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Annals of Surgical Oncology 8:198-203 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Serum TA90 Antigen-Antibody Complex as a Surrogate Marker for the Efficacy of a Polyvalent Allogeneic Whole-Cell Vaccine (CancerVax) in Melanoma

George J. Tsioulias, MD, DMSc, Rishab K. Gupta, PhD, Glenn Tisman, MD, Eddy C. Hsueh, MD, Richard Essner, MD, Leslie A. Wanek, PhD and Donald L. Morton, MD, FACS

From the Roy E. Coats Research Laboratories and the Sonya Valley Ghidossi Vaccine Laboratory, John Wayne Cancer Institute, Saint John’s Health Center, Santa Monica, California.

Correspondence: Address correspondence and reprint requests to: Donald L. Morton, MD, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404-2302; Fax: 310-582-7185; E-mail: mortond{at}jwci.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Introduction: TA90 is a tumor-associated 90-kD glycoprotein antigen expressed on most melanoma cells, including those of CancerVax, a polyvalent allogeneic whole-cell vaccine. Previous studies have shown that a TA90 antigen-antibody immune complex (IC) in the serum of patients with melanoma is a marker of subclinical tumor burden and a strong prognostic factor. We hypothesized that the induction of TA90-IC during postoperative adjuvant CancerVax therapy might indicate vaccine-mediated immune destruction of subclinical melanoma cells with release of TA90, and thereby serve as a surrogate marker of vaccine efficacy.

Methods: From 1993 to 1997, 219 melanoma patients were enrolled in a prospective phase II trial of CancerVax plus bacille Calmette-Guerin (BCG) after complete tumor resection. Coded serum samples were prospectively collected and analyzed for TA90-IC before and 2, 4, 8, 12, and 16 weeks after initiation of CancerVax therapy. TA90-IC seroconverters were those patients whose negative TA90-IC values (< .410) became positive (>= .410) after initiation of CancerVax treatment.

Results: Before CancerVax therapy, 51 patients had positive TA90-IC values and 168 patients had negative TA90-IC values. During CancerVax treatment, all 51 positive patients remained positive, 79 (47%) negative patients seroconverted to positive, and 89 (53%) negative patients remained negative. Seroconverters had higher 2-year rates of disease-free survival (59% vs. 32%; P < .006) and overall survival (78% vs. 63%; P < .02) than did patients whose TA90-IC values remained positive.

Conclusions: CancerVax induces TA90-IC in melanoma patients with subclinical disease. TA90-IC seroconverted patients have significantly improved disease-free and overall survival compared with TA90-IC positive patients. TA90-IC is an important prognostic factor that can serve as a surrogate marker for the clinical efficacy of CancerVax.

Key Words: Melanoma vaccine • Active immunotherapy • Prognosis • Immune response • Tumor marker


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lack of effective adjuvant chemotherapy protocols for melanoma and evidence that melanoma may be more immunogenic than other tumors have generated intense interest in active specific immunotherapies to induce or augment immunity against tumor-associated antigens (TAA) found in melanoma.1,2 CancerVax is an allogeneic polyvalent melanoma vaccine composed of irradiated but viable cells from three melanoma cell lines. Its high content of immunogenic TAA commonly shared among melanomas can induce a cross-reactive immune response against the patient’s melanoma.3 Phase II trials have shown that patients with American Joint Committee on Cancer (AJCC) stage II, III, or IV melanoma receiving adjuvant active specific immunotherapy with CancerVax after complete surgical resection have prolonged overall survival (OS) and disease-free survival (DFS) when compared with historical controls receiving other adjuvant treatments.3–5

The therapeutic effect of CancerVax requires induction of an immune response to TAA shared by vaccine cells and the patient’s melanoma cells. This response is characterized by activation of cell-mediated and humoral antibody responses reactive with the patient’s melanoma cells. Patients with high IgM antibody responses and/or cell-mediated immunity as defined by a delayed-type hypersensitivity skin test to CancerVax (immunologic responders) have improved prognosis.5–7

The most extensively researched antigen among the 20 known TAA in CancerVax is TA90. This tumor-associated 90-kD glycoprotein antigen is highly expressed in melanomas and melanoma cell lines.8 Because of its immunogenic nature, it induces the formation of endogenous antigen-antibody complexes.9 A high serum concentration of TA90 antigen-antibody immune complex (IC) in melanoma patients is a marker of subclinical tumor burden and a strong prognostic factor.10,11 Previous unpublished observations of negative TA90-IC patients becoming positive after adjuvant CancerVax treatment led us to hypothesize that the induction of TA90-IC in these patients might indicate immune destruction of subclinical melanoma cells, with release of TA90 antigen. Such a TA90-IC seroconversion could serve as a surrogate marker of CancerVax efficacy.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
From 1994 to 1997, 219 melanoma patients were enrolled in a prospective phase II trial of CancerVax plus bacille Calmette-Guerin (BCG). All study patients had undergone curative surgical resection of AJCC stage II, III, or IV melanoma. Postoperative clinical status was recorded prospectively until time of death or last follow-up, and data were transferred to the melanoma vaccine database stored in our statistical coordinating unit. Serum samples were prospectively collected before and 2, 4, 8, 12, and 16 weeks after initiation of CancerVax treatment. Complete sets of serum samples were available for all patients. These serum samples were coded and tested in a blinded manner, and the values were transferred to the statistical coordinating unit for analysis. Written consent for the CancerVax immunotherapy protocol was obtained from all patients, and the CancerVax protocol had been approved by the Institutional Review Board of the Saint John’s Health Center.

CancerVax Preparation and Administration
The preparation and administration of CancerVax have been described previously.3 In brief, cells were obtained from M10-V, M24-V, and M101-V allogeneic melanoma cell lines, which were established in our laboratory and selected for their high content of immunogenic TAA. Cells were grown in tissue culture medium, harvested, washed, and pooled (8 x 106 cells/line, or 24 x 106 total cells). The cells were then irradiated with 150 Gy and cryopreserved in liquid nitrogen. CancerVax was thawed, diluted with normal saline, and administered. The vaccine was given intradermally every 2 weeks for three courses, and then monthly during the first year of CancerVax therapy. Each injection contained 24 x 106 cells. During the first two treatments, CancerVax was administered with the Tice strain of BCG at 8 x 106 colony-forming units (cfu) per treatment.

TA90-IC Assay
Serum samples from each patient were assayed for TA90-IC using an antigen-specific ELISA as previously described.9 Briefly, sera were diluted 1:60 in .025 M phosphate-buffered saline and 1% Triton X-100, pH 7.2. Diluted sera were placed in the wells of ELISA plates coated with a murine anti-TA90 monoclonal antibody (AD1–40F4), incubated at 37°C for 45 minutes, and washed to remove unbound proteins. AD1–40F4 monoclonal antibody recognizes an epitope on TA90 that is distinct from those recognized by human anti-TA90 IgG. F(ab')2 fragment of goat antihuman IgG conjugated to alkaline phosphatase was added, and the plates were incubated at 37°C for 45 minutes and then washed to remove unbound congugate. The addition of 1 mM p-nitrophenyl phosphate in 10% diethanolamine produced a yellow color in the presence of the bound alkaline phosphatase conjugate. After incubation for 1 hour in the dark at room temperature, absorbance was measured at 405 nm using a microtiter ELISA plate reader (Titertek Multiscan, Dynatech Laboratory, Alexandria, VA). All samples were assayed in duplicate, corrected for nonspecific binding, and compared with standard positive and negative controls.

Sera were considered positive if the absorbance was >=.410 optical density (OD) at 405 nm. This value had previously been generated by testing 59 normal volunteers, none of whom had TA90-IC >=.410, which was the mean OD of this group plus 3 standard deviations.9 TA90-IC seroconverters were those patients whose negative TA90-IC values became positive after initiation of CancerVax treatment.

Clinical Correlation and Statistical Analysis
The OS and DFS curves were estimated by the Kaplan-Meier method. Duration of DFS and OS was defined as the interval between the initiation of CancerVax treatment and the first recurrence or the patient’s death, respectively. The significance of the association between TA90-IC status as a categorical variable and OS or DFS was determined with the log rank test. Analysis of categorical data was performed with {chi}2 testing. The paired t-test was used to compare TA90-IC as a continuous variable before and during CancerVax treatment. Data analysis was performed using a SAS statistical software program, version 6 (SAS Institute, Cary, NC). A Pvalue < .05 was required for statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The study population comprised 121 (55%) males and 98 (45%) females; median age was 52 years (range, 25–72 years). The median follow-up after the initiation of CancerVax therapy was 26 months (range, 2–38 months). The primary lesion was on the head and neck in 48 patients (22%), the trunk in 81 patients (37%), the extremities in 71 patients (32%), and other sites in 19 patients (9%). Fifty-five (25%) patients had AJCC stage II melanoma, 113 (52%) had stage III melanoma, and 51 (23%) had stage IV melanoma.

TA90-IC Before and During CancerVax Treatment
Before administration of CancerVax, TA90-IC values were positive in 51 patients (23%) and negative in 168 patients (77%); the median TA90-IC value was .22. Patients with stage IV melanoma had a significantly higher median pre-CancerVax TA90-IC value (.49) than did patients with stage II (.16, P < .007) or stage III (.19, P < .005) melanoma. Median pre-CancerVax TA90-IC levels were not significantly different between patients with stage II and III melanoma.

The TA90-IC levels of the entire cohort increased significantly after the start of CancerVax therapy (median, .79 post-CancerVax vs. .22 pre-CancerVax, P < .0001). During CancerVax treatment, median TA90-IC values were .72 for stage II melanoma, .76 for stage III melanoma, and .99 for stage IV melanoma; each value was significantly higher than its respective pre-CancerVax level (P < .0001) (Fig. 1). During CancerVax treatment, TA90-IC levels were significantly higher for patients with stage IV melanoma than patients with stage II (P < .007) or stage III melanoma (P < .02). Median TA90-IC values were not significantly different between patients with stage II and III melanoma.



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  		FIG. 1. TA90-IC ELISA values before and during CancerVax (C-Vax) treatment.

 
Using a cutoff point of .410 to define a positive TA90-IC value, 51 (23%) patients were TA90-IC positive and 168 (77%) were TA90-IC negative before the start of vaccine therapy. During CancerVax treatment, 79 (47%) of 168 TA90-IC negative patients seroconverted to TA90-IC positive (Fig. 2), and all initially positive patients remained positive. The rate of seroconversion was higher for stage IV melanoma (65%) than stage II and III melanoma (42% and 43%, respectively), but the difference was not statistically significant.



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  		FIG. 2. TA90-IC status (–, negative; +, positive) according to AJCC stage before and during CancerVax treatment.

 
TA90-IC Status and OS
The correlation between TA90-IC seroconversion and OS approached statistical significance (P < .08) (Fig. 3). Rates of 2-year OS were 78% for seroconverters, 72% for patients who remained TA90-IC negative (P = not significant), and 63% for patients who remained positive (P < .02) (Table 1). Pre-CancerVax TA90-IC levels also correlated with OS: rates of 2-year OS were 72% for TA90-IC negative patients and 63% for TA90-IC positive patients (P < .04). In patients with stage III melanoma, the rate of 2-year OS was significantly higher for seroconverters than for patients who remained positive (81% vs. 65%, P < .05). For patients with stage II or IV melanoma, no significant 2-year OS difference was observed among the three subgroups.



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  		FIG. 3. Overall survival and TA90-IC status after the start of CancerVax treatment.

 

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  		TABLE 1. TA90-IC status and overall survival in patients with stage II, III, or IV melanoma
 
TA90-IC Status and DFS
TA90-IC seroconversion significantly correlated with DFS (P < .02) (Fig. 4). Median DFS and rate of 2-year DFS were >36 months and 59%, respectively, for TA90-IC seroconverters; 24 months and 53%, respectively, for patients who remained negative (P = not significant); and 13 months and 32%, respectively, for patients who remained positive (P < .006) (Table 2). Pre-CancerVax TA90-IC levels also correlated with DFS: rates of 2-year DFS were 53% for TA90-IC negative patients and 32% for TA90-IC positive patients (P < .005). The rate of 2-year DFS was significantly higher for seroconverters than for patients who remained positive and had stage II melanoma (P < .02) or stage III melanoma (P < .05) but not stage IV melanoma. The DFS rate for seroconverters with stage II, III, or IV melanoma was also higher than that for patients of the same stage who remained negative, but this difference reached statistical significance only for patients with stage III melanoma (P < .05).



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  		FIG. 4. Disease-free survival and TA90-IC status after the start of CancerVax treatment.

 

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  		TABLE 2. TA90-IC status and disease-free survival in patients with stage II, III, or IV melanoma
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Phase II trial results have shown that active specific immunotherapy using CancerVax holds promise for patients with melanoma.3–5 Humoral and cellular immune responses to CancerVax have been demonstrated to correlate with survival.7,12 CancerVax may inhibit the metastatic process by activating cytotoxic T cells and antibodies to attack melanoma tumor cells that have entered the bloodstream or have formed subclinical metastases.

TA90-IC has been shown to be a useful prognostic marker for patients who have undergone complete resection of their melanoma,10,11 and it seems to be a more useful prognostic factor than other melanoma markers.13–15 As previously shown, and confirmed by the present study, TA90-IC positive patients with early or advanced melanoma have a higher incidence of recurrent disease and a worse prognosis.

Nevertheless, not all patients derive benefit from CancerVax, and a considerable number of TA90-IC negative patients succumb to their disease. Detection of increased levels of TA90-IC in the serum of melanoma patients after vaccination could indicate destruction of metastatic melanoma cells, and might be helpful as a surrogate marker for CancerVax efficacy. Early identification of patients not responding to CancerVax might allow a timely change to another type of adjuvant therapy.

The results of this study indicate that CancerVax can induce a 2- to 3-fold increase in TA90-IC among patients with early or advanced melanoma. Patients with stage IV melanoma had a higher rate of TA90-IC seroconversion than did patients with stage II or III melanoma, possibly indicating a higher incidence of subclinical melanoma deposits which were destroyed by the immune response to CancerVax. Almost a quarter of this patient cohort (23%) had positive TA90-IC values before CancerVax treatment, despite surgical resection of all detectable disease. This finding could indicate release of TA90 antigen into the circulation from occult metastases and formation of IC with IgG, or persistent presence of the TA90-IC even after excision of the melanoma.

TA90-IC seroconversion is associated with delayed disease relapse and prolonged overall survival. At 24 months of follow-up, significantly more TA90-IC seroconverters were alive (78%) and disease-free (59%) compared with TA90-IC positive patients (63% and 32%, respectively). The TA90-IC seroconverters had better OS and DFS than did TA90-IC negative patients, although this difference did not reach statistical significance. These results are particularly intriguing, because we have previously demonstrated a better prognosis for TA90-IC negative patients than TA90-IC positive patients.10,11 Patients who have negative TA90-IC values before CancerVax treatment are most likely a nonhomogenous group that includes patients with no residual disease or dormant residual disease, patients whose melanoma does not express TA90, and patients who do not generate an IgG response or produce IgG subclasses that cannot bind with TA90 antigen to form immune complexes. The mechanism of seroconversion after vaccination is unclear; an immune response to CancerVax might cause the destruction of residual occult disease, thereby releasing TA90 into the circulation.

Stratification of our data according to AJCC stage demonstrated a clear survival advantage for TA90-IC seroconversion among patients with stage III but not stage IV melanoma. It is conceivable, considering the higher rate of seroconversion in stage IV melanoma, that despite the destruction of melanoma deposits and release of TA90 antigen, the existing occult tumor burden eventually overwhelmed the immune response elicited by CancerVax. For patients with stage II melanoma, the relatively small sample size and low number of recurrences and deaths during the relatively short follow-up period may explain the absence of a detectable survival difference among the three TA90-IC subgroups.

In summary, our study demonstrates for the first time the prognostic significance of serum TA90-IC seroconversion in determining the outcome of adjuvant CancerVax treatment for melanoma. Its potential value as a surrogate marker of CancerVax efficacy may facilitate early identification of patients who should continue vaccine therapy - and patients who are not likely to benefit and should be considered for other adjuvant treatment regimens. Results of the ongoing multicenter randomized phase III CancerVax trials will be helpful in validating the role of TA90-IC as a surrogate marker of vaccine efficacy.


   Acknowledgments
 
Supported in part by grants from the National Cancer Institute (CA 12582) and by funding from the Wayne and Gladys Valley Foundation, Oakland, California. The authors thank Wei Ye, MS, for database queries and statistical analysis.


   Footnotes
 
Presented at the Society of Surgical Oncology Annual Meeting, New Orleans, Louisiana, March 16-19, 2000.

Received for publication March 29, 2000. Accepted for publication October 2, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barth A, Morton DL. The role of adjuvant therapy in melanoma management. Cancer 1995; 75: 726–34.[CrossRef][Medline]
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  5. Shen P, Foshag L, 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 1999; 18: 533a.
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  7. Hsueh EC, Gupta RK, Qi K, Morton DL. Correlation of specific immune responses with survival in melanoma patients with distant metastases receiving polyvalent melanoma cell vaccine. J Clin Oncol 1998; 16: 2913–20.[Abstract/Free Full Text]
  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 1989; 29: 247–54.[CrossRef][Medline]
  9. Gupta RK, Morton DL. Monoclonal antibody-based ELISA to detect glycoprotein tumor-associated-antigen-specific immune complexes in cancer patients. J Clin Lab Anal 1992; 6: 329–36.[Medline]
  10. Hsueh EC, Gupta RK, Qi K, Yee R, Leopoldo ZC, Morton DL. TA90 immune complex predicts survival following surgery and adjuvant vaccine immunotherapy for stage IV melanoma. Cancer J Sci Am 1997; 3: 364–70.[Medline]
  11. Kelley MC, Jones RC, Gupta RK, Yee R, Stern S, Wanek L, Morton DL. Tumor-associated antigen TA-90 immune complex assay predicts subclinical metastasis and survival for patients with early stage melanoma. Cancer 1998; 83: 1355–61.[CrossRef][Medline]
  12. Barth A, Hoon DSB, Foshag LJ, et al. Polyvalent melanoma cell vaccine induces delayed-type hypersensitivity and in vitro cellular immune response. Cancer Res 1994; 54: 3342–5.[Abstract/Free Full Text]
  13. Sirott MN, Bajorin DF, Wong GYC. Prognostic factors in patients with metastatic melanoma. Cancer 1993; 72: 3091–8.[CrossRef][Medline]
  14. Tartour E, Blay JY, Dorval T. Predictors of clinical response to interleukin-2-based immunotherapy in melanoma patients: a French multi-institutional study. J Clin Oncol 1996; 14: 1697–1703.[Abstract/Free Full Text]
  15. Viac J, Schmitt D, Claudy A. Circulating vascular endothelial growth factor (VEGF) is not a prognostic indicator in malignant melanoma. Cancer Lett 1998; 125: 35–8.[CrossRef][Medline]



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