes Present 8 Absent 6 2 Tumour mitotic rate (per mm ) v6 4 §6 10 Ulcerative state Present 11 Absent 3 No. of cases (%) 14 (31)
26–50%
51–75%
76–100%
Total
Significance of correlation of TRAIL expression with histopathological features P value
5 10
4 3
3 6
18 27
0.73
9 6
5 2
7 2
29 16
0.53
3 12
1 6
5 4
13 32
0.06
14 1 15 (33)
4 3 7 (16)
7 2 9 (20)
36 9 45
0.73
melanomas. In 12 lesions there were overlapping features between two or more subtypes. The expression of TRAIL did not correlate with the various histological subtypes of melanomas (data not shown). Relationship of TRAIL expression to important prognostic determinants in primary melanoma Recurrence and overall survival for melanoma patients are strongly related to various histological characteristics of the primary melanoma. Thickness, ulceration, and tumour dermal mitotic rate are the most important histological determinants. We reasoned that should TRAIL expression be related to prognosis, it might be expected to correlate with these determinants. A reduction in tumour-infiltrating lymphocytes (TILs) might also be expected in melanoma with strong TRAIL expression. These different histological parameters were examined by contingency table and regression analysis to determine their correlation with TRAIL expression (Table 2). Expression of TRAIL was not correlated with thickness or ulcerative state of the melanoma or the presence of tumour infiltrating lymphocytes. Only mitotic rate of the melanoma showed significant correlation with TRAIL expression using the x2-test (P~0.04; Fig. 2). However, when the level of TRAIL expression was divided into four groups (v25, 26–50, 51–75 and w75%), the association between TRAIL expression and mitotic rate was not statistically significant (P~0.06) using Fisher’s exact test (Table 2). Logistic regression analysis did not show that TRAIL expression was independently correlated with tumour mitotic rate. Relationship of TRAIL expression with clinical outcome In our series, 2-year overall and disease-free survival rates were 72 and 48%, respectively. During the follow-up period, 12 patients (26%) developed recurrent melanoma. Among those, eight cases (18%) were in transit or distant metastases. Despite the relatively short follow-up period of the cases included in this study, Breslow thickness showed a significant adverse correlation with disease-free survival (Pv0.02). There was no significant correlation of either the other histopathological factors or of TRAIL expression with clinical outcome (Fig. 3).
Fig. 2 Strong TRAIL expression in a case with multiple mitoses (arrowheads) (TRAIL immunohistochemical stain, original magnification, 6400).
Fig. 3 Disease-specific survival of patients with TRAIL-positive versus TRAIL-negative primary melanomas.
BRON et al.
Pathology (2004), 36(6), December
DISCUSSION
melanoma-reactive T cells may have allowed the overgrowth of more rapidly growing melanoma cells. A larger number of patients and longer follow-up time would be needed to assess whether TRAIL expression is associated with a decrease in disease-free survival.
564
This study was initiated to test the hypothesis that TRAIL expression in primary melanoma may be an adverse prognostic feature due to inhibition of effector T-cell responses against the tumour and the creation of immune privileged sites. If this was the case, an association with important histopathological prognostic features such as Breslow thickness, ulcerative state or dermal tumour mitotic rate in primary melanoma might be expected. The results above show that TRAIL expression was significantly correlated only to the mitotic rate of the primary melanoma (P~0.04) and, consequently, is most likely an adverse prognostic indicator in melanoma. The likely reason for the loss of significance of the correlation between TRAIL expression and mitotic rate when the level of TRAIL expression was divided into four groups is the small number of patients in some of the groups (Table 2). Even though it is a recognised adverse prognostic factor for survival,28 mitotic rate in our small group of patients was not significantly associated with disease-free survival (P~0.49). Several explanations could be proposed to account for the correlation of TRAIL expression with mitotic rate. One would be that the signal pathway and transcription factors acting to increase cell division in the tumour may also act on promoters controlling TRAIL expression. NF-KB is one factor involved in upregulation of TRAIL29 and activation of NF-KB in situ is associated with progression of melanoma.30 A second possibility is that TRAIL expression may have resulted in destruction (by apoptosis) of TRAIL-sensitive melanoma cells and selected TRAIL-resistant melanoma cells, and that resistance mechanisms involved pathways that activate cell division. Evidence for such a role was described in studies in leukaemia31 and other cancer cells.32 TRAIL expression on mastocytoma cells was reported to support their growth by destruction of macrophages.33 In either case, the result is consistent with an association between TRAIL expression and more aggressive melanoma. TRAIL expression in melanoma did not show any correlation with the presence of tumour infiltrating lymphocytes; however, it is possible that such an association may only be seen in the early stages of evolution of melanoma. At later stages, TRAIL-expressing melanoma cells could be expected to delete TRAIL death receptor-expressing lymphocytes, leaving only non-reactive lymphocytes infiltrating into the tumour. In several animal models, expression of TRAIL on tumour cells has been shown to be associated with reduced tumour growth. In one model, this was due to induction of apoptosis in the tumour cells.32 In another model, FasL was believed to induce an antitumour effect due to infiltration by neutrophils. It is not common to see neutrophils around primary melanoma so that the latter is unlikely in human melanoma. We conclude, on the evidence available, that TRAIL expression on primary melanoma appears to define a more aggressive/proliferative phenotype. It is not possible to conclude if this is secondary to other factors responsible for the development of such malignant cells or whether TRAIL is an initiator of the phenotype due to selection of apoptotic resistant cells. It is also not possible to exclude the possibility that TRAIL-mediated deletion of
ACKNOWLEDGEMENTS We wish to thank Ashley Young for technical assistance in cutting the melanoma sections, Dr Lawrence Li and the Department of Pathology, University of Sydney, for technical assistance, and the Sydney Melanoma Unit for the use of their computerised patients database. This work was supported by a Technical Assistance Grant from the Royal College of Pathologists of Australasia (Dr Richard Scolyer), a NSW State Cancer Council Grant (Professor Peter Hersey), the Melanoma and Skin Cancer Research Institute and the Melanoma Foundation of the University of Sydney. Dr Luc Bron was partially funded during his fellowship by a grant from the foundation of Decker and Emma Muschamp, Switzerland. Address for correspondence: Professor P. Hersey, Immunology and Oncology Unit, Room 443, David Maddison Clinical Sciences Building, Cnr King and Watt Street, Newcastle, NSW 2300, Australia. E-mail: [email protected]
References 1. Griffith TS, Lynch DH. TRAIL: a molecule with multiple receptors and control mechanism. Curr Opin Immunol 1998; 10: 559–63. 2. Pitti RM, Masters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumour necrosis factor cytokine family. J Biol Chem 1996; 271: 12687–90. 3. Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995; 3: 673–82. 4. Griffith TS, Chin WA, Jackson GC, Lynch DH, Kubin MZ. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 1998; 161: 2833–40. 5. Phillips TA, Ni J, Pan G, et al. TRAIL (Apo2L) and TRAIL receptors in human placentas: implication for immune privilege. J Immunol 1999; 162: 6053–9. 6. Rieger J, Naumann U, Glaser T, Ashkenazi A, Weller M. APO2 ligand a novel lethal weapon against malignant glioma? FEBS Lett 1998; 427: 124–8. 7. Thomas WD, Hersey P. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells. J Immunol 1998; 161: 2195–200. 8. Zhang XD, Franco A, Myers K, Gray C, Nguyen T, Hersey P. Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. Cancer Res 1999; 59: 2747–53. 9. Zhang XD, Nguyen T, Thomas WD, Sanders JE, Hersey P. Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types. FEBS Lett 2000; 482: 193–9. 10. Ashkenazi A. Targeting death and decoy receptors of the tumournecrosis factor superfamily. Nat Rev Cancer 2002; 2: 420–30. 11. Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumour activity of recombinant soluble Apo2 ligand. J Clin Invest 1999; 104: 155–62. 12. Gliniak B, Le T. Tumor necrosis factor-related apoptosis-inducing ligand’s antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Res 1999; 59: 6153–8. 13. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumour necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157–63. 14. Gura T. How TRAIL kills cancer cells but not normal cells. Cancer Res 1997; 277: 768. 15. Nguyen T, Thomas W, Zhang XD, Gray C, Hersey P. Immunologically-mediated tumour cell apoptosis: the role of TRAIL in T-cell and cytokine-mediated responses to melanoma. Forum 2000; 10: 243–52.
TRAIL EXPRESSION IN MELANOMA
16. Kayagaki N, Yamaguchi N, Nakayama M, Kawasaki A, Akiba H, Okumura KYH. Involvement of TNF-related apoptosis-inducing ligand in human T cell-mediated cytotoxicity. J Immunol 1999; 162: 2639–47. 17. Takeda K, Hayakawa Y, Smyth MJ. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells. Nat Med 2001; 7: 94–100. 18. Fanger NA, Maliszewski CR, Schooley K, Griffith TS. Human dendritic cells mediate cellular apoptosis via tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL). J Exp Med 1999; 190: 1155–64. 19. Griffith TS, Wiley SR, Kubin MZ, Sedger LM, Maliszewski CR, Fanger NA. Monocyte-mediated tumoricidal activity via the tumor necrosis factor related cytokine, TRAIL. J Exp Med 1999; 189: 1343–54. 20. Kemp TJ, Elzey BD, Griffith TS. Plasmacytoid dendritic cell-derived IFN-alpha induces TNF-related apoptosis-inducing ligand/Apo-2Lmediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation. J Immunol 2003; 171: 212–8. 21. Kayagaki N, Yamaguchi N, Nakayama M, Eto H, Okumura K, Yagita H. Type I (IFNs) regulate tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression on human T cells: A novel mechanism for the antitumor effects of type I IFNs. J Exp Med 1999; 189: 1451–60. 22. Gratas C, Tohma Y, Meir EGV, et al. Fas ligand expression in glioblastoma cell lines and primary astrocytic brain tumors. Brain Pathol 1997; 7: 863–9. 23. Niehans GA, Brunner T, Frizelle SP, et al. Human lung carcinoma express Fas Ligand. Cancer Res 1997; 57: 1007–12. 24. Shiraki K, Tsuji N, Shioda T, Isselbacher KJ, Takahashi H. Expression of Fas ligand in liver metastases of human colonic adenocarcinomas. Proc Natl Acad Sci USA 1997; 94: 6420–5.
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25. Hahne M, Rimoldi D, Schro¨ter P, et al. Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape. Science 1996; 274: 363–4. 26. O’Connell J, O’Sullivan GC, Collins JK, Shanahan F. The Fas counterattack: Fas-mediated T Cell killing by colon cancer cells expressing Fas ligand. J Exp Med 1996; 184: 1075–82. 27. Giovarelli M, Musiani P, Garotta G, et al. A ‘stealth effect’: Adenocarcinoma cells engineered to express TRAIL elude tumorspecific and allogeneic T cell reactions. J Immunol 1999; 163: 4886–93. 28. Azzola MF, Shaw HM, Thompson JF, et al. Tumor mitotic rate is more powerful prognostic indicator than ulceration in patients with primary cutaneous melanomas: an analysis of 3661 patients from a single center. Cancer 2003; 97: 1488–98. 29. Beatu TM, Kwon H, Sharma S, Garndvaux N, Hiscott J. Disruption of NF-kappaB signaling reveals a novel role for NF-kappaB in the regulation of TNF-related apoptosis-inducing ligand expression. J Immunol 2001; 167: 3164–73. 30. Dhawan P, Singh AB, Ellis DL, Richmond A. Constitutive activation of Akt/protein kinase B in melanoma leads to regulation of nuclear factor kappaB and tumor progression. Cancer Res 2002; 62: 7335–42. 31. Ehrhardt H, Fulda S, Schmid I, Hiscott J, Debatin KM, Jeremias I. TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-kappaB. Oncogene 2003; 22: 3842–52. 32. Griffith TS, Anderson RD, Davidson BL, Williams RD, Ratliff TL. Adenoviral-mediated transfer of the TNF-related apoptosis-inducing ligand/Apo-2 ligand gene induces tumor apoptosis. J Immunol 2000; 165: 2886–94. 33. Strebel A, Bachman F, Wernli M, Erb P. Tumor necrosis factorrelated apoptosis-inducing ligand supports growth of mouse mastocytoma tumors by killing tumor-infiltrating macrophages. Int J Cancer 2002; 100: 627–34.
ANATOMICAL PATHOLOGY
Histological expression of tumour necrosis factor-related apoptosisinducing ligand (TRAIL) in human primary melanoma LUC P. BRON*, RICHARD A. SCOLYER{{§, JOHN F. THOMPSON{§,
AND
PETER HERSEY{§}
*Sydney Head and Neck Cancer Institute, {Department of Anatomical Pathology, {Sydney Melanoma Unit and §the Melanoma and Skin Cancer Research Institute, Royal Prince Alfred Hospital, Sydney, ,Discipline of Surgery, Faculty of Medicine, University of Sydney, Sydney and }Oncology and Immunology Unit, Newcastle Mater Hospital, Newcastle, NSW, Australia
Summary Aims: Tumour necrosis factor-related apoptosis ligand (TRAIL) appears to selectively induce apoptosis in a wide range of cultured malignant cells, including melanoma. This study was designed to attempt to clarify the role of TRAIL in the biology of human melanoma. Methods: Tissue sections cut from formalin-fixed, paraffin-embedded tissue blocks of 45 primary cutaneous melanomas were tested for expression of TRAIL using immunohistochemistry. The intensity, pattern of staining and percentage of positively stained tumour cells were evaluated in each melanoma. Breslow thickness, ulcerative state, dermal mitotic rate and the presence of tumour infiltrating lymphocytes were measured/determined in each case. Median follow up for the cohort of patients was 10 months (range 1–18). Survival analysis was conducted using the Kaplan–Meier method. The level of expression of TRAIL was compared with the various histological determinants using the two-tailed Fisher’s exact test and the x2-test. Results: Overall and disease-free survival were 72 and 48%, respectively, and did not correlate with TRAIL expression. Among the pathological prognostic determinants, only mitotic rate showed a statistically significant correlation with TRAIL expression using the x2-test (P~0.04). Conclusion: We conclude that TRAIL expression in melanoma defines a more aggressive/proliferative phenotype, either through selection of apoptotic resistant cells or by secondary induction of other factors enhancing proliferation of more malignant cells. Analysis of a larger group of patients with longer follow-up is required to determine whether TRAIL expression correlates with survival of patients. Key words: Tumour necrosis factor-related apoptosis-inducing ligand, tumor necrosis factor-related apoptosis-inducing ligand, TRAIL, cutaneous melanoma, apoptosis, pathology. Received 12 January, revised and accepted 26 May 2004
INTRODUCTION Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that, like TNF-a and Fas ligand, is a type II membrane protein that can induce apoptotic cell death in a variety of cell types.1–3
TRAIL appears to be particularly important because it can induce apoptosis in a wide range of cultured malignant cells but not in normal tissues,4–9 with the possible exception of some human liver cells.10 The potential importance of TRAIL as an anticancer agent has been supported by studies in animal models showing selective toxicity to transplanted human tumours but not to normal tissues.11–13 Induction of apoptosis by TRAIL is believed to be mediated by interaction with two death receptors on cells referred to as TRAIL-R1 and TRAIL-R2 (see Wiley et al.3 for alternate nomenclature). Normal cells are postulated to be protected from TRAIL-induced apoptosis by their expression of TRAIL-R3 and TRAIL-R4, which lack cytoplasmic death domains and act to sequester TRAIL (decoy receptors) or to mediate anti-apoptotic signals.4–14 We have shown previously that receptors for TRAIL were expressed on cultured melanoma and were able to induce varying degrees of apoptosis in approximately twothirds of the melanoma cell lines tested.7–9 Sensitivity of melanoma cells to TRAIL-induced apoptosis showed an overall correlation with the level of death receptors, particularly TRAIL-R2 expression, but did not correlate with the level of expression of the decoy receptors, TRAIL-R3 and TRAIL-R4. In previous studies we15 and others16 have shown that TRAIL is expressed on activated lymphocytes and is upregulated on CD4- and CD8-positive T cells,15,16 NK cells,17 dendritic cells and monocytes18,19 by IFN-a, IL-2, IL-15 and IFN-c.20,21 TRAIL-induced apoptosis, therefore, appears to represent a second major cytotoxic mechanism used by lymphocytes in addition to or in place of the perforin granzyme system. Fas ligand (FasL) is another member of the TNF family that is expressed on lymphocytes and, like TRAIL, can induce apoptosis of certain cultured cancer cell lines that express receptors for Fas (CD95). A number of studies have shown that FasL is also expressed on tumour cells,22–24 including human melanoma.25 The latter authors also reported that the expression of FasL on melanoma cells could induce apoptosis of effector T lymphocytes and suggested that FasL could contribute to immune privilege of tumours. Similarly, human colon cancer cells were reported to express FasL and were capable of inducing apoptosis in Jurkat T cells but not colon carcinoma cells.26 Studies by others supported this concept of immune privilege.
ISSN 0031-3025 printed/ISSN 1465–3931 # 2004 Royal College of Pathologists of Australasia DOI: 10.1080/00313020400011268
562
Pathology (2004), 36(6), December
BRON et al.
TABLE 1 Clinical and pathological characteristics of the melanoma patients and their tumours Characteristics
No. of patients
Percentage
45
68 (23–83)*
29 16
64 36 3.5 (0.1–16)* 60 40
Age Gender Male Female Breslow thickness w1 mm v1 mm Ulcerative state Present Absent Dermal tumour mitotic rate (per mm2)
27 18 9 36 45
20 80 2 (0–22)*
Sydney. Five-mm thick sections were cut from a representative tissue block of each tumour. Sections were deparaffinised in xylene and then rehydrated through graded decreasing concentrations of alcohol. They were then incubated with rabbit antihuman TRAIL antibody (IgG isotype; Santa Cruz, USA) at appropriate concentration in PBS. Binding sites of the primary antibodies were visualised using the Vectastain ABC Kit (Vector Laboratories, USA) according to the manufacturer’s instructions. Finally, the sections were faintly counterstained with H&E and mounted with aqueous media. Negative controls were performed by omission of the primary antibody. Once stained, slides of melanoma were analysed by one pathologist (RAS), who had no prior knowledge of the clinical data. Percentage of positively stained cells (0–100%), intensity and pattern of staining, on a scale of 0–3 was estimated and recorded. The presence of tumour infiltrating lymphocytes was also evaluated in each case.
*Median (range). Statistical analysis
The similarity between the functional role of FasL and TRAIL raises the question as to whether TRAIL expression on tumours may also cause the death of immune effector cells. This appeared to be so in that murine adenocarcinoma cells engineered to express TRAIL were more resistant to immune killing both in vitro and in vivo.27 In view of these findings, we examined TRAIL expression in tissue sections taken from a series of primary melanomas to determine if TRAIL expression was correlated with important prognostic determinants in primary melanoma.
Statistical analysis was performed with JMP Statistics Made Visual software (SAS Institute Inc., USA). Correlation between TRAIL expression and other histological features of the melanomas were performed using the two-tailed Fisher’s exact test or the x2-test, as appropriate. The correlation of TRAIL expression to disease-free survival and overall survival was calculated by the Kaplan–Meier estimate. Multivariate analyses were performed using the Cox stepwise-regression analysis to determine the independent relationship between TRAIL expression and pathological prognostic determinants of primary melanoma.
RESULTS PATIENTS AND METHODS Patients Histological sections were cut from formalin-fixed, paraffin-embedded tissue blocks of primary cutaneous melanomas from 45 patients treated at the Sydney Melanoma Unit over the period from December 2000 to March 2002. Demographic details of the patients and histological features of their primary melanomas are summarised in Table 1. Depending on their Breslow thickness, primary melanomas were separated into thin (v1 mm) and thick (w1 mm) melanomas. All patients had regular follow up at the Sydney Melanoma Unit. Median follow-up was 10 months (range 1–18). Immunohistochemistry Archival paraffin blocks of the melanomas were retrieved from the Department of Anatomical Pathology, Royal Prince Alfred Hospital,
Fig. 1
TRAIL expression in primary melanomas Forty-five melanomas were examined for TRAIL expression. Patient demographics and histological characteristics are described in Table 1. Overall, 33 cases (74%) showed some TRAIL expression of varying intensity and 12 tumours (26%) were completely negative. Among the positive cases, two tumours (4%) showed focal expression of TRAIL (v25% of cells), 15 (33%) showed expression in 26–50% of cells, seven (16%) showed expression in 51–75% of tumour cells and nine (20%) had a high expression of TRAIL (w75% of cells). Examples of TRAIL expression in sections from primary melanoma are shown in Fig. 1. In our series of melanomas, there were 14 nodular melanomas, 10 superficial spreading melanomas, six Hutchinson’s melanotic freckles, three desmoplastic
Example of negative, mild and strong TRAIL expression in melanoma (TRAIL immunohistochemical stains, original magnification, 6400).
TRAIL EXPRESSION IN MELANOMA
TABLE 2
563
Summary of TRAIL expression among 45 cases of melanoma and its correlation with histopathological features of the primary tumour
TRAIL expression (% cells) Pathological feature of primary melanoma
0–25%
Breslow thickness v1 mm 6 w1 mm 8 Tumour infiltrating lymphocytes Present 8 Absent 6 2 Tumour mitotic rate (per mm ) v6 4 §6 10 Ulcerative state Present 11 Absent 3 No. of cases (%) 14 (31)
26–50%
51–75%
76–100%
Total
Significance of correlation of TRAIL expression with histopathological features P value
5 10
4 3
3 6
18 27
0.73
9 6
5 2
7 2
29 16
0.53
3 12
1 6
5 4
13 32
0.06
14 1 15 (33)
4 3 7 (16)
7 2 9 (20)
36 9 45
0.73
melanomas. In 12 lesions there were overlapping features between two or more subtypes. The expression of TRAIL did not correlate with the various histological subtypes of melanomas (data not shown). Relationship of TRAIL expression to important prognostic determinants in primary melanoma Recurrence and overall survival for melanoma patients are strongly related to various histological characteristics of the primary melanoma. Thickness, ulceration, and tumour dermal mitotic rate are the most important histological determinants. We reasoned that should TRAIL expression be related to prognosis, it might be expected to correlate with these determinants. A reduction in tumour-infiltrating lymphocytes (TILs) might also be expected in melanoma with strong TRAIL expression. These different histological parameters were examined by contingency table and regression analysis to determine their correlation with TRAIL expression (Table 2). Expression of TRAIL was not correlated with thickness or ulcerative state of the melanoma or the presence of tumour infiltrating lymphocytes. Only mitotic rate of the melanoma showed significant correlation with TRAIL expression using the x2-test (P~0.04; Fig. 2). However, when the level of TRAIL expression was divided into four groups (v25, 26–50, 51–75 and w75%), the association between TRAIL expression and mitotic rate was not statistically significant (P~0.06) using Fisher’s exact test (Table 2). Logistic regression analysis did not show that TRAIL expression was independently correlated with tumour mitotic rate. Relationship of TRAIL expression with clinical outcome In our series, 2-year overall and disease-free survival rates were 72 and 48%, respectively. During the follow-up period, 12 patients (26%) developed recurrent melanoma. Among those, eight cases (18%) were in transit or distant metastases. Despite the relatively short follow-up period of the cases included in this study, Breslow thickness showed a significant adverse correlation with disease-free survival (Pv0.02). There was no significant correlation of either the other histopathological factors or of TRAIL expression with clinical outcome (Fig. 3).
Fig. 2 Strong TRAIL expression in a case with multiple mitoses (arrowheads) (TRAIL immunohistochemical stain, original magnification, 6400).
Fig. 3 Disease-specific survival of patients with TRAIL-positive versus TRAIL-negative primary melanomas.
BRON et al.
Pathology (2004), 36(6), December
DISCUSSION
melanoma-reactive T cells may have allowed the overgrowth of more rapidly growing melanoma cells. A larger number of patients and longer follow-up time would be needed to assess whether TRAIL expression is associated with a decrease in disease-free survival.
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This study was initiated to test the hypothesis that TRAIL expression in primary melanoma may be an adverse prognostic feature due to inhibition of effector T-cell responses against the tumour and the creation of immune privileged sites. If this was the case, an association with important histopathological prognostic features such as Breslow thickness, ulcerative state or dermal tumour mitotic rate in primary melanoma might be expected. The results above show that TRAIL expression was significantly correlated only to the mitotic rate of the primary melanoma (P~0.04) and, consequently, is most likely an adverse prognostic indicator in melanoma. The likely reason for the loss of significance of the correlation between TRAIL expression and mitotic rate when the level of TRAIL expression was divided into four groups is the small number of patients in some of the groups (Table 2). Even though it is a recognised adverse prognostic factor for survival,28 mitotic rate in our small group of patients was not significantly associated with disease-free survival (P~0.49). Several explanations could be proposed to account for the correlation of TRAIL expression with mitotic rate. One would be that the signal pathway and transcription factors acting to increase cell division in the tumour may also act on promoters controlling TRAIL expression. NF-KB is one factor involved in upregulation of TRAIL29 and activation of NF-KB in situ is associated with progression of melanoma.30 A second possibility is that TRAIL expression may have resulted in destruction (by apoptosis) of TRAIL-sensitive melanoma cells and selected TRAIL-resistant melanoma cells, and that resistance mechanisms involved pathways that activate cell division. Evidence for such a role was described in studies in leukaemia31 and other cancer cells.32 TRAIL expression on mastocytoma cells was reported to support their growth by destruction of macrophages.33 In either case, the result is consistent with an association between TRAIL expression and more aggressive melanoma. TRAIL expression in melanoma did not show any correlation with the presence of tumour infiltrating lymphocytes; however, it is possible that such an association may only be seen in the early stages of evolution of melanoma. At later stages, TRAIL-expressing melanoma cells could be expected to delete TRAIL death receptor-expressing lymphocytes, leaving only non-reactive lymphocytes infiltrating into the tumour. In several animal models, expression of TRAIL on tumour cells has been shown to be associated with reduced tumour growth. In one model, this was due to induction of apoptosis in the tumour cells.32 In another model, FasL was believed to induce an antitumour effect due to infiltration by neutrophils. It is not common to see neutrophils around primary melanoma so that the latter is unlikely in human melanoma. We conclude, on the evidence available, that TRAIL expression on primary melanoma appears to define a more aggressive/proliferative phenotype. It is not possible to conclude if this is secondary to other factors responsible for the development of such malignant cells or whether TRAIL is an initiator of the phenotype due to selection of apoptotic resistant cells. It is also not possible to exclude the possibility that TRAIL-mediated deletion of
ACKNOWLEDGEMENTS We wish to thank Ashley Young for technical assistance in cutting the melanoma sections, Dr Lawrence Li and the Department of Pathology, University of Sydney, for technical assistance, and the Sydney Melanoma Unit for the use of their computerised patients database. This work was supported by a Technical Assistance Grant from the Royal College of Pathologists of Australasia (Dr Richard Scolyer), a NSW State Cancer Council Grant (Professor Peter Hersey), the Melanoma and Skin Cancer Research Institute and the Melanoma Foundation of the University of Sydney. Dr Luc Bron was partially funded during his fellowship by a grant from the foundation of Decker and Emma Muschamp, Switzerland. Address for correspondence: Professor P. Hersey, Immunology and Oncology Unit, Room 443, David Maddison Clinical Sciences Building, Cnr King and Watt Street, Newcastle, NSW 2300, Australia. E-mail: [email protected]
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