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Annals of Surgical Oncology 8:389-401 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

CancerVax, An Allogeneic Tumor Cell Vaccine, Induces Specific Humoral and Cellular Immune Responses in Advanced Colon Cancer

Nizar Habal, MD, Rishab K. Gupta, PhD, Anton J. Bilchik, MD, PhD, Reynold Yee, BS, Zacharias Leopoldo, DDM, Wei Ye, MS, Robert M. Elashoff, PhD and Donald L. Morton, MD

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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The immunogenicity of the polyvalent tumor cell vaccine CancerVax has been correlated with the survival of patients receiving active immunotherapy for melanoma. Because the various antigens expressed on the vaccine are common to colon adenocarcinoma cells, we examined the survival impact of immune responses elicited by CancerVax in patients with advanced colon cancer refractory to standard therapy.

Methods: Twenty-seven patients with American Joint Committee on Cancer (AJCC) stage IV colorectal adenocarcinoma were entered prospectively into the study. CancerVax was coadministered with bacille Calmette-Guerin (BCG) for the first 2 weeks of vaccine treatment. Blood was drawn at the start of therapy and every 2 weeks thereafter to measure serum titers of immunoglobulin (Ig)G and IgM against TA90 (a 90-kD immunogen common to colon cancer and CancerVax cells) and against purified protein derivative (PPD), a nontumor control antigen. Cellular immune responses were evaluated by delayed-type hypersensitivity (DTH) reaction to vaccine cells and to PPD. Mean follow-up time was 17.5 months.

Results: There was a significant (P = .0001) increase in anti-TA90 IgG and IgM titers and in DTH response to vaccine cells. Humoral and skin responses to TA90 did not correlate with responses to PPD (P = .199 for IgM, P = .958 for IgG, and P = .149 for DTH). This suggests that these responses are not a manifestation of general immune competence. The median overall survival (OS) was 21.9 months for the entire group. Overall survival was higher among patients whose IgMTA90 titer was >800 (P = .003) or whose disease-free interval exceeded 12 months (P = .031). Multivariate Cox regression analysis—using age, sex, disease-free interval, disease status, extent of metastasis, humoral responses, and DTH responses—found only peak IgMTA90 titer to be a significant predictor of overall survival (P = .0365).

Conclusions: CancerVax can induce measurable humoral and cellular immune responses to tumor-associated antigens in patients with advanced-stage colon cancer. These responses correlate with overall survival. This novel therapeutic regimen for patients with advanced colon cancer merits further investigation.

Key Words: TA90 antigen • Immune response • Colon cancer • Active specific immunotherapy • CancerVax


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CancerVax, an allogeneic tumor cell vaccine developed at the John Wayne Cancer Institute (JWCI), consists of three live human melanoma cell lines chosen for their wide range of tumor-associated antigens (TAA) and major histocompatibility complex antigens. The immune response to CancerVax can cross-react with nonvaccine tumor cells expressing some of the same immunogenic TAA, such as gangliosides (GD2, GM2, GD3, and GM3), glycoproteins (fetal antigen, TA90), and/or proteins (MAGE-1, MAGE-3).1 Many of these TAA induce both humoral and cell-mediated responses against vaccine cells and the host’s autologous cross-reacting tumor cells.2–4 The resulting immune-mediated attack on tumor cells appears to translate into clinical regression and improved survival.2–7

One strongly immunogenic TAA expressed on CancerVax cells is TA90, a 90-kD glycoprotein that was initially characterized in melanoma patients treated at John Wayne Cancer Institute.8 TA90 circulates in the serum as an IgG-bound immune complex (IC). Improvements in enzyme-linked immunosorbent assay (ELISA) have permitted measurement of a patient’s IgG or IgM humoral immune response to TA90. Indeed, the IgM response to TA90 has been shown to predict survival in patients receiving CancerVax adjuvant immunotherapy for AJCC stage III and stage IV melanoma.9–11 Many solid malignancies express the TA90 antigen, including colon cancer. In competitive inhibition ELISA experiments, we have found that approximately 80% of colon cancer tissue cells express TA90 (unpublished data). In fact, the incidence of TA90-IC in colon cancer patients (64.1%) approximates the incidence of TA90-IC in melanoma patients (58.5%).12 In a recent study, TA90-IC levels were elevated more frequently than CEA (carcinoembryonic antigen) levels in patients with late-stage colon cancer (83% vs. 70%).13

Based on preclinical evidence that CancerVax and colon carcinoma cells share immunogenic antigens such as TA90, we hypothesized that the vaccine could sensitize a patient with colon cancer to cross-reacting antigens present on the endogenous tumor. The vaccine would thereby induce an immune response with potential clinical efficacy. The primary aim of this study was to determine if CancerVax could induce measurable immune responses in patients with advanced colon adenocarcinoma. The secondary aim was to determine whether these specific immune responses were correlated with overall survival.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Population
Between 1991 and 1999, 27 patients with AJCC stage IV colon adenocarcinoma were entered into a prospective study of CancerVax as adjuvant therapy. Prior to administration of vaccine, informed consent was obtained from all patients. The Institutional Review Board of the John Wayne Cancer Institute and the Saint John’s Health Center approved the investigational protocol. All patients had histologically confirmed colorectal carcinoma and were judged to have good performance status (Eastern Cooperative Oncology Group status 0–1) prior to enrollment. Patients had isolated hepatic, pulmonary, or other metastases that could be controlled by resection and/or cryoablation, or they had minimal residual disease. Patients with multiple brain metastases, immunocompromising conditions, significant hepatic disease, or recent chemotherapy, radiation, or immunotherapy (within 30 days) were excluded.

The age of the patients ranged from 43 to 87 years (mean, 66 years). Nine patients were female and 18 were male. The primary site was the colon in 22 patients and the rectum in the remaining 5 patients. The carcinoma was metastatic to the liver in 16 patients (59%): five patients (18%) had metastases confined to the liver; seven patients (26%) had combined liver and lung metastases—three with a third site of disease (adrenal, diaphragm, or peritoneal implant); and four patients (15%) had liver disease combined with pelvic or peritoneal deposits. All patients with liver disease had <40% hepatic involvement or fewer than 10 hepatic metastases. The remaining 11 patients had pelvic/retroperitoneal (n = 3), retroperitoneal (n = 4), or lung (n = 4) metastases without liver involvement. The disease-free interval (DFI) between initial resection of the primary colorectal adenocarcinoma and the development of stage IV disease ranged from 0 to 73 months (mean, 12.6 months; median, 4.0 months). Ten patients (37%) had synchronous metastatic disease at the time of initial cancer diagnosis. Patient characteristics are outlined in Table 1.


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  		TABLE 1. Clinical characteristics of study patients
 
Before enrollment in the study, 23 patients (85%) had previously received systemic 5-fluorouracil (5-FU)-based adjuvant chemotherapy. Two patients had 5-FU alone. Fourteen patients had additional leucovorin (LV), five patients had additional levamisole (LM), and two patients had both LV and LM. Five patients who received 5-FU and LV had additional therapy in the form of an intrahepatic pump (three patients), intraperitoneal cisplatinum (one patient), or systemic mitomycin-C (one patient). Four patients (15%) had not received prior chemotherapy. Vaccine therapy was started an average of 16.9 months after the diagnosis of stage IV disease (range, 1 to 53 months). Fifteen patients (56%) were rendered grossly disease-free at operation prior to the start of vaccine therapy.

Treatment Protocol
CancerVax is a whole-cell preparation of three allogeneic melanoma cell lines (M10-V, M24-V, and M101-V) pooled in equal amounts to a total of 24 x 106 cells. For this study, the cells were grown, prepared, irradiated with 150Gy, and then cryopreserved in liquid nitrogen as described previously.14 The cells were thawed immediately before intradermal injection; the vaccination schedule was similar to the schedule used for patients with melanoma.10 The vaccine was administered at weeks 0, 2, 4, 6, 8, and every fourth week successively for 1 year. The Tice strain of bacille Calmette-Guerin (BCG) was co-injected on weeks 0 (3 x 106 organisms) and 2 (1.5 x 106 organisms) only. After the first year of vaccine therapy, patients were offered continued treatment if they had responded clinically or immunologically. All patients were followed prospectively during their treatment, with specific evaluation of disease status and adverse toxic events related to the therapy. Complete blood cell count and blood chemistries were performed monthly, and staging computed tomography (CT) was performed at 3-month intervals. Clinical disease status was recorded as no evidence of disease (NED) or alive with disease (AWD) at each follow-up visit. Adverse events throughout the study period were also noted and recorded at each follow-up visit. The adverse events were graded according to the National Cancer Institute’s common toxicity criteria adapted from the World Health Organization guidelines for reporting results of cancer treatment.15 Recurrent disease was confirmed by complete physical examination, chest X-ray, CT of the chest/abdomen or pelvis, magnetic resonance imaging, and/or positron emission tomography (PET). Patient charts were reviewed to determine dates of recurrence and death. The mean follow-up for the entire group was 17.5 months from the start of vaccine therapy.

Blood samples were drawn within 2 weeks prior to first vaccination, every 2 weeks for the first 2 months, and every month thereafter. All samples were coded blindly and stored at -35°C until they were thawed for the serologic assays.

DTH Response
The presence of a cell-mediated immune response to vaccine was determined by delayed-type hypersensitivity (DTH) skin testing at 0, 2, 4, 6, 8, 12, and 16 weeks. Each intradermal injection contained 2.4 x 106 vaccine cells. Skin testing with purified protein derivative (PPD) was used as a nonspecific antigen control. The width of palpable induration and visible erythema around the injection site were recorded. An area of induration >=7 mm at 48 hours after injection was considered a positive DTH response, regardless of surrounding erythema.

Anti-TA90 IgG and IgM ELISA
All blood samples were coded and tested for immune responses in a blinded fashion. Purified TA90 glycoprotein was used as a target to determine IgG and IgM levels. The preparation of the glycoprotein is described elsewhere.12 The antigen was adsorbed to polystyrene wells of ELISA plates at a concentration of 50 ng/well. One hundred microliters of patient serum at serial doubling dilutions was incubated in the wells. Goat antihuman IgG (1:500 dilution) or antihuman IgM (at 1:1000 dilution) conjugated to alkaline phosphatase was then added to the wells. After incubation and washing, 200 µl of chromogenic substrate (1 mg of P - nitrophenyl-phosphate per 1 ml of 10% diethanolamine at pH 9.6) was added. After a 90-minute incubation at room temperature, the absorbance at 405 nm was read. Antibody titer was defined as the highest dilution yielding an absorbance of 0.05.

The baseline preimmune titer was obtained from a serum sample drawn within 2 weeks prior to the start of vaccine. Postimmune titers were obtained within the first 2 months after vaccine start.

Antipurified PPD IgG and IgM Assay
PPD is a non-melanoma-associated antigen used in this assay to measure the patient’s response to BCG in order to assess the specificity of the humoral immune response. One hundred microliters of PPD solution at 2 IU/ml (Connaught Laboratories Inc., Toronto, Canada) in 0.06 M bicarbonate buffer at pH 9.6 was adsorbed to the polystyrene wells at 4°C for 16 hours. The remainder of the ELISA was performed as described above.

Statistical Analysis
Members of JWCI’s Statistical Coordinating Unit coded and entered patient data on clinical status, survival, DTH, and immune responses into one database. Survival was examined using a landmark analysis set at 2 months after the day of the first vaccine dose. Only peak titers obtained within this 2-month landmark period were considered for analysis. The starting date for the survival counts, T0, was 2 months after the first dose of vaccine. One patient whose serum analyses were not completed during the first 2 months was disqualified from the survival analysis. Overall survival curves were plotted using the nonparametric Kaplan-Meier method, and the differences in survival were estimated using the log-rank test. The Cox proportional hazards model was used to determine the survival impact of gender, the disease status at the start of vaccine therapy, and the degree of metastatic disease as binary variables; age, DFI, and all immune responses were analyzed as continuous variables. The immune responses considered in the analysis included anti-TA90 and anti-PPD humoral IgG and IgM peaks, and the DTH responses to tumor cells and PPD (as continuous or binary variables). Increases in titers and DTH responses before and after vaccine were analyzed using the signed rank-sum test. The relations between the TA90 and control PPD responses (IgG, IgM, and DTH) were analyzed using the Spearman correlation test. All statistical analyses were performed using the SAS computer statistical package (version 6.12, SAS Institute, Cary, NC). A two-tailed P-value <=.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical Course
Twelve patients had evidence of disease (alive with disease, AWD) at the start of vaccine therapy. Seven died an average of 17.3 months after vaccine (range, 4–35 months). The disease progressed at a mean of 7.3 months (range, 2–19 months) after vaccine in these seven patients. Five of the 12 AWD patients remain alive: two showed no evidence of disease progression at 6 and 10 months after vaccine, respectively, and the remaining three showed disease progression at 2, 6, and 8 months, respectively.

Fifteen patients were grossly free of disease (no evidence of disease, NED) at the start of vaccine. Five remain NED at a mean of 23.4 months after vaccine (range, 7–73 months). Four patients recurred at 9 months (range, 2–19 months) but remain alive. Six patients expired at 18.2 months after vaccine (range, 5–43 months), having recurred at an average of 7.8 months (range, 4–24 months). The median overall survival for the entire group was 21.9 months.

Humoral Immune Responses
The humoral immune responses are graphically represented in Fig. 1. Preimmune anti-TA90 IgM (IgMTA90) titers ranged from 100 to 800 (median, 200; mean, 256). Peak postimmune titers ranged from 100 to 1600 (median, 800; mean, 663); the increase was significant (P = .0001). Two patients (7.4%) had preimmune titers >800. Fourteen patients (52%) developed peak postimmune IgMTA90 titers >800 at a mean of 21 days after the first vaccine (range, 7–55 days). The peak postimmune IgMTA90 titers for these 14 patients ranged from 800 to 1600 (median, 1000; mean, 1086); their preimmune titers ranged from 100 to 500 (median, 200; mean, 321), reflecting a mean increase in titer of 765 points. The 13 remaining patients had postimmune IgMTA90 titers below 800 (range, 100–500; mean, 269); their preimmune titers ranged from 100 to 400 (mean, 176), reflecting a mean rise of 93. Of the 12 patients who were AWD at the start of vaccine, five (42%) developed a titer above 800. Of the 15 patients who were NED, nine (60%) developed a titer above 800.



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  		FIG. 1. Mean preimmune and postimmune IgG and IgM titers and DTH indurations for TA90 and for PPD. Lines represent standard deviation. *P = .0001 by signed rank-sum test.

 
The preimmune anti-TA90 IgG (IgGTA90) titers ranged from 100 to 3200 (median, 200; mean, 696) and the peak postimmune IgGTA90 titers ranged from 200 to 6400 (median, 800; mean, 1315). The increase in titer was significant (P = .0001). Nine patients (33.3%) had preimmune titers above 800. Nineteen patients (70.3%) developed a postimmune IgGTA90 titer above 800 (range, 800–6400; median, 1600; mean, 1810) an average of 26 days after the first vaccine. Their preimmune titers ranged from 200 to 3200 (median, 600; mean, 942), reflecting an average rise of 868 points. Nine of the 19 patients (47%) had concomitant IgMTA90 titers above 800.

Humoral responses to PPD were assayed to determine response to coadministered BCG. Preimmune IgGPPD titers ranged from 100 to 200 (median, 100) and the postimmune peaks ranged from 100 to 800 (median, 200; only two patients had titers at 800). The preimmune IgMPPD titers ranged from 100 to 400 (median, 100) and the postimmune peaks ranged from 100 to 2400 (median, 200; four patients had titers above 800). The average increases in IgGPPD and IgMPPD titers were 104 and 226, respectively. The increases in IgG and IgM titers after vaccine were both significant (P = .002 and .026, respectively). As shown in Fig. 2, there was no correlation between the peak postimmune IgGTA90 and IgGPPD (P = .199) or IgMTA90 and IgMPPD (P = .958). This finding confirms that the immune responses elicited by CancerVax were specific and unrelated to general humoral immune competence as measured by the IgG and IgM responses to BCG.



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  		FIG. 2. Correlation of peak anti-TA90 antibody levels with peak anti-PPD antibody levels. (A) IgG: P = .199; (B) IgM: P = .958; (C) DTH: P = .149. P values estimated using the Spearman test for correlation.

 
DTH Responses
The increases in DTH responses are graphically depicted in Fig. 1. Skin hypersensitivity responses to vaccine cells, DTHTCV, prior to immunization ranged from 0 to 24.5 mm (mean, 5.4; median, 5). After vaccine, the responses ranged from 2 to 37.7 mm (mean, 12.4; median, 10); the increase in response was significant (P = .0001). Five patients had preimmune DTHTCV >7 mm, compared to 21 patients after vaccine.

Two patients had DTHPPD >7 mm before vaccine (range, 0–17; mean, 2.3; median, 0). After vaccine, all but three patients had developed a DTHPPD above 7 mm (range, 0–31; mean, 11.1; median, 10). Here again, the increase in DTH response after vaccine was significant (P = .0002) and there was no correlation between postimmune DTHTCV and DTHPPD (P = .149), suggesting that the skin responses to tumor cells were unrelated to a general boost in immunity (Fig. 2).

Relationship Between the Immune Response and Survival
Nine of the 14 patients with elevated postimmune IgMTA90 titers were NED at the start of vaccine; only one patient expired 24 months after the start of vaccine. Five of the 14 patients had residual gross disease (AWD); two of them expired at 31 and 35 months, respectively. Thirteen of the 27 patients (48%) did not develop elevated IgMTA90 titers. Five of the six patients who were NED at the start of vaccine expired at 5, 5, 13, 18, and 43 months. Five of the seven patients who were AWD at the start of vaccine expired at 4, 7, 9, 15, and 21 months.

The statistical impact of several clinical and immune variables on survival in the 26 patients evaluated is shown in Table 2. Cox regression analysis confirmed that age (P = .826), sex (P = .894), clinical status at the start of vaccine (P = .391), diffuse metastases (P = .125), and DFI (P = .169) did not have an impact on survival. Analysis of the immune responses as continuous variables identified peak IgMTA90 after vaccine as the single significant factor in predicting overall survival (P = .0365). A higher IgMTA90 after the initiation of vaccine was associated with improved survival, but the same was not true for IgGTA90 and DTHTCV. The correlations between overall survival and the humoral immune responses to TA90 and cellular response to tumor cells are depicted graphically in Fig. 3. Although DFI was not significant when analyzed as a continuous variable on Cox analysis, a log-rank analysis using a 12-month cutoff showed that patients with a longer DFI did have an improved outcome (19.0 vs. 41.6 months, P = .042). However, there was no correlation between the DFI and IgMTA90 responses (P = .161).


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  		TABLE 2. Statistical impact of prognostic variables on overall survival
 


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  		FIG. 3. Correlation of peak anti-TA90 responses with overall survival. Open circles represent patients who remain alive, closed circles represent patients who have expired. (A) IgG: P = .426; parameter estimate = 0.00091; SE = 0.000243; (B) IgM: P = .0365; parameter estimate = -0.00127; SE = 0.000606; (C) DTH: P = .921; parameter estimate = 0.00380; SE = 0.038460. Pvalues estimated by Cox proportional hazards model for continuous variables.

 
To determine a clinically applicable cutoff point for the immune responses to TA90, a log-rank analysis of the responses as categorical variables using different cutoff points was performed (Table 2). No significant cutoff points were noted for either IgGTA90 or for DTHTCV. In contrast, a cutoff point of 800 for IgMTA90 did show a statistically significant difference: patients who developed titers >800 had a statistically better survival than those who did not (median OS, 31.06 and 12.74, respectively; P = .003). A DTHTCV response >7 mm did, however, also approach significance (P = .076).

The relationship between IgM and overall survival was specific to TA90, not to PPD. Both Cox analysis and log-rank analysis failed to show a significant impact on survival or a significant cutoff point with any immune response directed against PPD, including IgG, IgM, or DTH responses (Table 3). In addition, the relationship between overall survival and IgMTA90 was specific to postimmune but not preimmune titers (Table 3).


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  		TABLE 3. Statistical impact of preimmune responses to TA90 and postimmune responses to PPD
 
The Kaplan-Meyer estimates of overall survival comparing responders and nonresponders were significant when IgMTA90 response was considered (see above) but not when the IgMPPD response was considered (19.2 vs. 29.2, respectively; P = .61 by log-rank) (Fig. 4).



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  		FIG. 4. Overall survival estimated by the Kaplan-Meier method for patients with a humoral IgM response to TA90 (left) vs. a humoral immune response to PPD (right). IgMTA90 = anti-TA90 IgM titer; IgMPPD = anti-PPD IgM titer. Response is defined as peak postimmune titer >800. Respective median overall survivals for responders (R) and nonresponders (NR) were 31.06 and 12.74 months for IgMTA90 (P = .003) and 19.21 and 29.21 months for IgMPPD (P = .6127). P-values estimated using the log-rank test.

 
Toxicity Profiles
The majority of toxicities were graded II and I. The most common were fatigue (27 reports), site reaction and pain (19 and 17 reports, respectively), myalgias and headaches (16 reports each), nausea (15 reports), diarrhea (14 reports), and fever (11 reports). No patient sustained grade IV toxicities throughout the study period (Table 4). No reported deaths were directly attributable to the vaccine.


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  		TABLE 4. Adverse side effects during vaccine treatment
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Each year more than 50,000 patients receive treatment for metastasis of colorectal cancer to the liver and other distant sites. Because median survival is low without therapeutic intervention,16–21 several approaches have been used to control metastatic disease in an attempt to prolong survival. Surgical resection offers the best chance for prolonged survival and the only potential for cure. Series of selected nonrandomized patients undergoing complete hepatic metastasectomy have reported 5-year survival rates of 25% to 46%.16–17 Unfortunately, only 20%–25% of patients with hepatic metastases from colorectal cancer are candidates for resection, and postoperative recurrence rates can be as high as 60%.17

Chemotherapy for advanced or recurrent metastatic colon cancer includes systemic bolus/continuous intravenous infusion of 5-FU and leucovorin, widely accepted as the standard regimen, with or without the newer chemotherapy agent irinotecan (CPT-11). Although response rates vary between 10% and 30%, the survival time remains 11 to 17 months.18–19 Palliative therapies for advanced colorectal cancer also include regional hepatic arterial infusion of high-dose chemotherapy (5-FU or floxuridine) with or without noncurative tumor ablation techniques such as cryosurgery and radiofrequency ablation, and chemoembolization.20–22 Although most of these therapies can produce an objective response and induce some tumor regression, none has shown a significant survival advantage in conclusive randomized trials.17–19

A number of immune-based treatment strategies for colon cancer have been applied. The simplest approach is to inject concentrated cytokines that are known to stimulate T-cells and natural killer cells, such as interferon and interleukin 2 (IL2). Holcombe et al.23 reported eventual progression of disease regardless of immunostimulation in four patients treated weekly with IL2. Radiolabeled murine monoclonal antibodies such as T84.66 and COL-1, conjugated with toxic molecules or radioisotopes, are used to selectively direct lethal doses of radiation or toxic drugs to the carcinoma cells. Their use has been plagued in part by the development of human antimurine antibodies, which limits the number of repeat administrations.24,25 This problem has led to a growing interest in human-murine chimeras administered with or without immunomodulators.26,27

Active immunotherapeutic strategies based on CEA (carcinoembryonic antigens) have also been developed. CEA is a well-characterized molecule that is highly expressed by colon carcinoma cells.28 Direct injections of purified or recombinant antigen or vector-bound antigen packaged in viral proteins, as well as anti-idiotypic antibodies mimicking CEA, have been examined.29–31 The end point for these studies is the induction of a humoral and/or cellular immune response in the host against CEA expressed on the tumor. However, although CEA-directed T-helper cell (CD4) activation and specific antibody production has been documented, the initial clinical response rates have been low.32,33 Still, a recent study of 32 patients with Duke’s stages B, C, and D colorectal cancer reported that anti-idiotype monoclonal antibody immunization induced a polyclonal IgG humoral response to CEA in all cases,34 including 17 patients with AJCC stage IV disease. Seven of the eight patients with resected metastatic disease (similar to our NED group) remained alive at 12–33 months with stable disease. Eight of nine patients with unresectable metastatic disease (similar to our AWD group) progressed on therapy.

A more promising immune-based therapy is active specific immunotherapy using autologous live or attenuated tumor cells admixed with various adjuvants.35 A recent prospective randomized trial conducted by Vermorken et al.36 in the Netherlands showed a significant increase in recurrence-free survival following four doses of autologous vaccine versus no adjuvant therapy in 254 patients with stage II and III colon cancer (P = .032). The vaccine was composed of 107 irradiated autologous colon cancer cells administered in three weekly injections, with a booster dose at 6 months. Admixed BCG (107) was administered during the first two injections only. There was a trend towards improved survival mainly in the stage II cohort: at a median follow-up of 64 months, overall survival was 80% in 85 vaccine-treated patients versus 72.9% in 85 patients not receiving vaccine (P = .149). Unfortunately, the widespread application of autologous vaccines is plagued by difficulty in cell harvesting and in vitro culture yields. Patients must have metastatic tissue deposits that are easily accessible and that can be harvested for culture. Once harvested, the tissue must delivered in a timely fashion to specialized laboratories to prepare viable single-cell suspensions. However, tumor biopsies may not yield enough viable tumor cells. Also, the results of the larger Eastern Cooperative Oncology Group (ECOG) study E5283 have recently been reported to be negative. The study, initiated in 1983, randomized 412 patients with stage II and III colon cancer to observation or to treatment with three weekly injections of 107 irradiated autologous tumor cells, the first two of which were admixed equally with BCG. The treatment protocol was similar to that used in the Netherlands study, but after a median follow-up of 7.6 years, overall survival was not significantly better in the treatment arm (P = .12). The investigators argued that the poorer outcome in this ECOG study might be related to lower patient compliance, less optimal quality control, and/or shorter treatment schedules.37 Neither group examined the effects of autologous vaccine in stage IV colorectal carcinoma patients.

Our pilot study examined the efficacy of active specific immunotherapy using CancerVax, which consists of three allogeneic melanoma cell lines that are sublethally irradiated. CancerVax has been shown to prolong survival of melanoma patients by inducing both humoral and cellular immune responses.2 Many proposed mechanisms are believed to be responsible for these responses. First, whole cells may be degraded by macrophages and/or dendritic cells that then present a multitude of tumor-associated antigens to T-cells, triggering T-cell cytotoxic and antibody responses. Alternatively, the live cells of CancerVax may act as antigen-presenting cells, directly stimulating the T-cells under MHC class I restriction. In vitro studies have confirmed the induction of T-cell immune responses by the mixed lymphocyte-tumor cell reaction (MLTR) assay and the increase in cytotoxic T-cell activity,5,7 as well as enhanced complement-dependent cytotoxicity6 in patients immunized with vaccine. Linear regression analysis confirmed a significant correlation between in vivo DTH and in vitro MLTR assays. In addition, stimulated helper T-cells may boost a humoral response to tumor-associated antigens expressed on the surface of vaccine cells; this humoral response would cross-react with native tumor cells expressing the same antigens. Our approach in using CancerVax for colorectal cancer is based largely on the presence of tumor antigens common to melanoma and colon carcinoma cells. We believe that the mechanisms which lead to a host response against one cell type will likely stimulate a cross-reacting immune response against the tumor antigens present on the other cell type after vaccination. One such tumor-associated antigen is TA90.

TA90 is a strongly immunogenic 90-kD glycoprotein that was originally described as a tumor-associated antigen in melanoma.8 Its measurable serum immune complex, TA90-IC, can be found in several solid malignancies, including colorectal carcinoma, but is rarely detected in the serum of healthy volunteers (<5%).12 Indeed, we recently identified TA90-IC in 88% of 41 patients with stage IV colorectal cancer.13 Our group has studied the characteristics and properties of TA90 extensively, and we have previously offered evidence for a link between the humoral response to this immunogen and the survival of melanoma patients treated with CancerVax. Multivariate analyses have shown that the IgMTA90 response induced by CancerVax is a powerful independent predictor of survival for patients with AJCC stage IV melanoma (P =.032),10 stage III melanoma (P = .0053),9 and, more recently, stage II melanoma.38 The 5-year survival of vaccine-treated stage IV melanoma patients who develop both IgMTA90 (humoral) and DTHTCV (cellular) responses is 75%, compared with 8% for patients who develop neither response.11 This humoral response is not correlated with immune responses to PPD and therefore does not reflect a generalized, nonspecific boost in immunity.

This study supports our main hypothesis that the allogeneic tumor cell vaccine CancerVax can elicit measurable immune responses in colon cancer patients. Specific IgGTA90 and IgMTA90 antibody levels were above 800 in nine patients (33%) and two patients (7.4%), respectively, before vaccine; however, after vaccine 19 patients (70%) and 14 patients (52%), respectively, had developed elevated titers. Skin-test responses (DTHTCV) were seen in five patients (19%) before treatment, compared with 21 patients (78%) after treatment. By contrast, only two patients (7%) developed elevated IgGPPD and four patients (15%) developed elevated IgMPPD after treatment, whereas the majority (89%) had developed a skin test response to DTHPPD. There was no correlation between the peak IgG and IgM responses to the respective antigens or between the peak skin-test responses. The immune responses to CancerVax are therefore distinct from those due to the coadministered BCG, confirming that they are not simply a reflection of a generally heightened immune system.

Our second hypothesis is that those specific immune responses elicited by CancerVax can predict a better outcome. In other words, the immune responses elicited by the vaccine cells, not the BCG, have a clinically beneficial effect. In fact, when compared with several clinical and immune variables, the IgM-based humoral response to TA90 was the most powerful prognosticator of overall survival. It is important to note that the preimmune IgMTA90 titer alone had no impact on survival (P = .820). The prognostic effect of the peak postvaccine humoral response applied only to IgM, not IgG (P = .0365 and .426, respectively), and was specific to TA90, not PPD (P = .483) (Tables 2 and 3). In an effort to find a clinically useful prognostic cutoff point, several points were examined for each of the immune variables. A cutoff point of 1:800 for IgMTA90 was highly significant (P = .003) as a predictor of overall survival. No other cutoff point showed such a strong correlation with survival. The median OS of the 14 patients with a postimmune IgM titer above 800 was more than two-fold higher than the median OS for the 12 patients who did not respond (31.06 vs. 12.74 months, respectively). The Kaplan Meier estimates for survival for these two groups showed a wide separation between the curves (Fig. 4). The DFI, a known prognostic factor for colon cancer, did predict improved survival when it exceeded 12 months. However, the statistical significance was lost when DFI was treated as a continuous variable in a Cox model, suggesting that it may have been a statistical anomaly. In fact, there was no correlation between the DFI and the peak IgM titer (P = .169). The possibility that these findings are due to a time bias does exist, but we have tried to control for it by applying the landmark statistical analysis. In addition, we have observed that the nonresponders in our study had median survival times no different from those of similar-stage historical controls treated only with FU-based chemotherapy (12.74 months), which suggests that our patient population was not highly selected.

But why should an IgM-based response, as opposed to one that is IgG-based, confer better immune protection? The mechanisms by which an IgM humoral immune response leads to improved survival are not yet evident. Early studies on serum immunoglobulins in colorectal carcinoma supported an unfavorable prognostic association with serum IgM when compared with other serum immunoglobulins. High IgM levels have been associated with advanced colon cancer.39–41 These observational studies were based on nonspecific, polyclonal elevations of immunoglobulin M; there has since been a better understanding of how the various Ig classes and subclasses interplay with cancer cells in vivo.

One hypothetical mechanism for the clinical efficacy of an IgM response to TA90 is based on the unproven notion that cell-surface glycoproteins can function as cell-adhesion molecules. CEA, a much larger surface-expressed glycoprotein, is believed to have cell-adhesion properties that allow colorectal carcinoma cells shed in portal blood to bind to hepatic endothelium and hepatocytes.42 This attachment phase would be a key step in the development of metastasis. If TA90 proves to be involved in cell adhesion, we speculate that pentameric IgM antibodies might block the cell-adhesion epitopes of the molecule more efficiently than monomeric IgG antibodies.

A primary function mediated by antibodies in general is complement activation. The human immunoglobulin subtypes vary significantly in their ability to bind and activate complement. IgM antibodies are very effective binders and activators of this system, as are IgG1 and IgG3 isotypes to a lesser extent. This activation initiates a series of intricate pathways that lead to the formation of a membrane-attack complex that directly lyses the cell.43 Therefore, anti-TAA IgM can efficiently and specifically bind to the tumor cells that express the TAA (such as TA90) and subsequently initiate the cascade. Our own experiments have shown that polyclonal anti-TA90 IgM but not anti-TA90 IgG isolated from baboon serum can lyse 36% to 71% of several cultured cell lines, including melanoma, breast and neuroblastoma, in the presence of complement.44 This cell lysis could be inhibited by preadsorption with a different melanoma cell line, but not by lymphoblastoid cell lines.

The humoral response described herein is not isolated to TA90. Indeed, our group has recently reported the prognostic significance of the IgM-based humoral response to other tumor-associated antigens, namely the gangliosides GD2, GM2, GD3, and GM3, in 66 stage III melanoma patients treated with CancerVax.45 This confirms our similar results with an earlier tumor cell vaccine,46 and similar results obtained by Livingston,47 who immunized melanoma patients with purified ganglioside GM2. We have demonstrated a similar increase in anti-GM2 IgM antibodies in some of the patients with stage IV colorectal cancer included in this study (unpublished data, 2000). A prognostic model combining the immune responses to multiple tumor-associated immunogens may provide an even stronger association with survival.

In conclusion, this study suggests that CancerVax may have a beneficial impact, with minimal or no toxicity, in patients whose late-stage colorectal cancer is refractory to conventional treatments. The overall survival of the 27 patients was 21.9 months, which compares quite favorably with historical controls with metastatic colorectal cancer. The results of this study are significant and very encouraging despite the fact that this is a small and heterogeneous group of patients. To confirm these results, a prospective randomized trial properly stratified by the known prognostic factors for colorectal carcinoma is needed to definitively evaluate the efficacy of CancerVax in the treatment of colon cancer. The optimal population would represent three groups: patients whose hepatic metastases are amenable to resection, radiofrequency ablation, and/or cryotherapy; patients with minimal residual disease after surgical treatment; and patients with a long disease-free interval prior to diagnosis of metastases at multiple sites. These groups may represent up to 40% of all patients diagnosed with colon cancer.48 Stratification factors would include disease status (NED or AWD) prior to vaccine treatment, prior systemic chemotherapy, disease-free interval, CEA level, and other variables.


   Acknowledgments
 
This study was supported by grants P01 CA 12.582 and T32 CA 09.689 from the National Cancer Institute and by funding from the Wayne and Gladys Valley Foundation, Oakland, CA; the Rogovin-Davidow Foundation, Los Angeles, CA; and the Rod Fasone Memorial Cancer Research Fund, Indianapolis, IN.


   Footnotes
 
Disclosure: Donald L. Morton has an ownership interest in patents on the vaccine and in the company which has the rights to the vaccine. (Per copyright form date 7/25/00).

Received for publication July 3, 2000. Accepted for publication December 15, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
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