- Email: [email protected]
Keyhole limpet hemocyanin: an effective adjunct against melanoma in vivo
The American Journal of Surgery 194 (2007) 628 – 632
Presentation
Keyhole limpet hemocyanin: an effective adjunct against melanoma in vivo Irfan Rizvi, M.D., Dale R. Riggs, B.S., Barbara J. Jackson, B.A., David W. McFadden, M.D.* Department of Surgery, Robert C. Byrd Health Science Center, West Virginia University, Morgantown, WV 26506, USA Manuscript received May 23, 2007; revised manuscript August 7, 2007 Presented at the 31st Annual Surgical Symposium of the Association of VA Surgeons, Little Rock, AR, May 10 –12, 2007
Abstract Background: We have previously demonstrated the potent in vitro antiproliferative effects of keyhole limpet hemocyanin (KLH) against melanoma. Our prior studies directed us to hypothesize that KLH would be effective in vivo against melanoma, alone and in combination with conventional immunotherapy. Methods: Mice were inoculated with 2 ⫻ 107 HTB68 cells and randomized to 6 groups. Treatment groups consisted of control, KLH 200 g, alpha interferon (AIFN) 1000 IU, interleukin-2 (IL-2) 5000 IU, KLH ⫹ AIFN, and KLH ⫹ IL-2. Results: KLH ⫹ IL-2 exhibited the greatest reduction in tumor volume (30%) as compared to control (P ⫽ .014), followed by KLH ⫹ AIFN (28%, P ⫽ .031). Singly treated animals had less tumor inhibition: IL-2 (30%, P ⫽ .022), KLH (18%, not significant), and AIFN (16%, not significant). Conclusions: KLH augments the effects of AIFN, one of the standard immunotherapeutic agents against melanoma in vivo. Further in vivo and early clinical studies into the effects of KLH as both a single and combined agent are warranted. © 2007 Excerpta Medica Inc. All rights reserved. Keywords: Keyhole limpet hemocyanin; Melanoma; Immunotherapy; In vivo
An estimated 62,000 new cases of melanoma were diagnosed in the United States in 2006, with approximately 8,000 disease-related deaths [1]. Surgical resection of locoregional disease, followed by immunotherapy, chemotherapy, and or radiation therapy, is the standard treatment regimen for these patients [2,3]. However, many patients will not respond to therapy and others will have recurrence or progression of disease. For this reason, new and more effective forms of treatment are currently being investigated. Keyhole limpet hemocyanin (KLH) is a high-molecularweight, copper-containing protein found in the sea mollusk Megathura crenulata [4]. Its primary biological role is the uptake, transport, and release of oxygen during respiration. KLH has been shown to enhance the host immune response by interacting with monocytes, macrophages, polymorpho-
* Corresponding author. Department of Surgery, University of Vermont College of Medicine, Fletcher House 301, 111 Colchester Ave., Burlington, VT 05401. Tel.: ⫹1-802-847-5354; fax: ⫹1-802-847-5552. E-mail address: [email protected]
nuclear lymphocytes, and T cells [5] KLH appears to be a non-specific immune stimulant that induces both a cellmediated and humoral response. KLH has been used as a form of therapy for patients with superficial bladder cancer for more than 30 years. Olsson et al immunized patients with 5 mg of KLH and observed a marked reduction in the recurrence of superficial bladder cancer [6]. Riggs et al reported reduced tumor growth and prolonged survival in a MBT-2 murine model of transitional cell carcinoma in mice [7]. In a clinical trial of KLH and mitomycin C chemotherapy, KLH was superior in preventing bladder tumor recurrence with no adverse local or systemic side effects [8,9]. Multicenter clinical trials have confirmed the efficacy of KLH given intravesically for 6 weeks to patients with various stages of bladder cancer [10]. Based on these studies, KLH is regarded as a safe and effective immunotherapy for superficial bladder cancer. Our laboratory has previously shown that KLH inhibits cellular proliferation in vitro in human cancer cell lines of the breast, esophagus, pancreas, and prostate [11,12]. We have shown KLH to alter tumor cytokine production in both breast and pancreatic cancer in vitro, in part explaining its
0002-9610/07/$ – see front matter © 2007 Excerpta Medica Inc. All rights reserved. doi:10.1016/j.amjsurg.2007.08.005
I. Rizvi et al. / The American Journal of Surgery 194 (2007) 628 – 632
effectiveness in isolated cell culture systems [13]. KLH also has been shown to decrease the cellular proliferation of Barrett’s esophageal adenocarcinoma and enhance its apoptotic activity [14,15]. We previously reported a significant reduction in cellular proliferation with the treatment of KLH (range, 12%– 60%) in the HTB68 melanoma cell line in vitro [12]. Immunologic therapies, specifically alpha interferon (AIFN) and interleukin-2 (IL-2), have been used in melanoma treatment for several years. High-dose interferon alfa-2b has been shown to increase both times to recurrence and survival time as compared to patients receiving no further treatment [16]. Additionally, IL-2 has been shown to be effective in the treatment of metastatic melanoma. In an analysis of 270 patients who received high-dose IL-2 for treatment of melanoma, Atkins et al found that both diseasefree and survival time were increased [17]. Given the limited success of the current therapeutic options for patients with advanced melanoma and the high incidence of toxic side effects related to such therapies new forms of immunemediated treatments are being investigated. Recently, we have shown significant growth inhibition of melanoma in vitro induced by treatment with KLH, AIFN, and IL-2 when administered as single agents [18]. These effects were significantly enhanced, ie, a statistical additive effect was noted, when these agents were combined. This formed the premise for our present study where effects of these immune modulators were studied in vivo. Materials and Methods Animals An initial KLH dose-response titration study was performed. Forty-one nude female (nu/nu) 6- to 7-week-old athymic mice were housed in the West Virginia University Office of Laboratory Animal Research and allowed to acclimatize for a period of 2 weeks. Subsequently, on experimental day 0, the mice received HTB68 tumor inoculation. The tumor inoculation consisted of a single subcutaneous injection of HTB68 cells (2 ⫻ 107 cells) on the flank of the left thigh. Subsequently, mice in the saline group (negative control, N ⫽ 11), KLH groups (50 g [N ⫽ 10], 100 g [N ⫽ 10], and 200 g subcutaneously on the flank of the left thigh [N ⫽ 10]) received treatments on day 2, 4, 7, 9, 11, and 16 after tumor inoculation. Upon completion of this feasibility study, 60 additional mice were obtained, acclimatized, and housed under identical conditions and maintained by veterinarian and trained staff. Mice were also monitored throughout the length of the study by research staff. Tumor cells The HTB68 human melanoma cell line was purchased from ATCC (Manassas, VA) and is tumorigenic in the nude mouse model. HTB68 cells for transplantation were grown under standard conditions (Eagle’s minimum essential media supplemented with 10% fetal bovine serum). The cells, when confluent, were trypsinized and then counted in .02% Trypan Blue and percent cell viability and viable cell concentration was determined.
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Tumor transplant and evaluation A single cell suspension containing 2 ⫻ 107 HTB68 cells in .1 mL tissue culture media was injected subcutaneously into the left flank. The dose of tumor inoculum was determined by performing in vivo tumor titrations of tumor cell suspensions in our feasibility study and choosing the dosage that assured a tumor incidence of at least 90% in untreated animals. Tumor incidence, as indicated by the first identifiable presence of tumor growth; this is before it is measurable by calipers and was evaluated by inspection and palpation. Tumor dimensions (length and width) were measured twice a week with a Vernier caliper. Tumor volume was calculated using the formula V ⫽ .4(LW2) in mm3. Alpha interferon AIFN was supplied as a lyophilized powder from Schering Corporation (Kenilworth, NJ). AIFN was resuspended to the desired assay concentrations in tissue culture medium. The AIFN concentration tested was 1000 IU per animal. Interleukin-2 IL-2 was supplied as a lyophilized powder from Chiron Corp (Emeryville, CA). IL-2 was resuspended to the desired assay concentrations in tissue culture medium. The IL-2 concentration tested was 5000 IU per animal. Keyhole limpet hemocyanin KLH was provided by Stellar Technologies (Port Hueneme, CA) in suspension at a protein concentration of 23.88 mg/mL. The KLH concentration tested was 200 g per animal. KLH was combined with standard immunotherapies for melanoma (AIFN and IL-2) to evaluate possible additive and/or synergistic effects. Combined pmmunotherapy with KLH and standard immunotherapy Mice were inoculated with 2 ⫻ 107 HTB68 cells on the left flank and randomized to 6 groups. Experimental treatments began on experimental day 1 and were administered for the next 5 weeks, 3 times per week. This treatment schedule was adjusted based on the results of the initial KLH titration and will be discussed in the results. The experimental groups tested were as follows: control (N ⫽ 10), saline injections (.1 mL), KLH 200 g (N ⫽ 10) experimental therapy (.1 mL), AIFN 1000 IU (N ⫽ 10) experimental therapy (.1 mL), IL-2 5000 IU (N ⫽ 10) experimental therapy (.1 mL), KLH and AIFN 1000 IU (N ⫽ 10) experimental therapy (.1 mL), and KLH and IL-2 5000 IU (N ⫽ 10) experimental therapy (.1 mL). Tumors were measured in standard fashion and tumor volume was calculated according to the aforementioned formula for the volume of an ellipse. Statistical analysis Determination of statistical significance was performed by analysis of variance [19]. Post hoc comparison of individual concentration means with the control was completed using the Tukey-Kramer multiple comparison test [20]. All data are reported as means ⫾ SE.
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I. Rizvi et al. / The American Journal of Surgery 194 (2007) 628 – 632 Table 1 Statistical comparisons of tumor volume (mm3) on days 24 and 29 post tumor inoculation Tumor volume Day 24
Control KLH AIFN IL-2 KLH ⫹ AIFN KLH ⫹ IL-2 ANOVA Fig. 1. Effects of KLH (50, 100, and 200 g) on tumor incidence in the melanoma-induced in vivo mouse model. *Statistical significance, P ⬍ .001.
Results KLH dose-response titration Tumor Incidence: The KLH 200 g group exhibited a 70% reduction in tumor incidence compared to the control group on day 16 post tumor inoculation (P ⬍ .001). Neither of the lower doses, KLH 100 g (30% reduction in tumor incidence) and KLH 50 g (0% reduction in tumor incidence), exhibited significant reductions in tumor incidence As illustrated in Fig. 1, once treatment with 200 g of KLH was suspended, tumor incidence quickly approached that of the untreated control and the beneficial suppressive effect of KLH was overcome. Tumor Volume: As illustrated in Fig. 2, during the pilot tumor incidence study, KLH was ineffective in reducing tumor growth after treatment was completed on day 16. Thereafter, tumors actually grew at a pace that produced tumor volumes (mm3) that were higher than control (P ⬍ .05). Statistically significant differences were noted at days 23, 28, and 30 in the KLH 100 g group. Combined Immunotherapy with KLH and Standard Immunotherapy: Based on our observations from the tumor incidence study, the second experiment employed a dose of
Fig. 2. Effects of KLH (50, 100, and 200 g) on tumor volume (mm3) in the melanoma-induced in vivo mouse model. Data shown are means ⫾ SE. P values indicate statistical significance versus the control.
Day 29
Mean ⫾ SE
P value
Mean ⫾ SE
P value
2,418 ⫾ 222 1,808 ⫾ 150 1,941 ⫾ 185 1,799 ⫾ 185 1,879 ⫾ 115 1,630 ⫾ 96
— NS NS NS NS P ⫽ .015 P ⫽ .030
3,349 ⫾ 288 2,732 ⫾ 246 2,830 ⫾ 222 2,357 ⫾ 183 2,393 ⫾ 138 2,408 ⫾ 141
— NS NS P⫽ P⫽ P⫽ P⫽
.023 .025 .028 .011
ANOVA ⫽ analysis of variance.
200 g of KLH and the treatment schedule was further extended to allow for KLH to exert its anti-tumor properties initially observed in regards to reduction in tumor incidence. On day 24 post tumor inoculation the combination of KLH ⫹ IL-2 (1,630 ⫾ 96) significantly reduced tumor volume when compared to control (2,418 ⫾ 222, P ⫽ .015, Table 1). At day 29, tumor volume was significantly reduced by IL-2 (2,357 ⫾ 183, P ⫽ .023), KLH ⫹ AIFN (2,393 ⫾ 138, P ⫽ .025), and KLH ⫹ IL-2 (2,408 ⫾ 141, P ⫽ .028). Looking at the percent change in tumor volume when compared to the control, on day 29 after tumor inoculation, all single agents reduced tumor volume compared with the control group (Fig. 3). IL-2 exhibited the greatest degree of reduction (30% ⫾ 6%, P ⫽ .022), followed by KLH (18% ⫾ 7%, not significant [NS]) and AIFN (16% ⫾ 7%, NS). Combination therapy of KLH with IL-2 reduced tumor volume to the greatest extent (30% ⫾ 4%, P ⫽ .014). This was followed by the combination of KLH ⫹ AIFN (28% ⫾ 4%, P ⫽ .031). Single-treated animals had less tumor inhibition than the combination groups.
Fig. 3. Percent reduction in tumor volume at day 29 from the effects of KLH (200 g), IL-2 (5000 IU), and AIFN (1000 IU) as single agents and in combination when compared to controls in the melanoma-induced in vivo mouse model. Data shown are means ⫾ SE. P values indicate statistical significance versus the control.
I. Rizvi et al. / The American Journal of Surgery 194 (2007) 628 – 632
Comments Cutaneous melanoma is a malignant neoplasm with an unpredictable behavior, ranging from spontaneous regression to rapid progression. Early detection can ensure effective treatment. Most cases are diagnosed in adults, and treatment planned based on tumor grade and stage. Surgical resection remains the standard of care followed by immunotherapy, chemotherapy, and/or radiation therapy for advanced disease. Adjuvant treatment for melanoma includes chemotherapy, immunotherapy, and radiation therapy for more advanced disease. Many clinical research trials currently underway employ the use of immunotherapies, usually AIFN or IL-2, in an effort to improve treatment outcomes. Kirkwood et al found that interferon alfa-2b was the first agent to show a significant benefit in relapse-free and overall survival of high-risk melanoma patients as compared to those who received no further treatment [16]. The same investigators found, in a subsequent randomized trial that interferon alfa-2b increased both time to recurrence and survival when compared to patients receiving ganglioside vaccine [21]. While these results are both impressive and significant, treatment with highdose interferon alfa-2b is not without significant side effects and cost. Moreover, the efficacy of interferon alfa-2b has not been validated consistently by other groups [22]. In addition to AIFN, IL-2 has been shown to be effective in the treatment of metastatic melanoma. Atkins et al found in an analysis of 270 patients who received high-dose IL-2 for treatment of malignant melanoma, that both disease-free and survival time were increased [17]. Given the limited success of the current therapeutic options for patients with melanoma, the search for new treatments must be continued. These promising results have led us to investigate the effects of KLH in combination with other conventional immunotherapies administered as standard treatment regimens in diseased patients. In this study, melanoma was induced in athymic mice by inoculation with HTB68 melanoma cell line. In the initial feasibility study, we were able to demonstrate that KLH, at a dose of 200 g per animal, inhibited tumor appearance (P ⬍ .001) until injections were stopped at day 16 post implant. Subsequently, 60 mice were separated in groups and were treated with KLH, AIFN, and IL-2 alone and in combination. Tumor volumes were then calculated in different treatment groups. Our results show that, as single agents, IL-2 reduced tumor volume by 30%, KLH by 18%, and AIFN by 16%. Combination therapy with KLH and IL-2 reduced tumor volume by 30% followed by KLH and AIFN at 28%. Although the combination therapies exhibited a greater reduction in tumor volume, these results did not meet the statistical criteria for synergism. However, an additive effect was noted for KLH and AIFN in combination. This additive effect was not seen in the KLH ⫹ IL-2 animals, possibly related to the predominance of IL-2 effects on T cells, and our use of an athymic, T-cell– deficient, mouse model for our study. Additional studies employing doseresponse analysis will be performed to amplify these promising results.
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Biological agents like KLH and inositol hexaphosphate are being studied increasingly as adjuvant treatments to melanoma [23]. Given the clinical safety and efficacy of KLH in numerous trials in human bladder cancer and the promising initial results we have obtained in this very aggressive model of melanoma, further studies and trials are definitely warranted with KLH. Its administration may allow the use of lower doses of conventional immunotherapy against melanoma, thereby reducing costs and toxicity while enhancing the quality of life of patients suffering with this malignancy. References [1] Jemal A, Siegal R, Ward E, et al. Cancer Statistics, 2006. CA Cancer J Clin 2006;56:106 –30. [2] McGillis ST, Fein H. Topical treatment strategies for non-melanoma skin cancer and precursor lesions. Semin Cutan Med Surg 2004;23: 174 – 83. [3] Villa AM, Berman B. Immunomodulators for skin cancer. J Drugs Dermatol 2004;3:533–9. [4] Harris JR, Markl J. Keyhole limpet hemocyanin: Molecular structure of a potent marine immunoactivator. A review. Eur Urol 2000; 37(suppl 3):24 –33. [5] Harris JR, Markl J. Keyhole limpet hemocyanin (KLH): a biomedical review. Micron 1999;30:597– 623. [6] Olsson CA, Chute R, Rao CN. Immunologic reduction of bladder cancer recurrence rate. J Urol 1974;111:173– 6. [7] Riggs DR, Tarry WF, DeHaven JI, et al. Immunotherapy of murine transitional cell carcinoma of the bladder using alpha and gamma interferon in combination with other forms of immunotherapy. J Urol 1992;147:212– 4. [8] Jurincic CD, Engelmann U, Gasch J, et al. Immunotherapy in bladder cancer with keyhole-limpet hemocyanin: a randomized study. J Urol 1988;139:723– 6. [9] Jurincic-Winkler CD, Metz KA, Beuth J, et al. Keyhole limpet hemocyanin for carcinoma in situ of the bladder: a long-term follow-up study. Eur Urol 2000;37(suppl 3):45–9. [10] Lamm DL, DeHaven JI, Riggs DR. Keyhole limpet hemocyanin immunotherapy of bladder cancer: laboratory and clinical studies. Eur Urol 2000;37(suppl 3):41– 4. [11] Riggs D, Jackson B, Vona-Davis L, et al. In vitro anticancer effects of a novel immunostimulant: keyhole limpet hemocyanin. J Surg Res 2002;108:279 – 84. [12] Somasundar P, Riggs DR, Jackson BJ, et al. Inhibition of melanoma growth by hemocyanin occurs via early apoptotic pathways. Am J Surg 2005;190:713– 6. [13] Riggs DR, Jackson BJ, Vona-Davis L, et al. In vitro effects of keyhole limpet hemocyanin in breast and pancreatic cancer in regards to cell growth, cytokine production, and apoptosis. Am J Surg 2005; 189:680 – 4. [14] McFadden DW, Riggs DR, Jackson BJ, et al. Keyhole limpet hemocyanin, a novel immune stimulant with promising anticancer activity in Barrett’s esophageal adenocarcinoma. Am J Surg 2003;186:552–5. [15] Vona-Davis L, Vincent T, Zulfiqar S, et al. Proteomic analysis of SEG-1 human Barrett’s-associated esophageal adenocarcinoma cells treated with keyhole limpet hemocyanin. J Gastrointest Surg 2004;8: 1018 –23. [16] Kirkwood JM, Strawderman MH, Ernstoff MS, et al. Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group trial EST 1684. J Clin Oncol 1996;14:7–17. [17] Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol 1999; 17:2105–16. [18] McFadden DW, Riggs DR, Jackson BJ, et al. Keyhole limpet hemocyanin potentiates standard immunotherapy for melanoma. Am J Surg 2007;193:284 –7. [19] Ludbrook J. Multiple comparison procedures updated. Clin Exp Pharmacol Physiol 1998;25:1032–7.
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[20] Dixon WJ, Massey FJ. Introduction to Statistical Analysis. 4th ed. New York: McGraw-Hill; 1983. [21] Kirkwood JM, Ibrahim J, Lawson DH, et al. High-dose interferon alfa-2b does not diminish antibody response to GM2 vaccination in patients with resected melanoma: results of the Multicenter Eastern Cooperative Oncology Group phase II trial E2696. J Clin Oncol 2001;19:1430 – 6.
[22] Eggermont AM, Suciu S, Mackie R, et al. Post-surgery adjuvant therapy with intermediate doses of interferon alfa 2b versus observation in patients with stage IIb/III melanoma (EORTC 18952): randomized controlled trial. Lancet 2005;366:1189 –96. [23] Rizvi I, Riggs DR, Jackson BJ, et al. Inositol hexaphosphate (IP6) inhibits cellular proliferation in melanoma. J Surg Res 2006;133: 3– 6.
Presentation
Keyhole limpet hemocyanin: an effective adjunct against melanoma in vivo Irfan Rizvi, M.D., Dale R. Riggs, B.S., Barbara J. Jackson, B.A., David W. McFadden, M.D.* Department of Surgery, Robert C. Byrd Health Science Center, West Virginia University, Morgantown, WV 26506, USA Manuscript received May 23, 2007; revised manuscript August 7, 2007 Presented at the 31st Annual Surgical Symposium of the Association of VA Surgeons, Little Rock, AR, May 10 –12, 2007
Abstract Background: We have previously demonstrated the potent in vitro antiproliferative effects of keyhole limpet hemocyanin (KLH) against melanoma. Our prior studies directed us to hypothesize that KLH would be effective in vivo against melanoma, alone and in combination with conventional immunotherapy. Methods: Mice were inoculated with 2 ⫻ 107 HTB68 cells and randomized to 6 groups. Treatment groups consisted of control, KLH 200 g, alpha interferon (AIFN) 1000 IU, interleukin-2 (IL-2) 5000 IU, KLH ⫹ AIFN, and KLH ⫹ IL-2. Results: KLH ⫹ IL-2 exhibited the greatest reduction in tumor volume (30%) as compared to control (P ⫽ .014), followed by KLH ⫹ AIFN (28%, P ⫽ .031). Singly treated animals had less tumor inhibition: IL-2 (30%, P ⫽ .022), KLH (18%, not significant), and AIFN (16%, not significant). Conclusions: KLH augments the effects of AIFN, one of the standard immunotherapeutic agents against melanoma in vivo. Further in vivo and early clinical studies into the effects of KLH as both a single and combined agent are warranted. © 2007 Excerpta Medica Inc. All rights reserved. Keywords: Keyhole limpet hemocyanin; Melanoma; Immunotherapy; In vivo
An estimated 62,000 new cases of melanoma were diagnosed in the United States in 2006, with approximately 8,000 disease-related deaths [1]. Surgical resection of locoregional disease, followed by immunotherapy, chemotherapy, and or radiation therapy, is the standard treatment regimen for these patients [2,3]. However, many patients will not respond to therapy and others will have recurrence or progression of disease. For this reason, new and more effective forms of treatment are currently being investigated. Keyhole limpet hemocyanin (KLH) is a high-molecularweight, copper-containing protein found in the sea mollusk Megathura crenulata [4]. Its primary biological role is the uptake, transport, and release of oxygen during respiration. KLH has been shown to enhance the host immune response by interacting with monocytes, macrophages, polymorpho-
* Corresponding author. Department of Surgery, University of Vermont College of Medicine, Fletcher House 301, 111 Colchester Ave., Burlington, VT 05401. Tel.: ⫹1-802-847-5354; fax: ⫹1-802-847-5552. E-mail address: [email protected]
nuclear lymphocytes, and T cells [5] KLH appears to be a non-specific immune stimulant that induces both a cellmediated and humoral response. KLH has been used as a form of therapy for patients with superficial bladder cancer for more than 30 years. Olsson et al immunized patients with 5 mg of KLH and observed a marked reduction in the recurrence of superficial bladder cancer [6]. Riggs et al reported reduced tumor growth and prolonged survival in a MBT-2 murine model of transitional cell carcinoma in mice [7]. In a clinical trial of KLH and mitomycin C chemotherapy, KLH was superior in preventing bladder tumor recurrence with no adverse local or systemic side effects [8,9]. Multicenter clinical trials have confirmed the efficacy of KLH given intravesically for 6 weeks to patients with various stages of bladder cancer [10]. Based on these studies, KLH is regarded as a safe and effective immunotherapy for superficial bladder cancer. Our laboratory has previously shown that KLH inhibits cellular proliferation in vitro in human cancer cell lines of the breast, esophagus, pancreas, and prostate [11,12]. We have shown KLH to alter tumor cytokine production in both breast and pancreatic cancer in vitro, in part explaining its
0002-9610/07/$ – see front matter © 2007 Excerpta Medica Inc. All rights reserved. doi:10.1016/j.amjsurg.2007.08.005
I. Rizvi et al. / The American Journal of Surgery 194 (2007) 628 – 632
effectiveness in isolated cell culture systems [13]. KLH also has been shown to decrease the cellular proliferation of Barrett’s esophageal adenocarcinoma and enhance its apoptotic activity [14,15]. We previously reported a significant reduction in cellular proliferation with the treatment of KLH (range, 12%– 60%) in the HTB68 melanoma cell line in vitro [12]. Immunologic therapies, specifically alpha interferon (AIFN) and interleukin-2 (IL-2), have been used in melanoma treatment for several years. High-dose interferon alfa-2b has been shown to increase both times to recurrence and survival time as compared to patients receiving no further treatment [16]. Additionally, IL-2 has been shown to be effective in the treatment of metastatic melanoma. In an analysis of 270 patients who received high-dose IL-2 for treatment of melanoma, Atkins et al found that both diseasefree and survival time were increased [17]. Given the limited success of the current therapeutic options for patients with advanced melanoma and the high incidence of toxic side effects related to such therapies new forms of immunemediated treatments are being investigated. Recently, we have shown significant growth inhibition of melanoma in vitro induced by treatment with KLH, AIFN, and IL-2 when administered as single agents [18]. These effects were significantly enhanced, ie, a statistical additive effect was noted, when these agents were combined. This formed the premise for our present study where effects of these immune modulators were studied in vivo. Materials and Methods Animals An initial KLH dose-response titration study was performed. Forty-one nude female (nu/nu) 6- to 7-week-old athymic mice were housed in the West Virginia University Office of Laboratory Animal Research and allowed to acclimatize for a period of 2 weeks. Subsequently, on experimental day 0, the mice received HTB68 tumor inoculation. The tumor inoculation consisted of a single subcutaneous injection of HTB68 cells (2 ⫻ 107 cells) on the flank of the left thigh. Subsequently, mice in the saline group (negative control, N ⫽ 11), KLH groups (50 g [N ⫽ 10], 100 g [N ⫽ 10], and 200 g subcutaneously on the flank of the left thigh [N ⫽ 10]) received treatments on day 2, 4, 7, 9, 11, and 16 after tumor inoculation. Upon completion of this feasibility study, 60 additional mice were obtained, acclimatized, and housed under identical conditions and maintained by veterinarian and trained staff. Mice were also monitored throughout the length of the study by research staff. Tumor cells The HTB68 human melanoma cell line was purchased from ATCC (Manassas, VA) and is tumorigenic in the nude mouse model. HTB68 cells for transplantation were grown under standard conditions (Eagle’s minimum essential media supplemented with 10% fetal bovine serum). The cells, when confluent, were trypsinized and then counted in .02% Trypan Blue and percent cell viability and viable cell concentration was determined.
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Tumor transplant and evaluation A single cell suspension containing 2 ⫻ 107 HTB68 cells in .1 mL tissue culture media was injected subcutaneously into the left flank. The dose of tumor inoculum was determined by performing in vivo tumor titrations of tumor cell suspensions in our feasibility study and choosing the dosage that assured a tumor incidence of at least 90% in untreated animals. Tumor incidence, as indicated by the first identifiable presence of tumor growth; this is before it is measurable by calipers and was evaluated by inspection and palpation. Tumor dimensions (length and width) were measured twice a week with a Vernier caliper. Tumor volume was calculated using the formula V ⫽ .4(LW2) in mm3. Alpha interferon AIFN was supplied as a lyophilized powder from Schering Corporation (Kenilworth, NJ). AIFN was resuspended to the desired assay concentrations in tissue culture medium. The AIFN concentration tested was 1000 IU per animal. Interleukin-2 IL-2 was supplied as a lyophilized powder from Chiron Corp (Emeryville, CA). IL-2 was resuspended to the desired assay concentrations in tissue culture medium. The IL-2 concentration tested was 5000 IU per animal. Keyhole limpet hemocyanin KLH was provided by Stellar Technologies (Port Hueneme, CA) in suspension at a protein concentration of 23.88 mg/mL. The KLH concentration tested was 200 g per animal. KLH was combined with standard immunotherapies for melanoma (AIFN and IL-2) to evaluate possible additive and/or synergistic effects. Combined pmmunotherapy with KLH and standard immunotherapy Mice were inoculated with 2 ⫻ 107 HTB68 cells on the left flank and randomized to 6 groups. Experimental treatments began on experimental day 1 and were administered for the next 5 weeks, 3 times per week. This treatment schedule was adjusted based on the results of the initial KLH titration and will be discussed in the results. The experimental groups tested were as follows: control (N ⫽ 10), saline injections (.1 mL), KLH 200 g (N ⫽ 10) experimental therapy (.1 mL), AIFN 1000 IU (N ⫽ 10) experimental therapy (.1 mL), IL-2 5000 IU (N ⫽ 10) experimental therapy (.1 mL), KLH and AIFN 1000 IU (N ⫽ 10) experimental therapy (.1 mL), and KLH and IL-2 5000 IU (N ⫽ 10) experimental therapy (.1 mL). Tumors were measured in standard fashion and tumor volume was calculated according to the aforementioned formula for the volume of an ellipse. Statistical analysis Determination of statistical significance was performed by analysis of variance [19]. Post hoc comparison of individual concentration means with the control was completed using the Tukey-Kramer multiple comparison test [20]. All data are reported as means ⫾ SE.
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I. Rizvi et al. / The American Journal of Surgery 194 (2007) 628 – 632 Table 1 Statistical comparisons of tumor volume (mm3) on days 24 and 29 post tumor inoculation Tumor volume Day 24
Control KLH AIFN IL-2 KLH ⫹ AIFN KLH ⫹ IL-2 ANOVA Fig. 1. Effects of KLH (50, 100, and 200 g) on tumor incidence in the melanoma-induced in vivo mouse model. *Statistical significance, P ⬍ .001.
Results KLH dose-response titration Tumor Incidence: The KLH 200 g group exhibited a 70% reduction in tumor incidence compared to the control group on day 16 post tumor inoculation (P ⬍ .001). Neither of the lower doses, KLH 100 g (30% reduction in tumor incidence) and KLH 50 g (0% reduction in tumor incidence), exhibited significant reductions in tumor incidence As illustrated in Fig. 1, once treatment with 200 g of KLH was suspended, tumor incidence quickly approached that of the untreated control and the beneficial suppressive effect of KLH was overcome. Tumor Volume: As illustrated in Fig. 2, during the pilot tumor incidence study, KLH was ineffective in reducing tumor growth after treatment was completed on day 16. Thereafter, tumors actually grew at a pace that produced tumor volumes (mm3) that were higher than control (P ⬍ .05). Statistically significant differences were noted at days 23, 28, and 30 in the KLH 100 g group. Combined Immunotherapy with KLH and Standard Immunotherapy: Based on our observations from the tumor incidence study, the second experiment employed a dose of
Fig. 2. Effects of KLH (50, 100, and 200 g) on tumor volume (mm3) in the melanoma-induced in vivo mouse model. Data shown are means ⫾ SE. P values indicate statistical significance versus the control.
Day 29
Mean ⫾ SE
P value
Mean ⫾ SE
P value
2,418 ⫾ 222 1,808 ⫾ 150 1,941 ⫾ 185 1,799 ⫾ 185 1,879 ⫾ 115 1,630 ⫾ 96
— NS NS NS NS P ⫽ .015 P ⫽ .030
3,349 ⫾ 288 2,732 ⫾ 246 2,830 ⫾ 222 2,357 ⫾ 183 2,393 ⫾ 138 2,408 ⫾ 141
— NS NS P⫽ P⫽ P⫽ P⫽
.023 .025 .028 .011
ANOVA ⫽ analysis of variance.
200 g of KLH and the treatment schedule was further extended to allow for KLH to exert its anti-tumor properties initially observed in regards to reduction in tumor incidence. On day 24 post tumor inoculation the combination of KLH ⫹ IL-2 (1,630 ⫾ 96) significantly reduced tumor volume when compared to control (2,418 ⫾ 222, P ⫽ .015, Table 1). At day 29, tumor volume was significantly reduced by IL-2 (2,357 ⫾ 183, P ⫽ .023), KLH ⫹ AIFN (2,393 ⫾ 138, P ⫽ .025), and KLH ⫹ IL-2 (2,408 ⫾ 141, P ⫽ .028). Looking at the percent change in tumor volume when compared to the control, on day 29 after tumor inoculation, all single agents reduced tumor volume compared with the control group (Fig. 3). IL-2 exhibited the greatest degree of reduction (30% ⫾ 6%, P ⫽ .022), followed by KLH (18% ⫾ 7%, not significant [NS]) and AIFN (16% ⫾ 7%, NS). Combination therapy of KLH with IL-2 reduced tumor volume to the greatest extent (30% ⫾ 4%, P ⫽ .014). This was followed by the combination of KLH ⫹ AIFN (28% ⫾ 4%, P ⫽ .031). Single-treated animals had less tumor inhibition than the combination groups.
Fig. 3. Percent reduction in tumor volume at day 29 from the effects of KLH (200 g), IL-2 (5000 IU), and AIFN (1000 IU) as single agents and in combination when compared to controls in the melanoma-induced in vivo mouse model. Data shown are means ⫾ SE. P values indicate statistical significance versus the control.
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Comments Cutaneous melanoma is a malignant neoplasm with an unpredictable behavior, ranging from spontaneous regression to rapid progression. Early detection can ensure effective treatment. Most cases are diagnosed in adults, and treatment planned based on tumor grade and stage. Surgical resection remains the standard of care followed by immunotherapy, chemotherapy, and/or radiation therapy for advanced disease. Adjuvant treatment for melanoma includes chemotherapy, immunotherapy, and radiation therapy for more advanced disease. Many clinical research trials currently underway employ the use of immunotherapies, usually AIFN or IL-2, in an effort to improve treatment outcomes. Kirkwood et al found that interferon alfa-2b was the first agent to show a significant benefit in relapse-free and overall survival of high-risk melanoma patients as compared to those who received no further treatment [16]. The same investigators found, in a subsequent randomized trial that interferon alfa-2b increased both time to recurrence and survival when compared to patients receiving ganglioside vaccine [21]. While these results are both impressive and significant, treatment with highdose interferon alfa-2b is not without significant side effects and cost. Moreover, the efficacy of interferon alfa-2b has not been validated consistently by other groups [22]. In addition to AIFN, IL-2 has been shown to be effective in the treatment of metastatic melanoma. Atkins et al found in an analysis of 270 patients who received high-dose IL-2 for treatment of malignant melanoma, that both disease-free and survival time were increased [17]. Given the limited success of the current therapeutic options for patients with melanoma, the search for new treatments must be continued. These promising results have led us to investigate the effects of KLH in combination with other conventional immunotherapies administered as standard treatment regimens in diseased patients. In this study, melanoma was induced in athymic mice by inoculation with HTB68 melanoma cell line. In the initial feasibility study, we were able to demonstrate that KLH, at a dose of 200 g per animal, inhibited tumor appearance (P ⬍ .001) until injections were stopped at day 16 post implant. Subsequently, 60 mice were separated in groups and were treated with KLH, AIFN, and IL-2 alone and in combination. Tumor volumes were then calculated in different treatment groups. Our results show that, as single agents, IL-2 reduced tumor volume by 30%, KLH by 18%, and AIFN by 16%. Combination therapy with KLH and IL-2 reduced tumor volume by 30% followed by KLH and AIFN at 28%. Although the combination therapies exhibited a greater reduction in tumor volume, these results did not meet the statistical criteria for synergism. However, an additive effect was noted for KLH and AIFN in combination. This additive effect was not seen in the KLH ⫹ IL-2 animals, possibly related to the predominance of IL-2 effects on T cells, and our use of an athymic, T-cell– deficient, mouse model for our study. Additional studies employing doseresponse analysis will be performed to amplify these promising results.
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Biological agents like KLH and inositol hexaphosphate are being studied increasingly as adjuvant treatments to melanoma [23]. Given the clinical safety and efficacy of KLH in numerous trials in human bladder cancer and the promising initial results we have obtained in this very aggressive model of melanoma, further studies and trials are definitely warranted with KLH. Its administration may allow the use of lower doses of conventional immunotherapy against melanoma, thereby reducing costs and toxicity while enhancing the quality of life of patients suffering with this malignancy. References [1] Jemal A, Siegal R, Ward E, et al. Cancer Statistics, 2006. CA Cancer J Clin 2006;56:106 –30. [2] McGillis ST, Fein H. Topical treatment strategies for non-melanoma skin cancer and precursor lesions. Semin Cutan Med Surg 2004;23: 174 – 83. [3] Villa AM, Berman B. Immunomodulators for skin cancer. J Drugs Dermatol 2004;3:533–9. [4] Harris JR, Markl J. Keyhole limpet hemocyanin: Molecular structure of a potent marine immunoactivator. A review. Eur Urol 2000; 37(suppl 3):24 –33. [5] Harris JR, Markl J. Keyhole limpet hemocyanin (KLH): a biomedical review. Micron 1999;30:597– 623. [6] Olsson CA, Chute R, Rao CN. Immunologic reduction of bladder cancer recurrence rate. J Urol 1974;111:173– 6. [7] Riggs DR, Tarry WF, DeHaven JI, et al. Immunotherapy of murine transitional cell carcinoma of the bladder using alpha and gamma interferon in combination with other forms of immunotherapy. J Urol 1992;147:212– 4. [8] Jurincic CD, Engelmann U, Gasch J, et al. Immunotherapy in bladder cancer with keyhole-limpet hemocyanin: a randomized study. J Urol 1988;139:723– 6. [9] Jurincic-Winkler CD, Metz KA, Beuth J, et al. Keyhole limpet hemocyanin for carcinoma in situ of the bladder: a long-term follow-up study. Eur Urol 2000;37(suppl 3):45–9. [10] Lamm DL, DeHaven JI, Riggs DR. Keyhole limpet hemocyanin immunotherapy of bladder cancer: laboratory and clinical studies. Eur Urol 2000;37(suppl 3):41– 4. [11] Riggs D, Jackson B, Vona-Davis L, et al. In vitro anticancer effects of a novel immunostimulant: keyhole limpet hemocyanin. J Surg Res 2002;108:279 – 84. [12] Somasundar P, Riggs DR, Jackson BJ, et al. Inhibition of melanoma growth by hemocyanin occurs via early apoptotic pathways. Am J Surg 2005;190:713– 6. [13] Riggs DR, Jackson BJ, Vona-Davis L, et al. In vitro effects of keyhole limpet hemocyanin in breast and pancreatic cancer in regards to cell growth, cytokine production, and apoptosis. Am J Surg 2005; 189:680 – 4. [14] McFadden DW, Riggs DR, Jackson BJ, et al. Keyhole limpet hemocyanin, a novel immune stimulant with promising anticancer activity in Barrett’s esophageal adenocarcinoma. Am J Surg 2003;186:552–5. [15] Vona-Davis L, Vincent T, Zulfiqar S, et al. Proteomic analysis of SEG-1 human Barrett’s-associated esophageal adenocarcinoma cells treated with keyhole limpet hemocyanin. J Gastrointest Surg 2004;8: 1018 –23. [16] Kirkwood JM, Strawderman MH, Ernstoff MS, et al. Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group trial EST 1684. J Clin Oncol 1996;14:7–17. [17] Atkins MB, Lotze MT, Dutcher JP, et al. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol 1999; 17:2105–16. [18] McFadden DW, Riggs DR, Jackson BJ, et al. Keyhole limpet hemocyanin potentiates standard immunotherapy for melanoma. Am J Surg 2007;193:284 –7. [19] Ludbrook J. Multiple comparison procedures updated. Clin Exp Pharmacol Physiol 1998;25:1032–7.
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[20] Dixon WJ, Massey FJ. Introduction to Statistical Analysis. 4th ed. New York: McGraw-Hill; 1983. [21] Kirkwood JM, Ibrahim J, Lawson DH, et al. High-dose interferon alfa-2b does not diminish antibody response to GM2 vaccination in patients with resected melanoma: results of the Multicenter Eastern Cooperative Oncology Group phase II trial E2696. J Clin Oncol 2001;19:1430 – 6.
[22] Eggermont AM, Suciu S, Mackie R, et al. Post-surgery adjuvant therapy with intermediate doses of interferon alfa 2b versus observation in patients with stage IIb/III melanoma (EORTC 18952): randomized controlled trial. Lancet 2005;366:1189 –96. [23] Rizvi I, Riggs DR, Jackson BJ, et al. Inositol hexaphosphate (IP6) inhibits cellular proliferation in melanoma. J Surg Res 2006;133: 3– 6.