Annals of Surgical Oncology Sign the Guestbook
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Originally published as Ann Surg Oncol Early Release 10.1245/ASO.2004.02.018 on September 20, 2004

Annals of Surgical Oncology 11:892-899 (2004)
© 2004 Society of Surgical Oncology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hsueh, E. C.
Right arrow Articles by Morton, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hsueh, E. C.
Right arrow Articles by Morton, D. L.

Article

Peripheral Blood CD4+ T-Cell Response Before Postoperative Active Immunotherapy Correlates with Clinical Outcome in Metastatic Melanoma

Eddy C. Hsueh, MD, Estela Famatiga, MS, Sherry Shu, BS, Xing Ye, MS and Donald L. Morton, MD

From the Department of Surgery at Saint Louis University (ECH), St. Louis, Missouri, and the Roy E. Coats Research Laboratories of the John Wayne Cancer Institute of Saint John’s Health Center (EF, SS, XY, DLM), Santa Monica, California.

ABSTRACT

Background: Canvaxin polyvalent specific active immunotherapeutic (CancerVax Corp., Carlsbad, CA) is a minimally toxic adjuvant after resection of regional metastatic melanoma. Because Canvaxin immunotherapeutic requires induction of an immune response, we hypothesized that survival would be directly correlated with cellular immune responses to Canvaxin cells prior to immunization.

Methods: We randomly selected 54 patients from a study of Canvaxin therapy after complete resection of American Joint Committee on Cancer (AJCC) stage III melanoma. Peripheral blood lymphocytes (PBLs) collected before immunotherapy were co-cultured with Canvaxin cells; cellular response was determined by flow cytometric measurement of the production of intracellular interleukin 4 (IL4) or interferon gamma (IFN{gamma}) by CD4+ T-cells. Results were calculated as percent positive for double staining of CD4+ plus IL4+ or CD4+ plus IFN{gamma}+.

Results: The mean (± SD) increase in cytokine-producing CD4+ T-cells after Canvaxin stimulation was 4.8 ± 2.3% for an IFN response and 5.1 ± 2.0% for an IL4 response. Both increases were significantly correlated with overall survival by univariate analysis (P = .0471 for IFN{gamma} and 0.002 for IL4). There was no significant correlation between unstimulated IFN{gamma}/IL4 responses and overall survival. Multivariate analysis showed that a CD4+ T-cell IL4 response before Canvaxin therapy was a significant independent prognostic variable.

Conclusions: In vitro cellular immune response to Canvaxin cells directly correlates with survival after subsequent initiation of immunotherapy for AJCC stage III melanoma. This finding will be evaluated in a multicenter phase III trial of Canvaxin plus bacille Calmette-Guerin (BCG) versus placebo plus BCG after resection of stage III melanoma.

Key Words: Endogenous immune response • Immunotherapy • Melanoma

Because malignant melanoma is generally resistant to chemotherapy and radiation, various immunologic interventions have been tested. Interferon (IFN) {alpha} 2b and interleukin (IL) 21,2 have been approved by the U.S. Food and Drug Administration, but both are nonspecific immune enhancers. Although no specific immunotherapeutic agent has demonstrated clinical efficacy against melanoma in a randomized phase III trial, several promising immunotherapeutic approaches are being tested in clinical trials. These approaches range from monospecific immunotherapy, which targets only one specific tumor antigen,3–5 to polyvalent immunotherapy, which attempts to induce immune responses to multiple antigenic components,6–9 to gene-based therapy, which manipulates the immunogenicity of the tumor.10–13 While these immunotherapeutic approaches have been effective in animal models, the frequent disparity between animal findings and clinical results and the lack of reliable clinical immunologic assays have made the outcome of immunotherapeutic trials uncertain. Assays based on clinically relevant immunologic mechanisms are necessary to design potent immunotherapeutic agents against melanoma.

Since 1984, we have conducted phase I/II trials of Canvaxin polyvalent specific active immunotherapeutic (CancerVax Corporation, Carlsbad, CA) for patients with American Joint Committee on Cancer (AJCC) stage III and IV melanoma.8,9,14 Canvaxin immunotherapeutic is an irradiated preparation of whole melanoma cells from three allogeneic melanoma cell lines. Recent matched-pair analyses strongly suggest a significant survival benefit from adjuvant Canvaxin therapy after complete resection of metastatic melanoma.8,9 In stage III melanoma, median overall survival (OS) and 5-year rate of OS were 55.3 months and 48.8%, respectively, for 739 Canvaxin recipients, versus 31.6 months and 36.8%, respectively, for 739 patients not receiving Canvaxin immunotherapeutic (P = .0001).8 In stage IV melanoma, median OS and 5-year rate of OS were 38 months and 39%, respectively, for 107 Canvaxin recipients, versus 19 months and 20%, respectively, for matched nonrecipients (P = .0009).9 The cellular (skin test reaction) and humoral (increase in specific antibody titer) immune responses to Canvaxin immunotherapeutic correlated strongly with survival in the Canvaxin cohort.15,16

The irradiated, nonproliferating melanoma cells of Canvaxin immunotherapeutic do not have a direct in vivo cytotoxic effect on melanoma cells. Instead, this immunotherapeutic’s clinical efficacy putatively derives from the induction of immune responses to multiple immunogenic antigens found on the surface of Canvaxin cells and shared by the melanoma cells.8,14,17–21 Responses to these antigens have been identified in preoperative and postoperative peripheral blood samples.22–27 The mechanism underlying the initiation of an effective antimelanoma immune response is unclear, but multiple independent investigators recently have demonstrated that CD4+ T-cells may be pivotal.28–31

Because Canvaxin immunotherapeutic is believed to act by enhancing endogenous cellular and humoral immune responses, we hypothesized a correlation between survival and the in vitro CD4+ T-cell response to Canvaxin cells, as measured in peripheral blood lymphocytes (PBLs) obtained prior to immunization with Canvaxin immunotherapeutic.

PATIENTS AND METHODS

Study Subjects
The study population was drawn from patients diagnosed with AJCC stage III melanoma and enrolled in a protocol for adjuvant Canvaxin immunotherapy after complete regional lymph node dissection. Prior to protocol enrollment, absence of distant metastatic disease was confirmed by complete physical examination, chest x-ray, magnetic resonance imaging of the head, computed tomography of the chest/abdomen/pelvis, and/or whole-body positron emission tomography. Excluded were patients who had received other adjuvant therapy within 30 days prior to Canvaxin initiation. Informed consent for the Canvaxin protocol and for specimen use was obtained from all patients. The protocol for Canvaxin adjuvant therapy 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.

PBL samples were obtained after surgery but before immunotherapy. These PBL samples were processed by Ficoll-Hypaque separation and were aliquoted, program-frozen, and stored in a liquid nitrogen freezer in our lymphocyte specimen bank. Each patient’s clinical status was recorded prospectively until time of recurrence or the last follow-up date, and data were transferred to the melanoma immunotherapy database managed by our statistical coordinating unit.

From January 1, 1993, through October 31, 1997, 595 patients were enrolled in the protocol. We randomly selected 54 of these patients by computer. The time period was chosen to ensure that PBL specimens had been cryopreserved no more than 10 years and that the duration of follow-up was at least 4 years prior to testing. There was a slight female predominance (54%) in the study cohort (Table 1). Most patients (86%) received the first dose of Canvaxin immunotherapeutic within 3 months after lymph node dissection. Prior to initiation of Canvaxin therapy, 48 patients (89%) had a normal lactate dehydrogenase (LDH) value and 49 (91%) had a lymphocyte count >1000.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Patient demographics

 

PBLs were also obtained from four self-proclaimed healthy volunteers who had given informed consent for specimen use. These PBLs were processed, aliquoted, and stored in liquid nitrogen in the lymphocyte bank.

Preparation and Administration of Canvaxin Immunotherapeutic
The preparation and administration of Canvaxin immunotherapeutic have been described elsewhere.8,9 In brief, cells from M10-V, M24-V, and M101-V melanoma lines were grown in tissue culture medium, harvested, washed, and pooled (8.3 x 106 cells/line; 25 x 106 total cells) under sterile conditions. The cells were then irradiated with 150 Gy and cryopreserved in liquid nitrogen. Prior to April 30, 1996, 10% dimethylsulfoxide used in cryopreservation was washed out of all three lines after thawing, and the cells were reconstituted in RPMI 1640 prior to injection; each line was kept in a separate vial. After this date a new preparation (Canvaxin immunotherapeutic) was used: the same three lines were cryopreserved together in a smaller volume (0.5 mL) and reconstituted with the addition of 0.5 mL of saline. Canvaxin immunotherapeutic was administered immediately after thawing.

Patients received Canvaxin immunotherapeutic every 2 weeks (x 2) and then monthly for the first year of treatment. After year 1, the immunotherapeutic was administered every 3 months (x 4) and then every 6 months (x 6), for a total of 5 years. All patients received the Tice strain of bacille Calmette-Guerin (BCG), mixed with the first two doses of immunotherapeutic.

Assessment of Immunologic Response
Immediately prior to initiation of Canvaxin therapy and at each scheduled therapeutic dosing of Canvaxin immunotherapeutic, the delayed-type hypersensitivity (DTH) response to the immunotherapeutic was determined by injection of 2.4 x 106 Canvaxin cells (one-tenth of the therapeutic dose) at a separate intradermal site. DTH response to Canvaxin immunotherapeutic was determined 48 hours later. Control DTH response to nonmelanoma antigens was monitored by administering a purified protein derivative (PPD) skin test to each PPD-negative patient at monthly intervals until the patient tested positive.

CD8+ T-Cell Depletion
Cryopreserved PBL samples from the 54 melanoma patients and the four healthy donors were retrieved from the lymphocyte bank, coded, and tested in a blinded fashion. After thawing, PBLs were washed twice with a 1% solution of fetal bovine serum (FBS) in phosphate-buffered saline (PBS). Ten million PBLs were suspended in 1 mL PBS with 2% FBS. Dynabeads with antihuman CD8 antibody (Dynal Biotech, Oslo, Norway) were added per the manufacturer’s protocol; the cell suspension was incubated on a rotator for 30 minutes at 4°C and then placed against a magnetic particle concentrator (MPC) magnet for 2 minutes to collect the bead-rosetted cells (CD8+ T-cells). The CD8+ T-cell–depleted supernatant was collected, transferred into another sterile tube, and washed once with RPMI 1640/10% human antibody (AB) serum. After cell viability was confirmed by trypan blue exclusion, cells were counted and resuspended (1 x 106 cells/mL) in RPMI 1640/10% human AB serum for stimulation.

Intracellular Cytokine Assay
Cryopreserved irradiated Canvaxin cells (25 x 106 cells/mL) were thawed, washed with a 2% solution of FBS in PBS, and resuspended in media containing RPMI 1640/10% human AB serum at a concentration of 1 x 106 cells/mL. To determine optimal conditions for stimulation, PBLs from the four healthy donors and from four melanoma patients were cultured with Canvaxin cells. Ratios and duration of culture were varied, as described in the Results (see Establishment of Experimental Conditions).

After optimal conditions had been determined, 1 million irradiated Canvaxin cells were seeded in each well of 12-well polystyrene culture plates (Fisher Scientific) and incubated at 37°C for 8 hours in 5% CO2 at 95% humidity. After incubation the spent medium was carefully removed, and 1 million CD8+ T-cell–depleted PBLs (CD8- PBLs) were added to Canvaxin cells. CD8- PBLs stimulated with concanavalin A (Con-A; Sigma, St. Louis, MO), at 10 µg/mL, and CD8- PBLs stimulated with PBS (pH, 7.4) served as positive and negative controls, respectively. The reaction mixture was incubated for 72 hours. Monensin (GolgiStop; PharMingen, San Diego, CA) was added 6 hours prior to harvesting per the manufacturer’s protocol. After 72 hours of incubation, the reaction was harvested, washed, and resuspended in staining buffer (Dulbecco’s PBS/1% heat-inactivated FBS/0.09% w/v sodium azide). The phycoerythrin (PE)-conjugated anti-CD4 (mouse IgG1, clone: RPA-T4; PharMingen) was added to the suspension and incubated for 30 minutes at 4°C. Cells were then washed twice with the staining buffer and fixed and permeabilized with Cytofix/Cytoperm solution (PharMingen) according to manufacturer’s protocol. The fixed and permeabilized cells were incubated with FITC-conjugated mouse antihuman IFN{gamma} (mouse IgG1, clone: B27; PharMingen) or with FITC-conjugated rat anti-human IL4 (rat IgG1, clone: MP4–25D2; PharMingen). Included were CD8- PBLs stimulated with Con-A, CD8- PBLs stimulated with PBS buffer, CD8- PBLs stained with PE- and FITC-conjugated isotype controls, and positive control cells for intracellular IFN{gamma} staining (HiCK-1; human IL2+, IFN{gamma}+, TNF{alpha}+ PBMC; PharMingen) and IL4 (HiCK-2; human IL3+, IL4+, IL10+, IL13+, GM-CSF+ PBMC; PharMingen).

Flow cytometry was performed with FACScan and CellQuest software (Becton Dickinson, Mountain View, CA) with an argon laser tuned at 488 nm and run to 350 mW of power. Each sample was gated on forward light and right-angle scatter to exclude cell debris, clumps, and nonviable cells.

Statistical Analysis
All test results were given to the statistician (X.Y.) for data analysis. A Wilcoxon signed rank sum test was used to assess the difference in two related variables. Survival curves were estimated by the Kaplan-Meier method. Univariate analysis was performed by the log rank test. Cox proportional hazard regression was used for multivariate analysis. Survival analysis initially considered CD4+ T-cell intracellular cytokine responses as continuous variables. For comparison of subgroups, the median value of each cytokine response in the cohort was used as the cutoff level. When applicable, the Spearman test was used to assess the correlation between two variables. All statistical tests were two-tailed and performed on SAS software (SAS, Cary, NC).

RESULTS

Establishment of Experimental Conditions
To determine the optimal ratio of CD8- PBLs and Canvaxin cells, 1 x 106 CD8- PBLs from each of four healthy volunteers was co-cultured for 72 hours with 2.5 x 105, 5 x 105, or 1 x 106 irradiated Canvaxin cells or with 10 µg/mL Con-A (control). Stimulation of CD8- PBLs from healthy donors with Canvaxin cells under the current experimental conditions yielded minimal responses; the percentage of CD4+/IL4+ or IFN{gamma}+ double-staining cells ranged from 0 to 1.16%. In contrast, Con-A stimulation of CD8- PBLs from the same donors resulted in marked CD4+ T-cell intracellular IFN{gamma} and IL4 responses (range, 8.7%–10.1% double-staining cells) (Table 2). When the same experiment was repeated with CD8- PBLs from four melanoma patients, CD4+ T-cell intracellular cytokine responses were optimal, with 1 x 106 CD8- PBLs and 1 x 106 Canvaxin cells. To determine if the 72-hour co-culture period could be shortened, CD8- PBLs from the four melanoma patients were stimulated with 10 µg/mL Con-A or 1 x 106 Canvaxin cells for 24, 48, and 72 hours. CD4+ T-cell intracellular IFN{gamma} and IL4 responses peaked at 72 hours (Fig. 1). On the basis of these results, subsequent experiments co-cultured 1 x 106 Canvaxin cells with 1 x 106 CD8- PBLs (1:1 ratio) for 72 hours.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Optimization of Canvaxin cell:PBL ratio

 


View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1. Determination of optimal duration for co-culture. CD8+ T-cell–depleted peripheral blood lymphocytes (PBLs) from 4 melanoma patients were stimulated with 10 µg/mL of Con-A or 1 x 106 Canvaxin cells (37°C, 5% CO2, and 95% humidity) for 24, 48, and 72 hours. Monensin was added to the culture media 6 hours prior to harvest. For flow cytometry analysis, PBLs were stained with PE-conjugated anti-CD4 antibody, fixed, permeabilized, and then stained with FITC-conjugated anti-IL4 (left) or anti-IFN{gamma} antibodies (right).

 

Intracellular Cytokine Response Profile
The intracellular IFN{gamma} and IL4 cytokine responses were determined by subtracting the percent double-staining cells in the unstimulated reaction from the percent double-staining cells in the Canvaxin cell–stimulated reaction. A variable pattern of CD4+ T-cell intracellular cytokine responses was observed. The mean (± standard deviation) and median intracellular IFN{gamma} responses were 4.77% ± 2.25% and 4.61%, respectively. The mean and median IL4 responses were 5.05% ± 2.01% and 5.09%, respectively. Representative IFN{gamma} and IL4 responses to Canvaxin cells are shown in Figure 2. The mean (± standard deviation) Con-A IFN{gamma} and IL4 responses for the study cohort were 10.85% ± 1.67% and 10.85% ± 2.27%, respectively. Stimulation in terms of intracellular IFN{gamma} and IL4 responses was observed in all patients with Con-A.



View larger version (57K):
[in this window]
[in a new window]
  		FIG. 2. Representative flow cytometric diagram for study patients with low and high intracellular cytokine responses after co-culture of PBLs with Canvaxin cells. (A) 0.1% CD4/IL4 double-staining positive cells; (B) 25% CD4/IL4 double-staining positive cells; (C) 0.5% CD4/IFN{gamma} double-staining positive cells; (D) 28% CD4/IFN{gamma} double-staining positive cells.

 

Survival Analysis
On univariate analysis with intracellular cytokine response considered a continuous variable, there was a significant correlation between CD4+ T-cell intracellular IFN{gamma} response and survival (P = .0471) and between CD4+ T-cell intracellular IL4 response and survival (P = .0020) (Table 3). There was no significant correlation between Con-A stimulated cytokine responses and survival. Survival tended to increase with cytokine response.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Statistical impact of prognostic variables on overall survival

 

In a multivariate analysis that included important clinical prognostic variables, CD4+ T-cell intracellular IL4 response remained significant (Table 3). When patients were categorized as high or low responders on the basis of median percentage of CD4+ T-cells staining positive for IL4, the median survival time and 5-year survival rate were 59 months and 48%, respectively, for high responders, versus 12 months and 30%, respectively, for low responders (P = .0145) (Fig. 3).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3. Correlation of IL4 responses with OS. Black line represents high (> median) IL4 responders and gray line represents low IL4 responders (P = .0145). Median and 5-year overall survival rates were 59 months and 48%, respectively, for high responders and 12 months and 30%, respectively, for low responders.

 

Because the Canvaxin protocol required baseline (pretreatment) assessment of delayed-type hypersensitivity (DTH) response to Canvaxin immunotherapeutic, DTH and cytokine responses were compared. DTH showed no significant association with IL4 response to Canvaxin immunotherapeutic, IFN{gamma} response to Canvaxin immunotherapeutic, IL4 response to Con-A, or IFN{gamma} response to Con-A (P values = .5643, .3905, .1124, and .1325, respectively).

DISCUSSION

There was a significant correlation between CD4+ T-cell responsiveness to Canvaxin immunotherapeutic prior to immunotherapy and duration of survival after the first Canvaxin dosing. Interestingly, while both IL4 and IFN{gamma} responses correlated with survival on univariate analysis, only pre-Canvaxin CD4+ T-cell IL4 response independently predicted survival on multivariate analysis. The higher the IL4 response, the longer the survival.

Although the timing of the experiments appears to contradict earlier studies showing reactivation of T-cells and cytokine production within 24 to 48 hours,32,33 the difference may be attributed to use of different forms of antigens for restimulation. The antigens used in the current study were irradiated whole cells, whereas other investigators used peptides or recombinant proteins. The small percentage of cells that were stimulated corresponds to previous observations that only a small percentage of circulating lymphocytes would be sensitized to specific tumor antigens. The use of Con-A as a positive control antigen allowed assessment of general immunocompetence. In this study population, there was no correlation between the level of responsiveness to Con-A and the level of intracellular cytokine responses (data not shown). Thus, the small percentage of activated lymphocytes and the observed difference were not due to different levels of immunocompetence among study patients.

A successful specific active immunotherapeutic against cancer must induce proliferation and differentiation of memory T-cells with the desired antigen-specificity. The mechanism for induction of immune responses against a specific tumor antigen is complex. An antigen in the immunotherapeutic may augment the host’s immune memory response to the same antigen present on autologous cancer cells. Alternatively, the immunotherapeutic may induce responses against antigens that are recognized by the patient’s innate immunity. The induced immune responses may or may not be cytotoxic to the tumor cells.

Most "tumor antigens" are actually self-antigens expressed in limited amounts by certain normal tissues.17–19 The fact that peripheral blood may contain corresponding antibodies and memory T-cells suggests that autologous cancer cells can induce immune responses against self-antigens. Corroborative evidence includes the demonstration that serum from melanoma patients contains cytotoxic antibodies against cultured melanoma cells.34–36 In addition, antibody responses are directly correlated with survival after clinically complete surgical resection of metastatic melanoma.14,21–24 Also, the peripheral blood of melanoma patients contains peptide-specific CD8+ T-cells,25 and antigen-specific tumor-infiltrating lymphocytes have been demonstrated in melanoma metastases.22,23,27 Thus autologous melanoma cells are a source of immunogens and antigens.

If immunotherapy acts by inducing cytotoxic immune responses against antigens present on autologous melanoma cells, then its outcome should be predicted by the in vitro responses of autologous PBLs to the relevant immunogen(s). However, the nature of an effective antitumor immune response is unclear. Recognition of cell-surface or shed antigens by circulating antibodies can result in specific humoral (antibody) immune responses.34–36 Antigens are also taken up by antigen-presenting cells and presented to CD4+ and CD8+ T-cells for generation of cellular immune responses.37,38 The presentation of antigenic peptides to CD4+ T-cells can result in recruitment of more CD4+ T-cells for CD8+ T-cell help, enhancement of antibody responses, or generation of cytotoxic CD4+ T-cells.30,39

Classically, the generation of a CD8+ T-cell immune response is a Th1 response (IFN-{gamma}), and the generation of an antibody response is a Th2 response (IL4). In this study, IL4 CD4+ T-cell response was the most significant prognostic factor and the only variable that remained significant on multivariate analysis. IFN{gamma} CD4+ T-cell response was significant on univariate analysis but not on a multivariate analysis that considered other important clinical and immunological variables. This finding is not unexpected; most immunotherapy regimens rely on either the cellular or the humoral arm of the immune system, but our group has reported that both arms may be enhanced by Canvaxin immunotherapeutic.16,40 Furthermore, the humoral and cellular immune responses to Canvaxin immunotherapeutic appeared to be additive in their effects on survival.16 Patients with both types of response had longer survival than those with only one type of response (humoral or cellular), whereas patients with only one type of response did better than those with neither type. Other investigators have reported induction of both cellular and humoral responses to similar polyvalent immunization strategies.41,42 A temporal profile of the cytokine response to specific active immunotherapy would help clarify the relative importance of cellular versus humoral immune responses to tumor antigens, but it is beyond the scope of this paper.

Since the study patients were enrolled in a trial of an irradiated whole-cell immunotherapeutic that contains more than 30 tumor antigens identified to date, it is unlikely that measurement of cellular response to a specific antigen would correlate with outcome on this immunotherapy regimen. Thus, further evaluation of immune response to specific individual antigens of Canvaxin immunotherapeutic would not add utility to the assay for the purpose of this study. However, this study offers proof of the concept that patients previously sensitized to antigen(s) will likely have a better response to immunotherapy containing those antigen(s). Although we cannot eliminate the possibility of allogeneic responses against non-self-alloantigens under these experimental conditions (only 72 hours of co-culture), PBLs from healthy donors did not significantly respond to co-culture with Canvaxin cells. As a further refinement of this immunologic monitoring tool for specific antigen immunization, we are investigating specific recombinant protein antigens as stimulation agents.

In conclusion, this study demonstrated that PBL CD4+ T-cell responsiveness can be a potentially useful marker of response to immunotherapy. The augmentation of this type of response should be considered in designing future anticancer immunotherapy.

ACKNOWLEDGMENTS

Dr. Hsueh’s clinical research in melanoma immunotherapy is funded in part by a Career Development Award from the American Society of Clinical Oncology. Canvaxin and CancerVax are trademarks of CancerVax Corporation, Carlsbad, CA.

FOOTNOTES

Received February 17, 2004; accepted June 18, 2004.

Presented at the 55th Annual Cancer Symposium of the Society of Surgical Oncology, Denver, March 14–17, 2002.

Supported in part by grants CA87071, CA12582, and CA76489 from the National Cancer Institute and by funding from the Wayne and Gladys Valley Foundation (Oakland, CA) and Nancy and Carroll O’Connor (Los Angeles, CA).

Donald L. Morton has an ownership interest in CancerVax Corporation, the company that holds the rights to CanvaxinTM immunotherapeutic. Estella Famatiga has a consulting agreement with CancerVax Corporation.

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

REFERENCES

  1. 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.[Abstract/Free Full Text]
  2. Kirkwood JM, Strawderman MH, Ernstoff MS, Smith TJ, Borden EC, Blum RH. 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.[Abstract]
  3. Ferrone S. Human tumor-associated antigen mimicry by anti-idiotypic antibodies: immunogenicity and clinical trials in patients with solid tumors. Ann NY Acad Sci 1993;690:214–24.[Medline]
  4. Ayyoub M, Zippelius A, Pittet MJ, et al. Activation of human melanoma reactive CD8+ T-cells by vaccination with an immunogenic peptide analog derived from Melan-A/Melanoma Antigen Recognized by T Cells-1. Clin Cancer Res 2003;9:669–77.[Abstract/Free Full Text]
  5. Machiels JP, van Baren N, Marchand M. Peptide-based cancer vaccines. Semin Oncol 2002;29:494–502.[CrossRef][Medline]
  6. Berd D, Sato T, Cohn H, Maguire Jr HC, Mastrangelo MJ. Treatment of metastatic melanoma with autologous, hapten-modified melanoma vaccine: regression of pulmonary metastases. Int J Cancer 2001;94:531–9.[CrossRef][Medline]
  7. Sondak VK, Liu PY, Tuthill RJ, et al. Adjuvant immunotherapy of resected, intermediate-thickness, node-negative melanoma with an allogeneic tumor vaccine: overall results of a randomized trial of the Southwest Oncology Group. J Clin Oncol 2002;20:2058–66.[Abstract/Free Full Text]
  8. Morton DL, Hsueh EC, Essner R, et al. Prolonged survival of patients receiving active immunotherapy with Canvaxin therapeutic polyvalent vaccine after complete resection of melanoma metastatic to regional lymph nodes. Ann Surg 2002;236:438–48.[CrossRef][Medline]
  9. Hsueh EC, Essner R, Foshag LJ, et al. Prolonged survival following complete resection of disseminated melanoma and active immunotherapy with a therapeutic cancer vaccine. J Clin Oncol 2002;20:4549–54.[Abstract/Free Full Text]
  10. Sun Y, Jurgovsky K, Moller P, et al. Vaccination with IL-12 gene-modified autologous melanoma cells: preclinical results and a first clinical phase I study. Gene Ther 1998;5:481–90.[CrossRef][Medline]
  11. Soiffer R, Lynch T, Mihm M, et al. Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc Natl Acad Sci U S A 1998;95:13141–6.[Abstract/Free Full Text]
  12. Belli F, Arienti F, Sule-Suso J, et al. Active immunization of metastatic melanoma patients with interleukin-2-transduced allogeneic melanoma cells: evaluation of efficacy and tolerability. Cancer Immunol Immunother 1997;44:197–203.[CrossRef][Medline]
  13. Arienti F, Belli F, Napolitano F, et al. Vaccination of melanoma patients with interleukin 4 gene-transduced allogeneic melanoma cells. Hum Gene Ther 1999;10:2907–16.[CrossRef][Medline]
  14. Morton DL, Barth A. Vaccine therapy for malignant melanoma. CA Cancer J Clin 1996;46:225–44.[Medline]
  15. Barth A, Hoon DS, 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]
  16. 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]
  17. Sun Y, Paschen A, Schadendorf D. Cell-based vaccination against melanoma–background, preliminary results, and perspective. J Mol Med 1999;77:593–608.[CrossRef][Medline]
  18. Rosenberg SA. A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity 1999;10:281–7.[CrossRef][Medline]
  19. Renkvist N, Castelli C, Robbins PF, Parmiani G. A listing of human tumor antigens recognized by T-cells. Cancer Immunol Immunother 2001;50:3–15.[CrossRef][Medline]
  20. Berd D, Herlyn M, Koprowski H, Mastrangelo MJ. Flow cytometric determination of the frequency and heterogeneity of expression of human melanoma-associated antigens. Cancer Res 1989;49:6840–4.[Abstract/Free Full Text]
  21. Degiovanni G, Lahaye T, Herin M, Hainaut P, Boon T. Antigenic heterogeneity of a human melanoma tumor detected by autologous CTL clones. Eur J Immunol 1988;18:671–6.[Medline]
  22. Chen Q, Hersey P. MHC-restricted responses of CD8+ and CD4+ T-cell clones from regional lymph nodes of melanoma patients. Int J Cancer 1992;51:218–24.[Medline]
  23. Mackensen A, Carcelain G, Viel S, et al. Direct evidence to support the immunosurveillance concept in a human regressive melanoma. J Clin Invest 1994;93:1397–402.
  24. Hsueh EC, Gupta RK, Yee R, Leopoldo ZC, Qi K, Morton DL. Does endogenous immune response determine the outcome of surgical therapy for metastatic melanoma? Ann Surg Oncol 2000;7:232–8.[Abstract]
  25. Lee PP, Yee C, Savage PA, et al. Characterization of circulating T-cells specific for tumor-associated antigens in melanoma patients. Nat Med 1999;5:677–85.[CrossRef][Medline]
  26. Morton DL, Malmgren RA, Holmes EC, Ketcham AS. Demonstration of antibodies against human malignant melanoma by immunofluorescence. Surgery 1968;64:233–40.[Medline]
  27. Topalian SL, Solomon D, Rosenberg SA. Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 1989;142:3714–25.[Abstract]
  28. Janssen EM, Lemmens EE, Wolfe T, Christen U, Von Herrath MG, Schoenberger SP. CD4(+) T-cells are required for secondary expansion and memory in CD8(+) T lymphocytes. Nature 2003;421:852–6.[CrossRef][Medline]
  29. Gao FG, Khammanivong V, Liu WJ, Leggatt GR, Frazer IH, Fernando GJ. Antigen-specific CD4+ T-cell help is required to activate a memory CD8+ T-cell to a fully functional tumor killer cell. Cancer Res 2002;62:6438–41.[Abstract/Free Full Text]
  30. Jenkins MK, Khoruts A, Ingulli E, et al. In vivo activation of antigen-specific CD4 T-cells. Annu Rev Immunol 2001;19:23–45.[CrossRef][Medline]
  31. Nouri-Shirazi M, Banchereau J, Fay J, Palucka K. Dendritic cell based tumor vaccines. Immunol Lett 2000;74:5–10.[CrossRef][Medline]
  32. Maecker HT, Auffermann-Gretzinger S, Nomura LE, Liso A, Czerwinski DK, Levy R. Detection of CD4 T-cell responses to a tumor vaccine by cytokine flow cytometry. Clin Cancer Res. 2001;7(Suppl 3):902s–8s.
  33. Suni MA, Picker LJ, Maino VC. Detection of antigen-specific T-cell cytokine expression in whole blood by flow cytometry. J Immunol Methods 1998;212:89–98.[CrossRef][Medline]
  34. Lewis MG, Ikonopisov RL, Nairn RC, et al. Tumour-specific antibodies in human malignant melanoma and their relationship to the extent of the disease. BMJ 1969;3:547–52.
  35. Bodurtha AJ, Chee DO, Laucius JF, Mastrangelo MJ, Prehn RT. Clinical and immunological significance of human melanoma cytotoxic antibody. Cancer Res 1975;35:189–93.[Abstract/Free Full Text]
  36. Canevari S, Fossati G, Della Porta G, Balzarini GP. Humoral cytotoxicity in melanoma patients and its correlation with the extent and course of the disease. Int J Cancer 1975;16:722–9.[Medline]
  37. Pardoll DM. Cancer vaccines. Nat Med 1998;4(Suppl 5):525–31.[CrossRef][Medline]
  38. Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18:767–811.[CrossRef][Medline]
  39. Pardoll DM, Topalian SL. The role of CD4+ T-cell responses in antitumor immunity. Curr Opin Immunol 1998;10:588–94.[CrossRef][Medline]
  40. Jones RC, Kelley M, Gupta RK, et al. Immune response to polyvalent melanoma cell vaccine in AJCC stage III melanoma: an immunologic survival model. Ann Surg Oncol 1996;3:437–45.[Abstract]
  41. Reynolds SR, Zeleniuch-Jacquotte A, Shapiro RL, et al. Vaccine-induced CD8+ T-cell responses to MAGE-3 correlate with clinical outcome in patients with melanoma. Clin Cancer Res 2003;9:657–62.[Abstract/Free Full Text]
  42. Applebaum J, Reynolds S, Knispel J, Oratz R, Shapiro R, Bystryn JC. Identification of melanoma antigens that are immunogenic in humans and expressed in vivo. J Natl Cancer Inst 1998;90:146–9.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Ann. Surg. Oncol.Home page
M.-A. Perales and J. D. Wolchok
CD4 Help and Tumor Immunity: Beyond the Activation of Cytotoxic T Lymphocytes
Ann. Surg. Oncol., October 1, 2004; 11(10): 881 - 882.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hsueh, E. C.
Right arrow Articles by Morton, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hsueh, E. C.
Right arrow Articles by Morton, D. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS