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© 2002 American Society for Clinical Oncology Enhanced Humoral Immune Response Correlates With Improved Disease-Free and Overall Survival in American Joint Committee on Cancer Stage II Melanoma Patients Receiving Adjuvant Polyvalent VaccineByFrom the Sonya Valley Ghidossi Vaccine Laboratory of the Roy E. Coats Research Laboratories of the John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, CA. Address reprint requests to Donald L. Morton, MD, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404; email: mortond{at}jwci.org
PURPOSE: Although the improved overall survival (OS) of patients who receive Canvaxin (CancerVax Corp, Carlsbad, CA) polyvalent vaccine (PV) immunotherapy for metastatic melanoma has been correlated with cellular and humoral immune responses, the mechanisms of vaccine immunotherapy for early-stage melanoma are unclear. Specific immune responses to tumor-associated antigens might correlate with disease-free survival (DFS) and OS in patients receiving adjuvant PV therapy for primary melanoma. PATIENTS AND METHODS: Eighty-three patients received PV plus bacille Calmette-Guérin after wide excision of American Joint Committee on Cancer stage II melanoma. Humoral and cellular responses during the first 12 weeks of adjuvant immunotherapy were assessed by serum antibody titers to a tumor-associated 90-kd glycoprotein antigen (TA90) expressed by PV, and by delayed-type hypersensitivity (DTH) skin testing with PV (PV-DTH).
RESULTS: At a median follow-up period of 46.6 months (range, 10.7 to 93.6 months), an increased PV-DTH response seemed to be associated with improved 5-year DFS (54% v 20%) and 5-year OS (75% v 60%), but the correlations were not statistically significant. Anti-TA90 immunoglobulin (Ig) M levels CONCLUSION: These findings suggest that an increased IgM response in patients receiving PV therapy for stage II melanoma is associated with decreased recurrence and improved survival.
ALTHOUGH MELANOMA has long been considered the paradigm for examining immune responses to malignancy, both the application and mechanism of immunotherapy in patients with different stages of melanoma remain controversial. Whereas chemotherapy tends to produce rapid but infrequently durable clinical responses, active immunotherapy is believed to upregulate different components of the immune system.1-3 However, questions remain regarding which component of the immune system should be stimulated in order to produce a beneficial immune response. Since the mid-1980s, we have performed phase I/II clinical trials of active immunotherapy with an irradiated preparation of whole melanoma cells that express at least 20 immunogenic melanoma-associated and tumor-associated antigens.4 A retrospective study of patients with American Joint Committee on Cancer (AJCC) stage IB and II melanoma demonstrated that 186 patients who received the polyvalent vaccine (PV) had a significantly better survival than 1,218 historical controls who did not receive PV.5 Because PV has no direct cytotoxic effect on tumor cells, its therapeutic effects as an adjuvant agent may depend on the ability of its tumor-associated and melanoma-associated antigens to induce immune responses against cross-reactive antigens on the patient’s residual melanoma cells. TA90, a 90-kd glycoprotein tumor-associated antigen that is expressed in 72% of human melanomas, is one such cross-reactive antigen that has been extensively studied.6,7 Previously, immunization with PV has been demonstrated to elevate immunoglobulin (Ig) G and IgM antibody titers to the TA90 antigen.8 Additional work demonstrated that the improved overall survival (OS) of patients who received PV adjuvant therapy after curative resection of distant (AJCC stage IV) melanoma was correlated with both cellular and humoral immune responses.9 In early-stage melanoma, however, where tumor burden is relatively low compared with that of stage IV disease, the mechanisms underlying the clinical efficacy of melanoma vaccines have not been elucidated. We hypothesized that specific postinduction immune responses might correlate with both disease-free survival (DFS) and OS in patients who received PV immunotherapy after wide surgical excision of AJCC stage II melanoma.
The study population consisted of patients who received Canvaxin PV (CancerVax Corp, Carlsbad, CA) as adjuvant immunotherapy after wide excision of AJCC stage II melanoma at the John Wayne Cancer Institute between 1991 and 1998. Although this group included some patients who underwent elective lymph node dissection or sentinel lymph node dissection, none of these patients had evidence of metastases in a regional lymphatic basin. In addition, the absence of metastatic disease was confirmed in all patients by physical examination and chest x-ray. Before initiation of PV therapy, all patients provided written informed consent, and the PV protocol was in accordance with the ethical standards of the joint Saint John’s Health Center and John Wayne Cancer Institute institutional review board and in compliance with the Helsinki Declaration. Serum samples were obtained prospectively before initiation of PV therapy and at 2, 4, 8, and 12 weeks after the first dose of PV. These serum samples were processed, coded, and stored at -35°C until the time of analysis, when they were thawed and tested in a blinded fashion. Clinical status was recorded prospectively for all patients until the time of death, recurrence, or the end of the study period on October 1, 1998. At least 36 months of clinical follow-up was required in patients who did not experience recurrence of disease. The preparation and administration of PV has been described in detail elsewhere.3 Briefly, melanoma cells from the M10-V, M24-V, and M101-V cell lines were cultured in tissue medium and then collected, washed, and pooled to obtain 24 x 106 total cells (8 x 106 cells per line). The cells were then irradiated with 150 Gy and cryopreserved in liquid nitrogen until they were thawed in preparation for administration. Each injection consisted of 24 x 106 total cells. The vaccine was given intradermally every 2 weeks for four cycles, then monthly for the remainder of the first year. After year 1 of PV therapy, the vaccine was administered every 3 months for three cycles, and thereafter at 6-month intervals. During the first two treatments, PV was given in conjunction with the Tice strain of bacille Calmette-Guérin at a dose of 8 x 106 total organisms per treatment. Before immunization, general immune competence was assessed by skin tests to purified protein derivative (PPD), mumps, and Candida antigens. In addition, all subjects were assessed for a delayed-type hypersensitivity (DTH) response to PV. One tenth of the normal PV dose was given intradermally at the time of vaccine administration. After 48 hours, the PV-DTH response was measured in millimeters of induration; erythema was not recorded. As a nonspecific control, a PPD skin test was administered simultaneously and measured in an identical manner. PV-DTH and PPD-DTH responses were evaluated before initiation of PV therapy and at 2, 4, 8, and 12 weeks. The antigen TA90 was purified from the urine of a melanoma patient, as previously described.10 Antigen purity was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis before using TA90 as a target in an enzyme-linked immunosorbent assay (ELISA). The ELISA was performed in a standard fashion.8 Briefly, TA90 antigen was adsorbed to polystyrene wells of ELISA plates at 15 ng per well. Serum samples at dilutions of 1:100, 1:200, 1:400, 1:800, and 1:1,600 were added to the wells. After this, the bound immunoglobulins were reacted with an alkaline phosphatase–conjugated Fab fragment of goat anti-human IgG at a 1:500 dilution, or goat anti-human IgM at a 1:1,000 dilution (Sigma Chemical Co, St Louis, MO). The unreacted conjugate was washed off and p-nitrophenyl phosphate (1 mg/mL) in 10% diethanolamine buffer was added. Absorbance at 405 nm was assessed with a microtiter plate reader (Titertek Multiscan; Dynatech, Alexandria, VA). Controls on each ELISA plate consisted of nonspecific binding of goat antihuman IgG or IgM to TA90, as well as nonspecific binding of a patient’s immunoglobulins and the goat antihuman IgG or IgM to the polystyrene plate. For the purposes of this study, the antibody titer was defined as the reciprocal of the highest dilution that resulted in an absorbance of 0.05 optical density at 405 nm, after subtracting the absorbance values of the controls. With respect to statistical analysis, univariate analysis of continuous variables was performed by the Cox proportional hazard regression method. Survival was computed from the onset of PV administration. Multivariate analysis was performed by Cox proportional hazard regression as well. Survival curves were estimated by the nonparametric Kaplan-Meier method, and the log-rank test was used for univariate analysis of categorical variables to determine differences between curves. A P value less than .05 was considered significant. All statistical analyses were two tailed and were performed by SAS software (SAS Institute, Cary, NC).
Complete sets of serum samples were available for 83 patients with AJCC stage II melanoma. The characteristics of these 83 patients are listed in Table 1. Complete information on standard prognostic variables was available for 79 of the 83 patients (Tables 2 and 3). The median tumor thickness was 3.0 mm (range, 0.5 to 15.0 mm). The median follow-up time was 46.6 months (range, 10.7 to 93.6 months). The median age at diagnosis was 47 years (range, 15 to 84 years). Forty-six patients (55%) underwent wide local excision alone, and the remaining 37 patients underwent wide local excision followed by sentinel lymph node dissection or elective complete lymph node dissection. The median interval between primary surgical therapy and initiation of PV adjuvant therapy was 2.6 months (range, 0.9 to 14.2 months). Forty-one patients (49%) developed recurrent disease; the recurrences were equally distributed among skin and soft tissue sites, nodal sites, and distant sites.
PV induced both cellular and humoral immune responses in a significant majority of patients. The median anti-TA90 IgM titer of 1:400 (range, 1:100 to 1:3,200) at baseline increased to 1:600 postinduction. The range of increase for anti-TA90 IgM titers was 0 to 3,200. Fifty-eight patients (70%) experienced at least a two-fold increase in IgM antibody titers to TA90; 17 (20%) experienced an eight-fold increase; and nine (11%) experienced no increase (Fig 1).
There was a similar increase in median anti-TA90 IgG titers. The median anti-TA90 IgG titer of 1:200 (range, 1:100 to 1:2,400) at baseline increased to 1:400 after induction. The range of increase for anti-TA90 IgG titers was 0 to 3,200. Fifty-one patients (61%) experienced at least a two-fold increase in anti-TA90 IgG antibody titers; seven (8%) experienced an eight-fold increase; and 12 (14%) experienced no increase (Fig 1). The median PV-DTH of 3.0 mm (range, 0 to 50 mm) at baseline increased to 8.5 mm after induction, with a range of increase from 0 to 33 mm. Because patients received bacille Calmette-Guérin with the first two doses of PV, the PPD-DTH also increased, from a median of 0 mm (range, 0 to 35 mm) at baseline to 11 mm (range, 0 to 39.5 mm) after induction. OS was not significantly affected by age, sex, primary tumor site, treatment, or indicators of general immune competence such as PPD and lymphocyte count. Additionally, baseline anti-TA90 IgM titers were not correlated with improvement in OS. By multivariate analysis, OS was significantly associated with Breslow thickness and with maximum anti-TA90 IgM titers (Table 2). With respect to DFS, maximum anti-TA90 IgG and IgM titers both were associated with DFS on univariate analysis; however, maximum anti-TA90 IgG titer lost significance in the stepwise multivariate analysis (Table 3). The baseline anti-TA90 IgM titer was not associated with any improvement in DFS. The maximum anti-TA90 IgM titer remained significant in the multivariate analysis for DFS; in addition, Breslow thickness was significantly correlated with DFS (Table 3).
Five-year rates of OS and DFS were significantly higher when maximal anti-TA90 IgM was at least 1:800 (94% v 52% for OS, P = .0001; and 89% v 17% for DFS, P = .0001) (Figs 2 and 3). Five-year OS was not significantly affected by elevated anti-TA90 IgG antibody titers (71% for titers
The DTH response to PV was not associated with a significant improvement in either OS (Table 2) or DFS (Table 3). Five-year OS was 75% when PV-DTH  4 mm and 60% otherwise (Fig 5). Five-year DFS was 54% when PV-DTH 4 mm and 20% otherwise (Fig 6), a difference that approached but did not reach significance.
Because humoral immune response was significantly correlated with survival patterns, we combined maximum anti-TA90 IgG and IgM antibody titers to create a model for humoral immune response to PV. Patients were classified into four groups on the basis of their anti-TA90 antibody titers. Ten patients developed favorable humoral immune responses—that is, elevated titers of anti-TA90 IgM and low titers of anti-TA90 IgG. Twenty-eight patients were considered intermediate responders because both antibody titers were elevated. The characteristic temporal pattern of antibody response for patients with elevated anti-TA90 IgM is displayed in Fig 7. Thirty-six patients had unfavorable humoral immune responses characterized by elevations in anti-TA90 IgG titers but low anti-TA90 IgM titers. Finally, five patients did not mount a significant humoral immune response to PV; these patients had low anti-TA90 IgG and IgM titers and were considered nonresponders (Fig 8).
With respect to OS, there were no deaths among patients with favorable humoral immune responses (Fig 9). Patients with intermediate humoral immune responses had a similarly improved OS, with only two of 28 patients dying as a result of melanoma. However, those patients who were nonresponders and those patients who had unfavorable humoral immune responses had a significantly worse OS (Fig 9, P = .0013). Similarly, none of the patients with favorable humoral immune responses developed a melanoma recurrence (Fig 10). Only four (15%) of the 28 patients demonstrating intermediate humoral immune responses developed recurrent melanoma. Three of five nonresponders developed recurrent disease, and 89% of the patients with unfavorable humoral immune responses had recurrent melanoma (Fig 10).
Our findings are consistent with the hypothesis that the immune response to PV correlates with survival in patients who receive the vaccine as an adjuvant therapy after wide excision of stage II melanoma. Importantly, immune responses to PV were not associated with general immune competence, as indicated by parameters such as PPD and lymphocyte count. Also, baseline IgG or IgM antibody titers to TA90 were not correlated with improved survival, suggesting that enhancement of immune responses by PV administration, rather than the presence of preexisting autologous antibodies, might play a significant role in modulating survival in these patients. With respect to autologous antibodies, it is important to note that even if not all primary melanomas expressed TA90 in sufficient quantity to induce an endogenous immune response, all patients were exposed to TA90 as soon as they received PV because all three melanoma cell lines of the vaccine express this antigen. Although both OS and DFS were correlated with immune responses to PV, the specific immune responses were different than those observed in previous studies of patients with AJCC stage III and IV melanoma. In patients who received PV after surgical resection of stage III melanoma, a change in PV-DTH and an elevation in anti-TA90 IgM antibody titers were both associated with prolonged survival,11 whereas elevations in anti-TA90 IgG antibody titers were correlated with decreased OS. Similarly, in patients who underwent curative resection of stage IV melanoma and then received PV as adjuvant therapy, changes in postinduction PV-DTH and anti-TA90 IgM antibody titers were associated with prolonged survival,9 and elevated anti-TA90 IgG titers were associated with decreased OS. By contrast, in our current analysis of patients with stage II melanoma, changes in PV-DTH were not correlated with survival, and elevations in anti-TA90 IgG antibodies were correlated with diminished DFS but not OS. It is surprising that an increase in PV-DTH after induction was not significantly correlated with improved survival: several previous studies that used a number of different active specific immunotherapeutic agents have demonstrated the importance of the cellular immune response in destroying tumor cells.3,12-14 It is possible that the cellular immune response does not play as critical a role in the destruction of tumor cells when tumor burden is low. Alternatively, the lack of a significant correlation between PV-DTH and survival in this study might represent a statistical error. This is suggested by a trend toward improved DFS when the PV-DTH was at least 4 mm (Fig 5); notably, only five patients (6%) were unable to mount a DTH skin test of at least 4 mm. Perhaps larger numbers of patients with impaired ability to demonstrate a minimal DTH response to PV would reveal a statistically significant survival difference between the two groups. Interestingly, the study of Bystryn et al12 found that 44% of 94 patients who received a partially purified polyvalent formulation for stage II melanoma did not mount a DTH response to the vaccine, yet those patients who demonstrated a strong DTH response experienced a significant improvement in DFS. Logically, larger differences in the ability of a given tumor vaccine to produce an observable response are more likely to result in definable differences in clinical outcome. Increased levels of anti-TA90 IgG antibody in the absence of elevated IgM levels (Fig 10) were associated with decreased DFS by univariate but not multivariate analysis (Table 3) and were not associated with changes in OS. This means that other factors caused the difference in survival for patients with high versus low levels of IgG; anti-TA90 IgG did not have a causal relationship with decreased DFS. As shown in Figs 9 and 10, the major effect on survival was attributed to IgM titer, not IgG titer. Because there were four patterns of immunoglobulin immune response, we combined anti-TA90 IgM and IgG antibody titers to create a humoral immune response model that might further elucidate possible relationships between IgG and IgM antibodies. This immune response model (Figs 9 and 10) indicated that even in the setting of elevated anti-TA90 IgG titers, elevated anti-IgM titers were associated with a significant survival advantage. Further study is needed to better define the complicated interactions between anti-TA90 IgM and IgG antibodies, as well as interactions with other antigen-antibody complexes. Careful definition of the IgM-IgG antibody interaction might ultimately be of clinical value in determining methods to improve results of adjuvant vaccine therapy. In this analysis, an elevated anti-TA90 IgM antibody titer was a significant prognostic variable for both DFS and OS. This association between survival and IgM antibody responses to glycoprotein tumor antigens has been demonstrated previously in patients who received PV immunotherapy for stage III and IV melanoma.9,11 Additionally, IgM responses to ganglioside antigens have been correlated with prolonged survival in patients who have received other types of vaccine immunotherapy for melanoma.1,15 Furthermore, antitumor activity of IgM antibody has been correlated with improved survival in nonmelanoma malignancies.16,17 The present study supports the clinical importance of IgM antibody in the destruction of neoplastic cells. In summary, the humoral immune response to PV adjuvant therapy, particularly that of anti-TA90 IgM antibody, correlates with both DFS and OS patterns in patients with stage II melanoma who receive the vaccine after surgical excision. It is important to stress, however, that these data are preliminary and require further validation. Future studies will attempt to analyze the relationship between endogenous antibody titers and survival, as well as the evolution of the immune response in patients who do not have native anti-TA90 antibody but who mount an immune response to PV administration. It would also be of considerable value to determine whether isotype-specific immune responses to additional melanoma-associated antigens found on PV, such as tyrosinase, MART-1/Melan A, MAGE-1, and MAGE-3, correlate with survival in the setting of PV administration. Ultimately, the immune response to a combination of antigens found in a PV may have an even greater association with survival than the immune response to a single antigen.
Supported in part by grant nos. CA12582 and CA29605 from the National Cancer Institute, Bethesda, MD, by funding from the Wayne and Gladys Valley Foundation, Oakland, CA, and the Harold McAllister Charitable Foundation, Los Angeles, CA. L.A.D. is a recipient of the American Society of Clinical Oncology Merit Award for 1999. Canvaxin and CancerVax are trademarks of CancerVax Corporation.
Presented in part at the Thirty-Fifth Annual Meeting of the American Society of Clinical Oncology, Atlanta, GA, May 15-18, 1999.
1. Livingston PO, Wong GYC, Sucharita A, et al: Improved survival in stage III melanoma patients with GM2 antibodies: A randomized trial of adjuvant vaccination with GM2 ganglioside. J Clin Oncol 12: 1036-1044, 1994 2. Rosenberg SA, Lotze MT, Muul LM, et al: Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 in patients with metastatic melanoma. N Engl J Med 313: 1485-1492, 1985[Abstract] 3. Morton DL, Foshag LJ, Hoon DSB, et al: Prolongation of survival in metastatic melanoma after active specific immunotherapy with a new polyvalent melanoma cell vaccine. Ann Surg 216: 463-482, 1992[Medline] 4. DiFronzo LA, Morton DL: Melanoma vaccines: Current status of clinical trials. Adv Oncol 16: 23-29, 2000 5. Shen P, Foshag LJ, Essner R, et al: Postoperative adjuvant therapy using a polyvalent melanoma vaccine improves overall survival of patients with primary melanoma. Proc Am Soc Clin Oncol 18: 533a, 1999 (abstr 2059)
6. Kelley MC, Gupta RK, Hsueh EC, et al: Tumor-associated antigen TA90 immune complex assay predicts recurrence and survival after surgical treatment of stage I-III melanoma. J Clin Oncol 19: 1176-1182, 2001 7. Gupta RK: Circulating immune complexes in malignant melanoma. Dis Markers 6: 81-96, 1988[Medline] 8. Euhus DM, Gupta RK, Morton DL: Induction of antibodies to a tumor-associated antigen by immunization with a whole melanoma cell vaccine. Cancer Immunol Immunother 29: 247-254, 1989[CrossRef][Medline]
9. Hsueh EC, Gupta RK, Qi K, et al: Correlation of specific immune responses with survival in melanoma patients with distant metastases receiving polyvalent melanoma cell vaccine. J Clin Oncol 16: 2913-2920, 1998 10. Gupta RK, Morton DL: Monoclonal antibody based ELISA to detect glycoprotein tumor–associated antigen-specific immune complexes in cancer patients. J Clin Lab Anal 6: 329-336, 1992[Medline] 11. Jones RC, Kelley MC, Gupta RK, et al: Immune response to polyvalent melanoma cell vaccine in AJCC stage III melanoma: An immunologic survival model. Ann Surg Oncol 3: 437-445, 1996[CrossRef][Medline] 12. Bystryn J-C, Oratz R, Roses D, et al: Relationship between immune response to melanoma vaccine immunization and clinical outcome in stage II malignant melanoma. Cancer 69: 1157-1164, 1992[Medline] 13. Berd D, Maguire HC Jr, McCue P, et al: Treatment of metastatic melanoma with an autologous tumor-cell vaccine: Clinical and immunologic results in 64 patients. J Clin Oncol 8: 1858-1867, 1990[Abstract]
14. Barth A, Hoon DSB, Foshag LJ, et al: Polyvalent melanoma cell vaccine induces delayed-type hypersensitivity and in vitro cellular immune response. Cancer Res 54: 3342-3345, 1994 15. Jones PC, Sze LL, Liu PY, et al: Prolonged survival for melanoma patients with elevated IgM antibody to oncofetal antigen (OFA-1). J Natl Cancer Inst 66: 249-254, 1981[Medline]
16. Ollert MW, David K, Schmitt C, et al: Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells. Proc Natl Acad Sci USA 93: 4498-4503, 1996 17. MacLean GD, Reddish MA, Koganty RR, et al: Antibodies against mucin-associated sialyl-Tn epitopes correlate with survival of metastatic adenocarcinoma patients undergoing active specific immunotherapy with synthetic STn vaccine. J Immunother 19: 59-68, 1996[Medline] Submitted January 7, 2002; accepted February 18, 2002.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
