- Email: [email protected]
neu Peptide Breast Cancer Vaccines: Current Status and Future Directions
HER-2/neu Peptide Breast Cancer Vaccines: Current Status and Future Directions Elizabeth A. Mittendorf, MD, and George E. Peoples, MD The identification of tumor-associated antigens (TAAs) recognized by immune effectors has generated increased interest in the development of breast cancer vaccines. Peptide-based vaccines use antigenic epitopes derived from TAAs for the induction of peptide-specific cytotoxic T lymphocytes (CTLs) that recognize and lyse tumor cells expressing the immunogenic peptide or peptides on their surfaces. Such peptide vaccines are the purest way to stimulate an immune response to TAAs, and this response can be monitored precisely by using a tetramer or dimer assay combined with a functional assessment. Peptide vaccines, combined with an immunoadjuvant agent, can be injected without requiring a delivery system; therefore, they are simple, easily produced, and can be efficiently exported to the community. One example of a TAA is HER-2/neu, which is overexpressed in 30% of breast cancers.1 Since its identification, several peptides derived from this TAA have been investigated for use as anticancer vaccines. The most studied of the HER-2/neuderived peptides is E75. Promising preclinical studies of this peptide led to 3 clinical trials of E75 for patients with metastatic disease.2-4 These small trials, in which the number of subjects ranged from 4 to 14, demonstrated that E75 is safe and capable of inducing a peptide-specific immune response. Unfortunately, little has been reported regarding the clinical impact of this HER-2/neu immunity, perhaps because the enrolled patients had late-stage disease and large residual tumor burdens. Our group has pursued a different strategy in our clinical trials. We have shifted focus from the treatment of late-stage disease to the prevention of disease recurrence. In our initial clinical trial,5 patients with lymph node–positive breast cancer who had been rendered disease-free through conventional treatment with surgery, chemotherapy, and radiation when appropriate, were enrolled. Because the E75 peptide is restricted to the human leukocyte antigen (HLA) -A2 molecule, patients were HLA-typed after enrollment, and those who were HLA-A2positive were vaccinated and those who were HLA-A2-negative served as prospective controls. Interestingly, the HLA-A2+ group generally had worse prognostic characteristics, including larger tumors, higher histologic grade, and less hormonal sensitivity. In that study, patients were given the E75 peptide admixed with granulocyte-macrophage colony-stimulating factor (GM-CSF) according to a 2-stage design with escalating doses in the first stage and alterations in the vaccine schedule in the second. Interim results from this trial confirmed the vaccine to
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be safe and effective at inducing clonal expansion of E75specific CTLs capable of lysing HER-2/neu-expressing tumors. At a median follow-up of 22 months, the disease-free survival rate among vaccinated patients was 85.7% compared with 59.5% for those in the control group. In the vaccinated group, no deaths had occurred and 2 of 24 patients (8%) had developed a recurrence. In the control group, 2 patients had died and 6 of 29 (21%) had recurrent disease.5 Although these preliminary results did not reach statistical significance, they are compelling and indicate a trend towards fewer recurrences among vaccinated patients with high-risk breast cancer. This initial report included 53 patients. To date, 90 patients have enrolled in our trial of E75 for node-positive disease, and thus far we have observed similar rates (unpublished data). The fact that E75 is HLA-restricted allowed the inclusion of a natural control group in the trial; however, in reality, HLA restriction is a potential drawback to a peptide vaccine strategy, as it limits the number of patients who could benefit from vaccination. As success with HLA-A2–restricted peptides continues to be reported, other investigators have begun identifying peptides for other common HLA types. In the future, it is conceivable that every patient could receive an HLA-specific peptide vaccine. Ultimately we aim to “work backwards” to the point of immunizing cancer-naïve patients. After completing the safety portion of the node-positive E75 trial, we began enrolling patients with node-negative breast cancer into another peptide vaccine trial designed to identify the optimal biological dose of peptide, to investigate multiple dosing schedules, and to determine the feasibility of immunizing patients with minimal endogenous tumor burdens. In this node-negative trial, we have also enrolled patients with HER-2/neu-negative tumors to investigate the induction of an immune response in TAA-naïve patients. To date, 81 patients have been enrolled, and all vaccinated patients have demonstrated an immunologic response in vitro as well as a delayed-type hypersensitivity reaction in vivo. Optimal doses and schedules have been determined. Importantly, we have followed the vaccinated and HLAA2–negative, node-negative control subjects for clinical recurrence. Combining the node-positive and node-negative E75 trials, we currently have 171 patients enrolled. At a median follow-up of 24 months, the recurrence rate among the vaccinated patients is 5.6% compared with 14.8% for the observation group (P < 0.04) (unpublished data). Although our initial trial results are encouraging, we recognize the legitimate criticisms of a single-peptide vaccine and the limitations of such a strategy. One such limitation is the HLA restriction discussed above. Another is lack of antigen diversity, since a single peptide vaccine targets only 1 epitope from a TAA. The ability to generate an immune response to additional epitopes from the same TAA would help to diversify the resulting immune response. In a study by Disis and col-
leagues,6 patients were given a vaccine consisting of 3 long peptides from either the intra- or extracellular domain of the HER-2/neu protein. These investigators observed that patients developed an immune response to peptide fragments that were not part of their immunization mixture—a phenomenon known as epitope spreading. Among our vaccinated patients, we have observed epitope spreading to GP2, a subdominant epitope of the HER-2/neu protein, in all patients with node-positive disease. Importantly, more than 80% of patients with nodenegative disease also experienced epitope spreading to GP2, providing indirect evidence of some degree of occult disease. These findings also confirm that GP2 is an in vivo immunogenic peptide that may be useful in a vaccine (Abstract 4–55). We have subsequently completed preclinical work evaluating GP2 and have begun enrolling patients in a clinical trial involving the administration of GP2 mixed with GM-CSF.7 Additional preclinical work has shown that the cytolytic activity achieved by CTLs stimulated with a combination of E75 and GP2 is superior to either peptide alone.7 Once the GP2 safety trial has been completed, a trial in which E75 and GP2 are administered as a multiepitope vaccine will begin enrollment. In a study in which a similar strategy was used for patients with melanoma,8 the vaccine consisted of 4 peptides. Patients demonstrated immune responses against all 4 peptides, confirming their immunogenicity; however, substantial inter- and intrapatient variations were noted in the response to different peptides.8 We hypothesize that a multiepitope vaccine may be able to elicit a response against tumors that express lower levels of HER-2/neu protein and in patients with earlier stage disease. Another concern regarding peptide vaccines is the ability of a vaccination strategy that activates only the CTL immune effector system to mediate an adequate antitumor response. Evidence suggests that CTLs cannot be sustained without the concomitant activation of CD4+ T-helper cells. The promotion of a T-helper response requires loading major histocompatibility complex (MHC) class II molecules with peptides. Ii-Key is a 4-amino-acid modification of peptides that can enhance their occupancy of MHC class II molecules and improve the T-helper response.9 We are completing a phase I clinical trial with an Ii-Key–HER-2/neu–MHC class II peptide (AE37). Preliminary results are encouraging, as AE37 seems to be safe and well tolerated with minimal systemic toxicity. Robust local reactions have been documented that correlate with peptide-specific in vitro assays. Future studies will address a multiepitope vaccine designed to stimulate both the cell-mediated and humoral immune systems. Finally, it is anticipated that peptide-based vaccines will ultimately be incorporated into treatment strategies using standard therapeutic modalities. Another area of interest, therefore, is combination immunotherapy. Trastuzumab (Herceptin,
Genentech BioOncology) is a monoclonal antibody approved for treatment of HER-2/neu–overexpressing breast cancer. Preclinical data demonstrate that breast cancer cells treated with trastuzumab are more susceptible to lysis by E75-specific CTLs from vaccinated patients with breast cancer.10 Importantly, we found that even breast cancer cells expressing low levels of HER-2/neu can be significantly sensitized to E75stimulated lymphocytes from patients receiving the E75 peptide vaccine. This finding may expand the clinical utility and potential therapeutic benefit of trastuzumab used in combination with HER-2/neu peptide vaccines. In conclusion, HER-2/neu peptide vaccines show promise, particularly when administered to prevent disease recurrence. Confirming our initial E75 peptide vaccine trial results requires further investigation in a phase III randomized clinical trial, which is currently being pursued. To maximize the clinical benefit of peptide vaccines, a strategy involving multiepitope vaccination, combination immunotherapy, or both will likely be important. Phase I/II trials have been instituted to address these issues. Results from these trials will help to define the role of peptidebased vaccines in the treatment and prevention of breast cancer. Acknowledgement: The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.
References 1. Slamon DJ, Godolphin W, Jones LA, et al: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989. 2. Knutson KL, Schiffman K, Cheever MA, et al: Immunization of cancer patients with a HER-2/neu, HLA-A2 peptide, p369377, results in short-lived peptide-specific immunity. Clin Cancer Res 8:1014-1018, 2002. 3. Murray JL, Gillogly ME, Przepiorka D, et al: Toxicity, immunogenicity, and induction of E75-specific tumor-lytic CTLs by HER-2 peptide E75 (369-377) combined with granulocyte macrophage colony-stimulating factor in HLA-A2+ patients with metastatic breast and ovarian cancer. Clin Cancer Res 8:3407-3418, 2002. 4. Zaks TZ, Rosenberg SA: Immunization with a peptide epitope (p369-377) from HER-2/neu leads to peptide-specific cytotoxic T lymphocytes that fail to recognize HER-2/neu+ tumors. Cancer Res 58:4902-4908, 1998. 5. Peoples GE, Gurney JM, Hueman MT, et al: Clinical trial results of a HER2/neu (E75) vaccine to prevent recurrence in high-risk breast cancer patients. J Clin Oncol 23:7536-7545, 2005. 6. Disis ML, Grabstein KH, Sleath PR, et al: Generation of immunity to the HER-2/neu oncogenic protein in patients with
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breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res 5:1289-1297, 1999. 7. Mittendorf EA, Storrer CE, Foley RJ, et al: Evaluation of the HER2/neu-derived peptide GP2 for use in a peptidebased breast cancer vaccine trial. Cancer 106:2309-2317, 2006. 8. Slingluff CL Jr, Petroni GR, Yamshchikov GV, et al: Immunologic and clinical outcomes of vaccination with a multiepitope melanoma peptide vaccine plus low-dose interleukin-2 admin-
Neoadjuvant Therapy for HER2/neu–Expressing Ductal Carcinoma in Situ Using Type 1 Polarized Dendritic Cell Vaccines Brian J. Czerniecki, MD, PhD, Min Xu, MD, and Gary Koski, PhD With the increasing use of screening mammography, the diagnosis of breast cancer will be made at an early disease stage with ductal carcinoma in situ (DCIS) in about 20% to 25% of cases. Although surgery (mastectomy or lumpectomy) and radiation therapy remain the highly successful mainstays of therapy for DCIS, patients with DCIS, especially younger ones, are still at significant risk of developing invasive breast cancer. Fortunately, the development of novel therapies to reduce the size of large areas of DCIS, to prevent recurrence, or to prevent invasive breast cancer may be obtainable goals. Although some overlap exists, breast cancers can, generally speaking, be divided into estrogen-dependent and estrogen-independent phenotypes. Clinical trials involving use of anti-estrogen therapies in the neoadjuvant setting are underway.1 Because HER-2/neu overexpression may have a critical biological role in a significant number of DCIS lesions, targeting this pathway may also be useful for treating DCIS. Trastuzumab can be used as passive therapy to inhibit cell growth; however, an alternative approach is active, specific therapy to induce an immune response against HER-2/neu. Such a response may not only treat DCIS but could also protect against recurrence. In this overview, we discuss the rationale for an ongoing clinical study involving the use of HER-2/neu–pulsed dendritic cells (DCs) in the neoadjuvant treatment of DCIS.
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istered either concurrently or on a delayed schedule. J Clin Oncol 22:4474-4485, 2004. 9. Kallinteris NL, Powell D, Blackwell CE, et al: Ii-Key/MHC class II epitope peptides as helper T cell vaccines for cancer and infectious disease. Front Biosci 11:46-58, 2006. 10. Mittendorf EA, Storrer CE, Shriver CD, et al: Investigating the combination of trastuzumab and HER2/neu peptide vaccines for the treatment of breast cancer. Ann Surg Oncol 13:10851098, 2006.
ROLE OF HER-2/NEU IN DCIS DEVELOPMENT In murine transgenic models, overexpression of HER-2/neu leads to the development of invasive breast cancer. This observation strongly suggests that HER-2/neu has a critical role in the pathogenesis of at least some breast cancers.2,3 Also, HER2/neu expression has been demonstrated in 50% to 70% of high-grade DCIS lesions,4 and some evidence, albeit controversial, exists to suggest that HER-2/neu overexpression in DCIS lesions may result in higher recurrence rates.3,5,6 In addition, blocking estrogen pathways in patients with breast cancer may lead to tumor escape through a mechanism or mechanisms involving HER-2/neu signaling pathways. Of particular interest is evidence that the HER-2/neu pathways and estrogen production may be related through a cyclooxygenase (COX) -2 intermediary. Overexpression of COX-2 seems to induce prostaglandin E2 production in breast ductal cells, which leads to increased aromatase activity in the breast stroma cells and subsequently to enhanced estrogen production.7 If HER-2/neu plays such a substantial role in breast carcinogenesis, then it stands to reason that targeting this pathway at the earliest possible moment in tumor development may be a useful strategy for both the treatment and prevention of breast cancer. HUMORAL IMMUNITY TO HER-2/NEU Clear evidence exists to show that some patients with breast or ovarian cancer develop anti-HER-2/neu antibodies, and several groups have identified antigenic determinants on the HER2/neu protein.8-10 Of possibly great significance, studies of HER-2/neu transgenic mice have shown that complementfixing anti-HER-2/neu antibodies can retard or prevent the development of breast cancer, suggesting an important role for antibodies in controlling malignancies of the breast.11 Interestingly, CD4+ T cells are required in this model for class
318 318
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be safe and effective at inducing clonal expansion of E75specific CTLs capable of lysing HER-2/neu-expressing tumors. At a median follow-up of 22 months, the disease-free survival rate among vaccinated patients was 85.7% compared with 59.5% for those in the control group. In the vaccinated group, no deaths had occurred and 2 of 24 patients (8%) had developed a recurrence. In the control group, 2 patients had died and 6 of 29 (21%) had recurrent disease.5 Although these preliminary results did not reach statistical significance, they are compelling and indicate a trend towards fewer recurrences among vaccinated patients with high-risk breast cancer. This initial report included 53 patients. To date, 90 patients have enrolled in our trial of E75 for node-positive disease, and thus far we have observed similar rates (unpublished data). The fact that E75 is HLA-restricted allowed the inclusion of a natural control group in the trial; however, in reality, HLA restriction is a potential drawback to a peptide vaccine strategy, as it limits the number of patients who could benefit from vaccination. As success with HLA-A2–restricted peptides continues to be reported, other investigators have begun identifying peptides for other common HLA types. In the future, it is conceivable that every patient could receive an HLA-specific peptide vaccine. Ultimately we aim to “work backwards” to the point of immunizing cancer-naïve patients. After completing the safety portion of the node-positive E75 trial, we began enrolling patients with node-negative breast cancer into another peptide vaccine trial designed to identify the optimal biological dose of peptide, to investigate multiple dosing schedules, and to determine the feasibility of immunizing patients with minimal endogenous tumor burdens. In this node-negative trial, we have also enrolled patients with HER-2/neu-negative tumors to investigate the induction of an immune response in TAA-naïve patients. To date, 81 patients have been enrolled, and all vaccinated patients have demonstrated an immunologic response in vitro as well as a delayed-type hypersensitivity reaction in vivo. Optimal doses and schedules have been determined. Importantly, we have followed the vaccinated and HLAA2–negative, node-negative control subjects for clinical recurrence. Combining the node-positive and node-negative E75 trials, we currently have 171 patients enrolled. At a median follow-up of 24 months, the recurrence rate among the vaccinated patients is 5.6% compared with 14.8% for the observation group (P < 0.04) (unpublished data). Although our initial trial results are encouraging, we recognize the legitimate criticisms of a single-peptide vaccine and the limitations of such a strategy. One such limitation is the HLA restriction discussed above. Another is lack of antigen diversity, since a single peptide vaccine targets only 1 epitope from a TAA. The ability to generate an immune response to additional epitopes from the same TAA would help to diversify the resulting immune response. In a study by Disis and col-
leagues,6 patients were given a vaccine consisting of 3 long peptides from either the intra- or extracellular domain of the HER-2/neu protein. These investigators observed that patients developed an immune response to peptide fragments that were not part of their immunization mixture—a phenomenon known as epitope spreading. Among our vaccinated patients, we have observed epitope spreading to GP2, a subdominant epitope of the HER-2/neu protein, in all patients with node-positive disease. Importantly, more than 80% of patients with nodenegative disease also experienced epitope spreading to GP2, providing indirect evidence of some degree of occult disease. These findings also confirm that GP2 is an in vivo immunogenic peptide that may be useful in a vaccine (Abstract 4–55). We have subsequently completed preclinical work evaluating GP2 and have begun enrolling patients in a clinical trial involving the administration of GP2 mixed with GM-CSF.7 Additional preclinical work has shown that the cytolytic activity achieved by CTLs stimulated with a combination of E75 and GP2 is superior to either peptide alone.7 Once the GP2 safety trial has been completed, a trial in which E75 and GP2 are administered as a multiepitope vaccine will begin enrollment. In a study in which a similar strategy was used for patients with melanoma,8 the vaccine consisted of 4 peptides. Patients demonstrated immune responses against all 4 peptides, confirming their immunogenicity; however, substantial inter- and intrapatient variations were noted in the response to different peptides.8 We hypothesize that a multiepitope vaccine may be able to elicit a response against tumors that express lower levels of HER-2/neu protein and in patients with earlier stage disease. Another concern regarding peptide vaccines is the ability of a vaccination strategy that activates only the CTL immune effector system to mediate an adequate antitumor response. Evidence suggests that CTLs cannot be sustained without the concomitant activation of CD4+ T-helper cells. The promotion of a T-helper response requires loading major histocompatibility complex (MHC) class II molecules with peptides. Ii-Key is a 4-amino-acid modification of peptides that can enhance their occupancy of MHC class II molecules and improve the T-helper response.9 We are completing a phase I clinical trial with an Ii-Key–HER-2/neu–MHC class II peptide (AE37). Preliminary results are encouraging, as AE37 seems to be safe and well tolerated with minimal systemic toxicity. Robust local reactions have been documented that correlate with peptide-specific in vitro assays. Future studies will address a multiepitope vaccine designed to stimulate both the cell-mediated and humoral immune systems. Finally, it is anticipated that peptide-based vaccines will ultimately be incorporated into treatment strategies using standard therapeutic modalities. Another area of interest, therefore, is combination immunotherapy. Trastuzumab (Herceptin,
Genentech BioOncology) is a monoclonal antibody approved for treatment of HER-2/neu–overexpressing breast cancer. Preclinical data demonstrate that breast cancer cells treated with trastuzumab are more susceptible to lysis by E75-specific CTLs from vaccinated patients with breast cancer.10 Importantly, we found that even breast cancer cells expressing low levels of HER-2/neu can be significantly sensitized to E75stimulated lymphocytes from patients receiving the E75 peptide vaccine. This finding may expand the clinical utility and potential therapeutic benefit of trastuzumab used in combination with HER-2/neu peptide vaccines. In conclusion, HER-2/neu peptide vaccines show promise, particularly when administered to prevent disease recurrence. Confirming our initial E75 peptide vaccine trial results requires further investigation in a phase III randomized clinical trial, which is currently being pursued. To maximize the clinical benefit of peptide vaccines, a strategy involving multiepitope vaccination, combination immunotherapy, or both will likely be important. Phase I/II trials have been instituted to address these issues. Results from these trials will help to define the role of peptidebased vaccines in the treatment and prevention of breast cancer. Acknowledgement: The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.
References 1. Slamon DJ, Godolphin W, Jones LA, et al: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989. 2. Knutson KL, Schiffman K, Cheever MA, et al: Immunization of cancer patients with a HER-2/neu, HLA-A2 peptide, p369377, results in short-lived peptide-specific immunity. Clin Cancer Res 8:1014-1018, 2002. 3. Murray JL, Gillogly ME, Przepiorka D, et al: Toxicity, immunogenicity, and induction of E75-specific tumor-lytic CTLs by HER-2 peptide E75 (369-377) combined with granulocyte macrophage colony-stimulating factor in HLA-A2+ patients with metastatic breast and ovarian cancer. Clin Cancer Res 8:3407-3418, 2002. 4. Zaks TZ, Rosenberg SA: Immunization with a peptide epitope (p369-377) from HER-2/neu leads to peptide-specific cytotoxic T lymphocytes that fail to recognize HER-2/neu+ tumors. Cancer Res 58:4902-4908, 1998. 5. Peoples GE, Gurney JM, Hueman MT, et al: Clinical trial results of a HER2/neu (E75) vaccine to prevent recurrence in high-risk breast cancer patients. J Clin Oncol 23:7536-7545, 2005. 6. Disis ML, Grabstein KH, Sleath PR, et al: Generation of immunity to the HER-2/neu oncogenic protein in patients with
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Breast Diseases: A Year Book Quarterly Vol 17 No 4 2007
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breast and ovarian cancer using a peptide-based vaccine. Clin Cancer Res 5:1289-1297, 1999. 7. Mittendorf EA, Storrer CE, Foley RJ, et al: Evaluation of the HER2/neu-derived peptide GP2 for use in a peptidebased breast cancer vaccine trial. Cancer 106:2309-2317, 2006. 8. Slingluff CL Jr, Petroni GR, Yamshchikov GV, et al: Immunologic and clinical outcomes of vaccination with a multiepitope melanoma peptide vaccine plus low-dose interleukin-2 admin-
Neoadjuvant Therapy for HER2/neu–Expressing Ductal Carcinoma in Situ Using Type 1 Polarized Dendritic Cell Vaccines Brian J. Czerniecki, MD, PhD, Min Xu, MD, and Gary Koski, PhD With the increasing use of screening mammography, the diagnosis of breast cancer will be made at an early disease stage with ductal carcinoma in situ (DCIS) in about 20% to 25% of cases. Although surgery (mastectomy or lumpectomy) and radiation therapy remain the highly successful mainstays of therapy for DCIS, patients with DCIS, especially younger ones, are still at significant risk of developing invasive breast cancer. Fortunately, the development of novel therapies to reduce the size of large areas of DCIS, to prevent recurrence, or to prevent invasive breast cancer may be obtainable goals. Although some overlap exists, breast cancers can, generally speaking, be divided into estrogen-dependent and estrogen-independent phenotypes. Clinical trials involving use of anti-estrogen therapies in the neoadjuvant setting are underway.1 Because HER-2/neu overexpression may have a critical biological role in a significant number of DCIS lesions, targeting this pathway may also be useful for treating DCIS. Trastuzumab can be used as passive therapy to inhibit cell growth; however, an alternative approach is active, specific therapy to induce an immune response against HER-2/neu. Such a response may not only treat DCIS but could also protect against recurrence. In this overview, we discuss the rationale for an ongoing clinical study involving the use of HER-2/neu–pulsed dendritic cells (DCs) in the neoadjuvant treatment of DCIS.
320 320
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istered either concurrently or on a delayed schedule. J Clin Oncol 22:4474-4485, 2004. 9. Kallinteris NL, Powell D, Blackwell CE, et al: Ii-Key/MHC class II epitope peptides as helper T cell vaccines for cancer and infectious disease. Front Biosci 11:46-58, 2006. 10. Mittendorf EA, Storrer CE, Shriver CD, et al: Investigating the combination of trastuzumab and HER2/neu peptide vaccines for the treatment of breast cancer. Ann Surg Oncol 13:10851098, 2006.
ROLE OF HER-2/NEU IN DCIS DEVELOPMENT In murine transgenic models, overexpression of HER-2/neu leads to the development of invasive breast cancer. This observation strongly suggests that HER-2/neu has a critical role in the pathogenesis of at least some breast cancers.2,3 Also, HER2/neu expression has been demonstrated in 50% to 70% of high-grade DCIS lesions,4 and some evidence, albeit controversial, exists to suggest that HER-2/neu overexpression in DCIS lesions may result in higher recurrence rates.3,5,6 In addition, blocking estrogen pathways in patients with breast cancer may lead to tumor escape through a mechanism or mechanisms involving HER-2/neu signaling pathways. Of particular interest is evidence that the HER-2/neu pathways and estrogen production may be related through a cyclooxygenase (COX) -2 intermediary. Overexpression of COX-2 seems to induce prostaglandin E2 production in breast ductal cells, which leads to increased aromatase activity in the breast stroma cells and subsequently to enhanced estrogen production.7 If HER-2/neu plays such a substantial role in breast carcinogenesis, then it stands to reason that targeting this pathway at the earliest possible moment in tumor development may be a useful strategy for both the treatment and prevention of breast cancer. HUMORAL IMMUNITY TO HER-2/NEU Clear evidence exists to show that some patients with breast or ovarian cancer develop anti-HER-2/neu antibodies, and several groups have identified antigenic determinants on the HER2/neu protein.8-10 Of possibly great significance, studies of HER-2/neu transgenic mice have shown that complementfixing anti-HER-2/neu antibodies can retard or prevent the development of breast cancer, suggesting an important role for antibodies in controlling malignancies of the breast.11 Interestingly, CD4+ T cells are required in this model for class