| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Portland Medical Center [H-M. H., C. H. P., W. J. U., B. A. F.], Portland, Oregon 97213, and Departments of Biochemistry and Molecular Biology [H-M. H., B. A. F.] and Molecular Microbiology and Immunology [B. A. F.], Oregon Health and Science University, Portland, Oregon 97201
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
4-fold in reconstituted lymphopenic hosts compared with normal hosts. Concomitantly, vaccination-induced Th1 T cells were also dramatically increased in vaccinated, reconstituted RAG1 hosts. T cells derived from reconstituted RAG1 hosts exhibited a higher level of melanoma-specific cytotoxicity in vitro. These cells were significantly more potent at mediating tumor regression in vivo after adoptive transfer into mice bearing established pulmonary metastases. Vaccination is best performed concomitantly with reconstitution; delayed vaccination resulted in T cells with less therapeutic activity. |
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
production, and CTL activity (5, 6, 7, 8, 9)
. Recent data also suggested a subset of naïve CD4+ T cells acquired memory-like function (secretion of IL3
-2 and IFN-) after expansion in RAG1 mice (10)
. Because reconstitution of the T-cell compartment in lymphopenic hosts is regulated by the peptides occupying MHC class I and II molecules at the time of T-cell recovery, there may be an opportunity to skew the T-cell repertoire during T-cell recovery by engaging the available MHC class I and class II molecules with peptides of particular interest. If, as it appears, naïve T cells are more sensitive to activation by weak self-antigens during reconstitution of lymphopenic hosts, there may be a window during which immune tolerance may be broken. If MHC class I and class II molecules presented self peptides, which had been shown to serve as tumor-associated antigens, during a lymphopenic episode, the host may be repopulated with tumor-reactive T cells that could lead to better tumor control. To test this hypothesis, we monitored tumor-specific CD4 and CD8 T cells in TVDLNs from RAG1 mice vaccinated with melanoma cells after i.v. reconstitution with naïve spleen cells. Adoptive immunotherapy was performed using effector T cells generated from the TVDLNs of RLP hosts. |
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
200 ng/ml/106 cells/24 h of GM-CSF. MCA 310 is a chemically induced fibrosarcoma from C57BL/6J mice. B16F10 melanoma is from American Type Culture Collection; B16F10-CIITA.28 is a stable clone that expresses the human MHC class II transactivator, which also exhibits high level I-Ab expression on the cell surface. CL8-1 is a stable clone of B16 melanoma that was transfected with a plasmid that encodes H-2 Kb. Tumor cells were cultured in CM, which consisted of RPMI 1640 (BioWhittaker, Walkersville, MD) containing 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 µg/ml of gentamicin sulfate. This was further supplemented with 50 µM 2-mercaptoethanol (Aldrich, Milwaukee, WI) and 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). Tumor cells were harvested two to three times per week by brief trypsinization and were maintained in T-150 or T-225 culture flasks. Hamster antimouse CD3 (clone 2c11) and CD28 (clone PV1) mAbs (American Type Culture Collection) were purified from culture supernatant by ammonium sulfate precipitation and ion exchange column chromatography.
Reconstitution, Vaccination, and in Vitro Sensitization.
RAG1 mice were reconstituted with 20 million naive spleen cells from wild-type B6 mice and then vaccinated with the D5-G6 tumor vaccine as described previously (11)
. Briefly, four aliquots of 1 x 106 tumor cells each were injected into both the fore and hind flanks of recipient mice. Eight days after vaccination, two enlarged inguinal and two auxillary TVDLNs were collected, and single cell suspensions were prepared by pressing lymph nodes between two sterile glass slides. The TVDLN cells were cultured at 1 x 106 cells/ml of CM in 24-well plates with 5 µg/ml 2c11 antibody (anti-CD3) and 5 µg/ml anti-CD28 mAb. After 2 days of activation, the T cells were harvested and subsequently expanded at 0.1 x 106 cells/ml in CM containing 60 IU/ml IL-2 (Chiron Co., Emeryville, CA) in Lifecell tissue culture flasks (Nexell Therapeutics, Inc., Irvine, CA) for 3 additional days. These in vitro activated and expanded cells are referred to as effector T cells.
Adoptive Immunotherapy.
Effector T cells were washed twice in HBSS and injected i.v. into B6 mice in which pulmonary metastases were established 3 days earlier by tail vein injection of 0.2 x 106 D5 tumor cells. Starting on the day of T-cell infusion, mice received 90,000 IU IL-2 i.p. once per day for 3 days. Animals were sacrificed at 13 days after tumor inoculation by CO2 narcosis. Lungs were resected and fixed in Fekete’s solution. The number of macroscopic pulmonary metastases was counted, and metastases that were too numerous to count accurately were assigned a value of 250.
Intracellular Cytokine Staining and Fluorescence-activated Cell Sorter Analysis.
For intracellular IFN- release assays, four million effector T cells were either not stimulated or stimulated with 2 x 105 D5, MCA 310, F10, F10-CIITA.28 tumor cells, or anti-CD3 antibody in 2 ml of CM in 24-well plates. Brefeldin A was added, and the cells were incubated at 37°C for 14–16 h. Cells were harvested and stained with FITC-labeled anti-CD8 antibody, Cy-chrome labeled anti-CD3 antibody, and PE-labeled anti-IFN- antibody after fixing and permeabilization (Cytofix/Cytoperm kit; PharMingen, San Diego, CA). Fifty thousand gated events based on forward and light scatter were collected and analyzed, and all of the analysis was gated on CD3+ cells.
Cytotoxicity Assay.
In vitro cytotoxicity of effector T cells was determined as described previously (11)
. Tumor cells were incubated with 100 mCi of Na251CrO4 (DuPont NEN, Boston, MA) for 90 min, washed twice, and plated into round-bottomed, 96-well plates with 1 x 104 target cells/well in triplicate. The target cells were incubated with effector T cells at the indicated E:T ratios in a total volume of 200 µl of CM at 37°C in a CO2 incubator. The supernatant was harvested and counted, and the percentage of specific lysis was calculated as described previously (11)
. Maximum lysis was determined by incubating target cells with 2% Triton X-100 detergent.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
. As expected, the control tumor, MCA 310, failed to stimulate effector T cells from either normal or reconstituted RAG1 hosts. However, the background was significantly higher in RLP RAG1 hosts (Fig. 2)
. In normal hosts, the percentage of TVDLN CD8 T cells that produced IFN- ranged from 1.12 to 2.61%, depending on the tumor cells used for stimulation (Fig. 2A)
. The percentage of CD8 T cells stimulated to release IFN- in response to the different cell lines is as follows: control MCA-310 (0.88%); D5 (1.83%); F10 (1.12%); F10-CIITA.28 (1.79%); and CL8-1 (2.61%; Fig. 2A
). There appears to be a direct correlation between MHC class I expression by the stimulator cells and the percentage of IFN--producing CD8 T cells. The percentage of IFN- producing cells at baseline was higher in effector T cells obtained from RLP vaccinated RAG1 mice: 2.24% and 2.5% CD8+ T cells producing IFN- without stimulation or stimulated with MCA-310 in RLP hosts, compared with 0.55% with no stimulation and 0.88% with control MCA-310 in normal hosts. There was also a large increase in the percentage of melanoma-specific IFN--secreting CD8 T cells after stimulation of TVDLN effector T cells from RLP RAG1 hosts with different cell lines derived from B16 melanoma: D5 (6.64%); F10 (6.91%); F10-CIITA.28 (10.29%); and CL8-1 (8.16%). Although the "background" stimulation by the control tumor MCA-310 was greater in RAG1 mice compared with normal mice, the increase in melanoma-specific T cells also appeared to be greater in RAG1 mice. Interestingly, although <1% of CD4 T cells (CD3+CD8-) from D5-G6 vaccinated normal hosts responded to F10 or F10-CIITA (expressing low and high level of I-Ab molecules, respectively), a much larger proportion of CD4 T cells from vaccinated RLP RAG1 mice were stimulated to release IFN- by F10 (3.62%) and F10-CIITA.28 (13.13%; Fig. 2B
). A summary of these data are also presented as a bar graph for easy comparison (Fig. 2, E and G)
.
|
|
by CD4 and CD8 tumor-specific T cells in vaccinated normal and RLP RAG1 hosts was also examined. Results similar to those for IFN- production were obtained. CD8 T cells from normal hosts exhibited an increased TNF- response after stimulation with each of the melanoma cell lines but not the MCA-310 cell line (Fig. 2C)
. Although baseline or nonspecific TNF- production was higher, the largest increase in melanoma-specific TNF--producing CD4 T cells was found in RAG1 RLP compared with normal hosts (Fig. 2, C and D)
. A summary of these data is also presented as a bar graph for easy comparison (Fig. 2, F and H)
.The significantly higher number of melanoma-specific T cells observed in RLP RAG1 mice supports our hypothesis that antigen-specific T cells can be expanded preferentially in reconstituted lymphopenic hosts if appropriate antigen is presented during immune reconstitution.
To determine whether effector T cells generated from RLP RAG1 mice exhibited tumor-specific cytotoxicity, a 6-h 51Cr-release assay was performed. Although effector T cells from both normal and RLP RAG1 mice failed to kill control tumor MCA 310, effector T cells generated from RLP RAG1 mice, but not normal mice, were highly cytolytic to D5 tumor cells in vitro (Fig. 3)
. These data demonstrate that vaccination during reconstitution of a lymphopenic host can result in a strong type 1 (both Th1 and Tc1) immune response.
|
. Results from two independent experiments demonstrated that 15 x 106 effector T cells generated from reconstituted RAG1 KO mice mediated complete tumor regression (no metastases), whereas the same number of T cells from normal mice failed to mediate complete tumor regression in three independent experiments. In experiment 1, as few as 5 x 106 effector T cells from RAG1 KO mice were able to mediate complete tumor regression, whereas 15 x 106 effector T cells from normal mice failed to eradicate tumor. In experiment 2, 15 x 106 effector T cells from vaccinated RLP RAG1 hosts mediated complete tumor regression, whereas 35 x 106 effector T cells from normal mice were not able to do so. In three of three experiments, 5 x 106 T cells from RLP mice mediated significantly better tumor regression than three times as many, 15 x 106, T cells from normal hosts (P < 0.05). Data from these three independent experiments were combined and represented in a graphic format (Fig. 4A)
. These results demonstrated that there is direct correlation between in vivo therapeutic activity and the percentage of melanoma-specific T cells measured by intracellular cytokine staining. This is consistent with our previous observation that CD8+ T cells are the effector T cells that mediate the regression of pulmonary metastases in this animal model (11)
.
|
|
. Vaccination after adoptively transferred naïve T cells have reached their peak of proliferation is less effective than vaccination at time of T-cell transfer. Although concomitant vaccination experiments were not performed at the same time, T cells from RLP RAG1 mice with delayed vaccination were less therapeutic. In contrast to T cells from RLP RAG1 mice with concomitant vaccination, 5 million effector T cells from mice with delayed vaccination were not more effective than 15 million effector T cells from normal hosts and actually were not therapeutic compared with mice without T-cell transfer (Table 2
and Fig. 4B
). These results indicate that vaccination efficacy in RLP mice is influenced by both proliferation rate of naïve T cells and space available for expansion of both antigen-specific and nonspecific T cells.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
For many cancers, the tumor-associated antigens are unknown, and whole tumor cells are often used as vaccines. One way to enhance an antitumor immune response would be to devise a situation in which tumor-specific T cells are "tuned" to become more sensitive to activation by weak tumor antigens. It has been reported that a naïve T-cell repertoire could be skewed to increase the relative percentage of antigen-specific T cells if antigen was given during immune reconstitution (14) . Borrelle et al. (15) recently demonstrated enhanced efficacy of a GM-CSF gene-modified lymphoma vaccine in lethally irradiated mice reconstituted with T-cell-depleted bone morrow. Our findings extend the report of Borrelle et al. (15) in several important ways:
(a) Because vaccination is augmented in reconstituted RAG-1 mice that are not irradiated, it documents that the cytokine storm induced by lethal irradiation is not required for the increased antitumor effect.
(b) Because we used spleen cells and vaccinated immediately after reconstitution, it suggests that the augmented antitumor response does not require differentiation of new T cells from bone marrow stem cells. These two findings are likely the most important and clinically relevant observations of our study, because lethal whole body irradiation and bone marrow transplant will likely prove to be roadblocks to widespread clinical application of this vaccine strategy for solid tumors.
(c) In contrast to their use of TCR transgenic T cells and a foreign model tumor antigen, influenza hemagglutinin, to detect an increase in antigen-reactive T cells, we were able to show strong CD4 and CD8 T-cell responses to native antigens presented by tumor cells. These results also infer that vaccination during a time of natural lymphocyte recovery from lymphopenia could increase the magnitude of the immune response to a vaccine. Because lymphocyte recovery occurs regularly in treated cancer patients (16) , our data suggest that the development of transient lymphopenia after chemotherapy or irradiation may be exploited by vaccinating patients during these lymphopenic episodes to boost immunity and possibly induce tumor regression and prevent tumor recurrence.
Currently, little is known about the mechanisms that regulate the homeostasis of naïve T cells in lymphopenic hosts. Two nonexclusive explanations have been proposed to explain this phenomenon. One is the "fill up the space" hypothesis (17) , which states that naive and memory T cells occupy specific survival niches. The induction of lymphopenia empties these niches and allows naïve cells to expand and fill the empty "space." The other, regulatory, or suppressor cell hypothesis, proposes that regulatory cells counter self-reactive T cells and thereby prevent the development of organ-specific autoimmunity (18) . Of course, the same process would inhibit the priming of tumor immunity (19) . It is possible that regulatory cells were not transferred in sufficient number when RAG-1 mice were reconstituted with normal spleen cells to prevent the expansion and eradication of autoreactive T cells. The spleen cells used for reconstitution in our experiments were unfractionated; therefore, additional transfer experiments with spleen cells depleted of CD4+CD25+ regulatory T cells would be necessary to determine their role in priming therapeutic T cells during reconstitution of a lymphopenic host.
Neither hypothesis directly addresses the factors that drive naïve T cells to proliferate in the absence of exogenous antigenic stimulation. Our results can be explained best by the T-cell activation threshold tuning hypothesis proposed by Grossman and Paul (20) . Their hypothesis states that the immune system is designed to be moderately self-reactive and that self-reactivity is controlled by the activation threshold. It proposes that the immune system responds to episodes of "system perturbation," such as episodes of infection or lymphopenia. In our experimental model, there are two system perturbations, lymphopenia and a tumor vaccine. As a population, T cells respond to lymphopenia by tuning their activation threshold down to a level at which a subgroup of naïve T cells are driven to proliferate, even in the absence of foreign antigens. If tumor antigens are also present, T-cell responses could be focused on these peptides, and tumor-specific T cells may be preferentially driven to proliferate to reach a level sufficient to confer resistance to tumor growth. Repopulation of both CD4 and CD8 niches with a T-cell repertoire skewed in favor of melanoma-reactive T cells after vaccination in reconstituted RAG1 mice would explain our results.
Previously, we demonstrated that priming of therapeutic CD8 effector T cells in normal hosts is CD4 independent and that CD8, but not CD4 effector T cells, mediated tumor regression (11) . We observed a large increase in the number of Th1 CD4 effector T cells that appear to be MHC class II restricted and tumor specific in RLP RAG1 mice compared with normal hosts after vaccination with D5-G6. Because a large percentage of CD4 T cells were primed in vaccinated RLP RAG1 mice, we are currently reexamining the role of CD4 T cells during the priming and effector phases of adoptive immunotherapy. Preliminary experiments suggest that the adoptive transfer of either CD4 T cells derived from vaccinated RLP RAG1 mice but not from normal mice are highly therapeutic.4 We also plan to determine whether these CD4 T cells are melanoma specific and identify the antigens they recognize. Future experiments will determine whether tolerance in melanoma-specific CD4 T cells was broken by vaccination during reconstitution of lymphopenic hosts. Most interestingly, we observed both enhanced tumor protection and the rapid development of vitiligo after tumor challenge with the B16F10 melanoma cell line in reconstituted lymphopenic mice that had been vaccinated with irradiated unmodified B16F10 tumor cells.5 This supports our hypothesis that vaccination of RLP hosts with whole tumor cell vaccines can break immune tolerance to tumor antigens. In conclusion, our study suggests that active vaccination of T-cell reconstituted lymphopenic hosts can jump start or reboot the immune system in favor of strong immune responses to self-antigens and promote protection against tumor outgrowth. This strategy will be tested in future clinical trials.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This study was supported by grants from the Chiles Foundation, the M. J. Murdock Charitable Trust, and NIH Grant RO1 CA 80964 (to B. A. F.). 
2 To whom requests for reprints should be addressed, at Providence Portland Medical Center, 4805 NE Glisan Street, Portland, OR 97213. Phone: (503) 215-6531; Fax: (503) 215-6841; E-mail: hhu{at}providence.org
3 The abbreviations used are: IL, interleukin; RLP, reconstituted lymphopenic; TVDLN, tumor vaccine draining lymph node; GM-CSF, granulocyte/macrophage-colony stimulating factor; CM, complete medium; mAb, monoclonal antibody; TNF, tumor necrosis factor.
4 C. H. Poehlein and H-M. Hu, unpublished results.
5 H-M. Hu. A poorly immunogenic tumor induces protective antitumor immunity in mice subjected to sublethal irradiation and immune reconstitution, manuscript in preparation.
Received 4/30/02. Accepted 6/ 3/02.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. Jing, J. A. Gershan, and B. D. Johnson Depletion of CD4 T cells enhances immunotherapy for neuroblastoma after syngeneic HSCT but compromises development of antitumor immune memory Blood, April 30, 2009; 113(18): 4449 - 4457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Shvets, R. Chakrabarti, R. Gonzalez-Quintial, R. Baccala, A. N. Theofilopoulos, and G. J. Prud'homme Impaired negative regulation of homeostatically proliferating T cells Blood, January 15, 2009; 113(3): 622 - 625. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Pegram, J. T. Jackson, M. J. Smyth, M. H. Kershaw, and P. K. Darcy Adoptive Transfer of Gene-Modified Primary NK Cells Can Specifically Inhibit Tumor Progression In Vivo J. Immunol., September 1, 2008; 181(5): 3449 - 3455. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-J. Lin, A. T. Chen, and R. M. Welsh Immune system derived from homeostatic proliferation generates normal CD8 T-cell memory but altered repertoires and diminished heterologous immune responses Blood, August 1, 2008; 112(3): 680 - 689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Voehringer, H.-E. Liang, and R. M. Locksley Homeostasis and Effector Function of Lymphopenia-Induced "Memory-Like" T Cells in Constitutively T Cell-Depleted Mice J. Immunol., April 1, 2008; 180(7): 4742 - 4753. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kobayashi, H. Hara, M. Ohashi, T. Nishimoto, K. Yoshida, N. Ohkohchi, T. Yoshida, and K. Aoki Allogeneic MHC Gene Transfer Enhances an Effective Antitumor Immunity in the Early Period of Autologous Hematopoietic Stem Cell Transplantation Clin. Cancer Res., December 15, 2007; 13(24): 7469 - 7479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-X. Wang, R. Li, G. Yang, M. Lim, A. O'Hara, Y. Chu, B. A. Fox, N. P. Restifo, W. J. Urba, and H.-M. Hu Interleukin-7-Dependent Expansion and Persistence of Melanoma-Specific T Cells in Lymphodepleted Mice Lead to Tumor Regression and Editing Cancer Res., November 15, 2005; 65(22): 10569 - 10577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhang and G. G. Meadows Chronic alcohol consumption in mice increases the proportion of peripheral memory T cells by homeostatic proliferation J. Leukoc. Biol., November 1, 2005; 78(5): 1070 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-X. Wang, S. Shu, and G. E. Plautz Host Lymphodepletion Augments T Cell Adoptive Immunotherapy through Enhanced Intratumoral Proliferation of Effector Cells Cancer Res., October 15, 2005; 65(20): 9547 - 9554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Emens and E. M. Jaffee Leveraging the Activity of Tumor Vaccines with Cytotoxic Chemotherapy Cancer Res., September 15, 2005; 65(18): 8059 - 8064. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Baccala, D. Witherden, R. Gonzalez-Quintial, W. Dummer, C. D. Surh, W. L. Havran, and A. N. Theofilopoulos {gamma}{delta} T Cell Homeostasis Is Controlled by IL-7 and IL-15 Together with Subset-Specific Factors J. Immunol., April 15, 2005; 174(8): 4606 - 4612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L A Emens, R T Reilly, and E M Jaffee Breast cancer vaccines: maximizing cancer treatment by tapping into host immunity Endocr. Relat. Cancer, March 1, 2005; 12(1): 1 - 17. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Mihalyo, A. D. H. Doody, J. P. McAleer, E. C. Nowak, M. Long, Y. Yang, and A. J. Adler In Vivo Cyclophosphamide and IL-2 Treatment Impedes Self-Antigen-Induced Effector CD4 Cell Tolerization: Implications for Adoptive Immunotherapy J. Immunol., May 1, 2004; 172(9): 5338 - 5345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-X. Wang, J. Kjaergaard, P. A. Cohen, S. Shu, and G. E. Plautz Memory T Cells Originate from Adoptively Transferred Effectors and Reconstituting Host Cells after Sequential Lymphodepletion and Adoptive Immunotherapy J. Immunol., March 15, 2004; 172(6): 3462 - 3468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Peacock, S.-K. Kim, and R. M. Welsh Attrition of Virus-Specific Memory CD8+ T Cells During Reconstitution of Lymphopenic Environments J. Immunol., July 15, 2003; 171(2): 655 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Prlic, B. R. Blazar, M. A. Farrar, and S. C. Jameson In Vivo Survival and Homeostatic Proliferation of Natural Killer Cells J. Exp. Med., April 21, 2003; 197(8): 967 - 976. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
