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Keywords:

  • ductal carcinoma in situ;
  • breast cancer;
  • cancer vaccine;
  • dendritic cell vaccine;
  • HER-2/neu

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

BACKGROUND:

HER-2/neu overexpression plays a critical role in breast cancer development, and its expression in ductal carcinoma in situ (DCIS) is associated with development of invasive breast cancer. A vaccine targeting HER-2/neu expression in DCIS may initiate immunity against invasive cancer.

METHODS:

A HER-2/neu dendritic cell vaccine was administered to 27 patients with HER-2/neu–overexpressing DCIS. The HER-2/neu vaccine was administered before surgical resection, and pre- and postvaccination analysis was conducted to assess clinical results.

RESULTS:

At surgery, 5 of 27 (18.5%) vaccinated subjects had no evidence of remaining disease, whereas among 22 subjects with residual DCIS, HER-2/neu expression was eradicated in 11 (50%). When comparing estrogen receptor (ER)neg with ERpos DCIS lesions, vaccination was more effective in hormone-independent DCIS. After vaccination, no residual DCIS was found in 40% of ERneg subjects compared with 5.9% in ERpos subjects. Sustained HER-2/neu expression was found in 10% of ERneg subjects compared with 47.1% in ERpos subjects (P = .04). Postvaccination phenotypes were significantly different between ERpos and ERneg subjects (P = .01), with 7 of 16 (43.8%) initially presenting with ERposHER-2/neupos luminal B phenotype finishing with the ERposHER-2/neuneg luminal A phenotype, and 3 of 6 (50%) with the ERnegHER-2/neupos phenotype changing to the ERnegHER-2/neuneg phenotype.

CONCLUSIONS:

Results suggest that vaccination against HER-2/neu is safe and well tolerated and induces decline and/or eradication of HER-2/neu expression. These findings warrant further exploration of HER-2/neu vaccination in estrogen-independent breast cancer and highlight the need to target additional tumor-associated antigens and pathways. Cancer 2012. © 2012 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

Cancer vaccines have had limited success largely because previous cancer vaccine trials have focused on later stages of disease, when tumor burden is at its maximum and when even standard therapies, at times, have minimal benefit.1, 2 Analyzing the history of vaccination strategies clearly demonstrates the prophylactic use of vaccination before the progression of disease. Thus, if cancer vaccines were to target early stages of disease, vaccination may prove to be more effective. Furthermore, most experimental cancer vaccines have targeted tumor-associated antigens that are not critical to tumor survival and disease progression; therefore, tumor cells can readily escape immunity and persist or metastasize. Finally, most cancer vaccines have not been used to target cancer stem cells or molecular pathways involved in specific cancer genotypes and phenotypes. A vaccination strategy that provides an integrated solution to each of these obstacles is therefore likely to meet with increased success.

The use of antiestrogen therapy in both primary and secondary prevention has resulted in significant benefits to patients developing estrogen-dependent breast cancers.3-5 However, prevention for estrogen-independent breast cancer remains a significant issue.5 When considering breast cancer in particular, novel treatment or prevention strategies must take into account that breast cancer develops from several different progenitors, each leading to distinct tumor phenotypes, which may vary characteristically in the ultimate course of disease and susceptibility to various therapies. These phenotypes include the luminal A and B subtypes, HER-2, and basal phenotypes.6, 7 Although overlaps clearly do exist, each of these subtypes generally possesses a set of distinguishing features that allows for their convenient identification. The luminal subtypes express estrogen receptors (ERs), whereas the basal and HER-2 and luminal HER-2 phenotypes often overexpress HER family members, including epidermal growth factor receptor (EGFR; HER-1) and HER-2/neu.6, 8, 9 Whereas antiestrogen therapy can be used for primary and secondary prevention of the luminal tumors, there are currently no such therapies available for prevention of the high-grade HER-2 luminal, HER-2, and basal tumors. Thus, targeting the HER family might be an ideal target for a breast cancer prevention.

With respect to breast cancer and potential intervention with a cancer vaccine, ductal carcinoma in situ (DCIS) is an ideal stage during which to test cancer vaccination and potentially halt disease progression. DCIS is an early preinvasive malignancy of the breast and represents an intermediary between normal breast tissue and invasive breast cancer; thus, implementation of a breast cancer vaccine, in the early stages of disease such as DCIS, would allow for a setting in which the tumor burden is quite low. Furthermore, the impact of treatment in a neoadjuvant setting can be rapidly assessed on tumors scheduled for timely surgical resection, rather than by waiting for disease development or progression in asymptomatic patients.10 Such an approach not only speeds development of novel therapies, but also offers a window into the biology of breast cancer development.

Dendritic cells (DCs) were activated with cytokines and a clinical-grade bacterial lipopolysaccharide (LPS; a TLR4 ligand) to induce a unique battery of chemokines and cytokines, including interleukin (IL)-12, which can condition T cells for superior antitumor immunity and memory.11-13 The DCs are pulsed with synthetic peptides based on the HER-2/neu sequence that are capable of sensitizing T helper cells (Th) in most individuals.14, 15 HER-2/neu was selected as the target because its overexpression in breast cancer is associated with enhanced invasiveness,16 metastatic potential, greater probability of local and systemic recurrence, and resistance to chemotherapy.17-20 It is also a defining phenotypic feature of some breast cancer stem cells associated with the capacity for self-renewal21 and critically expressed in at least 1 non–ER-dependent breast cancer phenotype.22 Vaccines were administered before surgical therapy, so that the effects of immunization could be assessed on residual tumor at the time of surgical resection. We have previously reported some of the early results from this study.14 This article represents the final analysis of the trial. The primary objectives were safety and feasibility, and the secondary objectives were immune response and clinical response to vaccination, including the assessment of HER-2 expression in response to vaccination. We report the final analysis of the trial, exclusive of the immune response, which is reported elsewhere.23 The trial suggests that vaccination in this early disease setting is safe, effective in inducing long-term stable immune responses23 and eliminating HER-2/neu–expressing tumor cells in residual DCIS lesions, and furthermore hints at possible new therapy options for patients with phenotypes underserved by currently available therapies.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

Clinical Trial Design

We conducted a pilot trial of a HER-2/neu–based DC vaccination strategy for patients with HER-2/neu–overexpressing DCIS (NCT001070211). The primary objectives were to evaluate the feasibility, safety, and efficacy of HER-2/neu vaccination. The secondary objectives were to assess immune response to vaccination and response to vaccine, including assessment of expression of HER-2 in remaining DCIS. HER-2/neu peptide-loaded DC were administered intranodally once per week for 4 weeks and followed by surgical resection. A maximum of 30 patients were to be enrolled to establish safety and have adequate power to evaluate pre- versus postvaccination T-cell sensitization status by McNemar test. If no occurrence of unacceptable toxicity was observed in the 30 patients, then the rate of unacceptable toxicity was no higher than 7% based on the upper bound of the 1-sided exact 90% confidence interval.

Feasibility was defined as the rate of patients who received all 4 immunizations on schedule. Toxicities were graded using the National Cancer Institute Common Toxicity Grading Scale version 3.0. Immunogenicity was based on tetramer, enzyme-linked immunospot, and in vitro sensitization assays. CD4pos and CD8pos sensitization of T cells was assessed in pre- and postvaccination peripheral blood samples and in sentinel lymph nodes where available. HER-2/neu protein expression was evaluated in pre- and postvaccination tissue specimens by a dedicated pathologist who oversees immunohistochemistry laboratory.

Eligible patients were older than 18 years, signed informed consent, had Eastern Cooperative Oncology Group performance status 0 or 1, had biopsy-proven DCIS, and had not yet received definitive treatment. Patients whose DCIS was eliminated by excisional biopsy at diagnosis were not eligible. HER-2/neu positivity was defined as >5% cells expressing 2+ or 3+ intensity of the HER-2/neu protein. These criteria were used because the majority of DCIS studies considered 2+ HER-2/neu expression as positive, and because there were no other current pathologic standards for HER-2 expression in DCIS when this trial was conceived, 2+ staining was considered HER-2/neu positive. These were all secondarily reviewed by our pathologist P.Z. for eligibility. Response to vaccination was defined as any decline in HER-2/neu protein expression ≥20% on postvaccination immunohistochemical staining. These were secondarily reviewed blinded for vaccine by our pathologist P.Z. Patients with areas suspicious for invasive disease were evaluated by magnetic resonance imaging (MRI). All subjects underwent cardiac evaluation with multigated acquisition (MUGA) scan or echocardiography to document adequate baseline cardiac function. Scans were performed before immunization and within 2 weeks of the final vaccine treatment. All patients underwent human leukocyte antigen (HLA) class I tissue typing pre-enrollment and had routine history, physical exams, electrocardiography, blood work, and urinalysis before immunization. After informed consent, all subjects underwent prevaccination leukapheresis to obtain sufficient monocytes for vaccine preparation; in a few cases, a second apheresis was required for additional monocytes. Postvaccination leukapheresis was performed usually within 2 weeks of the final immunization. All patients underwent postvaccination mammogram, MRI, and surgical resection of DCIS with either lumpectomy or mastectomy. Patients were followed for 30 days after vaccination to assess safety and to undergo second leukapheresis. Long-term immunologic surveillance (ie, serial blood sampling) was not mandatory and was conducted on a voluntary basis only.

Materials and Reagents

HER-2/neu peptides were purchased from American Peptide Corporation (Sunnyvale, Calif), monocyte-macrophage medium (serum-free medium [SFM]) and Iscove medium from Invitrogen (Carlsbad, Calif), lymphocyte separation medium from ICN Biomedical Inc. (Aurora, Ohio), human AB serum and fetal calf serum from Sigma Chemical (St Louis, Mo), and granulocyte-macrophage colony-stimulating factor (GM-CSF) from Amgen (Newbury Park, Calif). Reagents for enzyme-linked immunosorbent assays were obtained from Pharmingen (San Jose, Calif), and clinical grade interferon (IFN)-γ from Intermune (Brisbane, Calif). Clinical grade LPS was a generous gift from Dr. Anthony Suffredini at the National Institutes of Health (NIH).

Immunization Procedure

Vaccine treatments were administered in the NIH-designated General Clinical Research Center at the Hospital of the University of Pennsylvania. They consisted of 10 million to 20 million HER-2/neu–pulsed DCs suspended in 1 mL sterile saline. DCs were administered under ultrasound guidance into a single lymph node in each groin as previously described.23, 24 Half of each DC vaccine dose was placed into each lymph node with a 22-ga needle. The first 9 subjects were observed for 2 hours postimmunization with routine vital signs obtained at 15-minute intervals. Subsequent subjects were observed for 1 hour. Immunizations were administered once weekly for 4 weeks. All subjects completed all 4 scheduled vaccine treatments.

Preparation of HER-2/neu Vaccine

Interdigitating DCs were produced as described previously.12, 14, 23 Briefly, monocytic DC precursors were obtained from subjects via tandem leukapheresis/countercurrent centrifugal elutriation. Cells were cultured overnight at 37°C in SFM with GM-CSF and IL-4. The next day, DCs were pulsed with 6 HER-2/neu major histocompatibility complex (MHC) class II promiscuous-binding peptides (42-56, 98-114, 328-345, 776-790, 927-941, 1166-1180).25, 26 After 8 of 12 hours of incubation, IFN-γ (1000 U/mL) was added, and the next day, 6 hours before harvest, NIH reference standard LPS was added (10 ng/mL) to achieve full DC activation. For HLA-A2.1pos subjects, cells were also pulsed with MHC class I binding peptides 369-377 and 689-697. Harvested cells were washed and lot release criteria of >70% viability, negative Gram stain, and endotoxin <5 EU/kg confirmed.

Immunohistochemical Staining of DCIS Lesions

Formalin-fixed, paraffin-embedded tissue blocks were sectioned and stained via HERcepTest (Dako, Carpinteria, Calif), as well as with anti-CD4, anti-CD8 T lymphocyte, anti-CD20 (B cell), anti-CD56 (NK cell), anti-Fox-P3 (Treg) markers as described previously.14

Statistical Methods

Event rates and exact 95% confidence intervals (CIs) were calculated. Percentage change in HER-2/neu–expressing cells pre- and postvaccine treatment was defined as ([percentage of HER-2/neu–expressing cells post-treatment minus the percentage pretreatment] divided by the percentage pretreatment) multiplied by 100. Postvaccine HER-2/neu expression was defined as negative if the percentage of cells staining HER-2/neu 2+ to 3+ was <5%. Clinical response to immunization was defined as either no residual DCIS at surgery or >20% decrease in the percentage of HER-2/neu–expressing cells after vaccination. The percentage of HER-2/neu–expressing cells pre- and postimmunization was compared within patients by nonparametric Wilcoxon signed ranks test. The percentage change in HER-2/neu–expressing cells was compared between the study population (ie, pre- vs postvaccine change) and concurrent untreated controls (ie, diagnosis vs surgical specimen) by nonparametric Wilcoxon rank sum test. Association between the prevaccine percentage of HER-2/neu–expressing cells (a continuous predictor) and likelihood of response to immunization was evaluated by logistic regression analysis. Associations between prevaccine phenotype (eg, ERposHER-2/neupos or ERnegHER-2/neupos) and both the postvaccine response status (eg, no residual DCIS at surgery, responder, nonresponder) and phenotype (eg, luminal A, luminal B, HER-2/neu–driven, basal) were tested by Fisher exact test. All significance values were 2-sided. Statistical analyses were performed either with SPSS 15.0 software (SPSS Inc., Chicago, Ill) or StatXact software (Cytel Inc., Cambridge, Mass).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

Patient Characteristics

Between September 2003 and May 2008, 38 eligible patients were identified, of whom 29 patients gave written informed consent and enrolled on the institutional review board-approved trial. Of the 29 patients who initially enrolled, 2 were deemed ineligible on re-review of HER-2 staining after consent; a third underwent leukapheresis, insufficient cells were obtained, and the patient was withdrawn (Fig. 1). The mean age was 53 years (range, 38-87). A listing of the patient and tumor characteristics is shown in Table 1. Feasibility was 100%, because all patients who initiated immunization completed the 4 weekly vaccine treatments. Eighty-five percent of patients demonstrated evidence of anti-HER-2/neu CD4 and CD8 T-cell responses and are reported elsewhere.14, 23 Seventeen patients were ERposHER-2/neupos, and 10 patients were ERnegHER-2/neupos.

thumbnail image

Figure 1. A flow diagram outlines the patient population participating in the clinical trial. Of the 38 patients eligible for the trial, 27 patients gave written informed consent and enrolled on the institutional review board-approved trial. Five patients had no residual disease after vaccination, whereas 22 patients had residual disease, thus allowing for analysis of postvaccine immune response. DCIS, ductal carcinoma in situ; NED, no evidence of disease.

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Table 1. Tabulated Data Summary Displaying Characteristics of All (n=29) Eligible Patients as Well as Tumor Characteristics
SubjectAge, yHLADCIS ExtentBiopsySurgeryPrevaccination, Cells Staining 2+ to 3+
      ER Status%PR Status%HER-2/neu Status%
  1. Abbreviations: C, core biopsy; CALCS, calcifications; DCIS, ductal carcinoma in situ; ER, estrogen receptor; HLA, human leukocyte antigen; LUMP, lumpectomy; MAST, mastectomy; PR, progesterone receptor; S, surgical biopsy.

144A23 × 3 × 3SMAST00+30
264A222 × 12 × 20CLUMP+90+70+65
348A219 × 50 × 44SMAST00+60
467A2NoneCMAST00+30
545 15 × 20 × 13CLUMP+90+90+20
645A25 × 5 × 5CLUMP00+100
855 22 × 4 × 6CLUMP+50+100
953A228 × 45 × 35CMAST00+100
1048A2None (palpable)SLUMP+10+10+100
1151A21 × 1 × 2CLUMP+90+40+50
1258A239 × 38 × 18CLUMP00+10
1343 34 × 75 × 68CMAST+80+35+100
1438A2NoneC, SMAST+500+50
1559A2No significant calcsSMAST00+10
1669A2NoneCMAST00+90
1849 2.5 × 1.7 × 1.1CMAST+300+100
1987 18 × 18 × 14CMAST+90+30+20
2044A212 mm clusterSLUMP+95+10+10
2157 24 × 20 × 33CLUMP+100+90
2243 No studyCMAST00+10
2357 No studyCMAST+90+5+80
2464 17 × 12 ×18CLUMP+300+30
2564 47 × 32 × 22CMAST00+30
2640 Small cluster of calcsCMAST+90+85+25
2757 14.9 × 19.8 × 24.0CMAST+95+90+60
2847A23.4 × 5.8 × 11.7CLUMP+40+10+90
2945 Scattered benign calcsCLUMP+100+25+80

HER-2/neu Vaccine Safety and Toxicity

HER-2/neu vaccination treatments were well tolerated, with only grade 1 and 2 toxicities observed. The most common toxicities encountered include general malaise (72.4%, 21 of 29), injection site soreness (58.6%, 17 of 29), chills/rigors (37.9%, 11 of 29), fever (27.5%, 8 of 29), and headaches (24%, 7 of 29). Three patients demonstrated an asymptomatic depression in cardiac ejection, with 15% to 18% declines of grades 1 and 2.27 None of the ejection fractions was <50%. In 1 of the patients, a repeat MUGA scan returned to baseline within 30 days. Because no cases of unacceptable toxicity were observed in the final 27 patients who were evaluated in the trial, the upper bound of the 1-sided exact 90% CI for the toxicity rate is 8.2%, indicating that the vaccine is safe, with a population toxicity rate no greater than 8%.

Elimination of HER-2/neu Expression After HER-2/neu Vaccination

HER-2/neu expression between initial biopsy and surgical resection specimens is relatively constant.14, 28 For 5 of 27 (18.5%; exact 95% CI, 6.3-38.1) immunized subjects, no residual DCIS could be identified at the time of surgical resection; the subjects were considered complete responses. In the remaining 22 patients, the median percentage change in HER-2/neu expression after DC vaccine was −88% (ie, a decline of 88%), with a range of −100% to 700%. This pre- to postvaccine difference was statistically significant (Wilcoxon signed ranks test, P = .023). Furthermore, 13 of 22 (59.1%; exact 95% CI, 36.3-79.2) subjects demonstrated >20% decline in HER-2/neu expression after vaccination, and 11 (50%; exact 95% CI, 28.2-71.8) subjects actually achieved complete loss of detectable HER-2/neu expression as determined by HercepTest (Table 2). By logistic regression analysis, we also found that subjects with higher prevaccine HER-2/neu expression appeared less likely to respond to immunization (estimated odds ratio, 0.97; exact 95% CI, 0.94-1.00; P = .05), with a 3% lower risk of response for each percentage increase in HER-2/neu expression before vaccine treatment.14, 23, 26 Eleven DCIS patients who were contemporaneously screened with those in the trial, but declined to participate, served as untreated controls. Percentages of HER-2/neu–expressing cells in diagnosis and surgical resection specimens ranged from 20% to 100%. Percentage change in HER-2/neu expression between initial biopsy and surgical specimens was −10%, −10%, 0%, 0%, 0%, 0%, +21%, +25%, +29%, +29%, and +88%. The percentage change in HER-2/neu expression was statistically significantly different between the 11 untreated controls (median, 0%; range, −10% to 88%) and 22 treated patients who had their tumor evaluable after vaccination (median, −88%; range, −100% to 700%; Wilcoxon rank sum test, P = .005; Table 2). Therefore, these data suggest that HER-2/neu vaccination treatment results either in the destruction of HER-2/neu–expressing cells or suppression of their capacity to express HER-2/neu.

Table 2. Tabulated Data Summary Displaying Percentage of HER-2/neu–Overexpressing Cells Enumerated Before and After Vaccination With Percentage Change in Expression Noted
Vaccinated DCIS Study Patients, n=27a
Prevaccine, %Postvaccine, %Change, %
10NED
30NED
30NED
90NED
90NED
100−100b
100−100b
200−100b
200−100b
250−100b
300−100b
300−100b
600−100b
650−100b
800−100b
502−96b
10020−80
10060−40
10090−10
80800
90900
100>900
1001000
1001000
6070+17
5095+90
1080+700
DCIS Controls, n = 11c
Diagnosis Specimen, %Surgical Specimen, %Change, %
  • Abbreviations: DCIS, ductal carcinoma in situ; NED, no evidence of disease.

  • a

    Change in HER-2/neu expression before and after vaccination among trial participants.

  • b

    Patient scored as HER-2/neuneg.

  • c

    HER-2/neu expression status among the control population.

10090−10
10090−10
90900
90900
1001000
1001000
7085+21
2025+25
7090+29
7090+29
4075+88

HER-2/neu Vaccination Is More Effective in Hormone-Independent DCIS

Ten of the 27 patients were of the hormone-independent ERnegHER-2/neupos phenotype, and of these, 9 (90%) demonstrated clinical response to HER-2/neu vaccination. Four of these patients (40%) had no detectable residual disease at the time of surgery, and 5 others demonstrated the requisite declines in HER-2/neu expression after HER-2/neu vaccination. Of the 17 ERpos subjects, only 5.9% had no detectable disease at surgery. However, 47.1% of those ERpos subjects had a decline in HER-2/neu expression after vaccination. The difference in response rates between subjects with ERneg versus ERpos tumors was statistically significant (Fisher exact test, P = .04; Table 3). Even patients who had significant HER-2/neu expression on 100% of tumor cells before vaccination with the ERneg phenotype demonstrated a response after vaccination, with significant declines in HER-2/neu expression. In contrast, in the same subgroup within ERpos subjects, comparable declines were not observed. Interestingly, the fraction of subjects who did not demonstrate a decline in HER-2/neu expression after vaccination was greater among ERpos subjects compared with ERneg subjects, 47.1% versus 10%. Thus, it appears that tumors with hormone-independent phenotypes are more susceptible to this immunization regimen than their ERpos counterparts.

Table 3. Tabulated Data Summary Displaying Vaccine Response Based on Prevaccination and Postvaccination Hormone Receptor Status
Prevaccine PhenotypeNo.No Tumor at SurgeryHER-2/neu ResponderaHER-2/neu NonresponderFisher Exact P
  • Abbreviation: ER, estrogen receptor.

  • Patients who were ERnegHER-2/neupos had a greater response to vaccination than those who were ERposHER-2/neupos.

  • a

    Responders achieve >20% decrease in percentage of cells staining HER-2/neu 2+ to 3+ postvaccination.

ERposHER-2/neupos171 (5.9%)8 (47.1%)8 (47.1%).04
ERnegHER-2/neupos104 (40.0%)5 (50.0%)1 (10.0%) 

HER-2/neu Vaccination Alters the Ultimate Phenotype of DCIS Lesions

The selection of DCIS to test the effectiveness of DC-pulsed HER-2/neu vaccination offered the opportunity to rapidly assess effects on tumors in an early disease setting. In this regard, HER-2/neu vaccination appeared to alter tumor phenotype, causing ERposHER-2/neupos DCIS to become ERposHER-2/neuneg in 43.8% of patients, and ERnegHER-2/neupos DCIS to become triple-negative (6.3%), as shown in Table 4. A depiction of the postvaccination HER-2/neu and the hormone receptor staining is demonstrated in Figure 2. In total, 4 of the 22 (18.2%) patients with residual DCIS became triple-negative; this is an unusually high proportion, because this phenotype tends to account for only 3% to 5% of DCIS.29 It can be reasonably concluded that the observed losses of HER-2 expression in about half of the patients are a result of vaccination, because similar alterations were not observed in nonimmunized controls. These clinical alterations in tumor protein expression suggest that the vaccination treatment rapidly induced an immune response that may have ultimately impacted the overall phenotype of the tumor.

Table 4. Data Summary Comparing Prevaccine Phenotype to Postvaccine Phenotype of DCIS Lesions
Prevaccine PhenotypeNo.Postvaccination Phenotype
ERpos HER-2/neunegERpos HER-2/neuposERneg HER-2/neuposERneg HER-2/neunegFisher Exact P
  • Abbreviations: DCIS, ductal carcinoma in situ; ER, estrogen receptor. Of the patients who initially started with the ERposHER-2/neupos phenotype, 43.8% were converted to the ERposHER-2/neuneg phenotype due to the loss of HER-2/neu after vaccination. Interestingly, 50% of the patients with an initial HER-2/neuposERneg phenotype were transformed into the triple-negative phenotype by the loss of HER-2/neu antigen after vaccination.

  • a

    No change from prevaccination phenotype.

ERposHER-2/neupos167 (43.8%)5 (31.3%)a3 (18.8%)1 (6.3%).01
ERnegHER-2/neupos60 (0.0%)0 (0.0%)3 (50.0%)a3 (50.0%) 
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Figure 2. Vaccination induces decline in HER-2/neu expression. Photographs depict slides from a representative subject stained for HER-2/neu (HercepTest) and estrogen receptor (ER). Note the dramatic reduction in HER-2/neu staining intensity in the postvaccination tissue sample and the decline in ER staining after vaccination.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

In this trial, we activated peptide-pulsed DCs using a special clinical grade bacterial LPS that induced a unique set of soluble factors, including high levels of IL-12 and TH1 chemokines not elicited through traditional DC vaccine production methods.12, 14 TH1 responses are associated with good clinical outcomes in breast cancer.30 These DCs were then pulsed with HER-2/neu peptides and administered to patients with DCIS in the neoadjuvant setting in an effort to attack the early stage of disease. This clinical DC HER-2/neu vaccine was successful in inducing strong long-lasting immune responses23 and either reducing or eliminating HER-2/neu expression in most vaccinated subjects. These results suggest that this strategy holds considerable promise, and could be further explored for treating early breast cancer, for preventing recurrence, or for the outright prevention of primary disease in high-risk populations.

The finding that vaccination resulted in significantly more complete responses in ER-independent compared with ER-dependent tumors is interesting and continues to persist in our current study (unpublished observation). This may be biologically relevant, as ERposHER-2/neupos invasive breast cancer (IBC) seems to be less sensitive than ERnegHER-2/neupos tumors, with fewer complete responses noted in patients treated with immune-based trastuzumab and chemotherapy or chemotherapy alone.31, 32 We observed no difference in immune response development between ERposHER-2/neupos compared with ERnegHER-2/neupos subjects (not shown), and it has been suggested that ER signaling can result in other HER family members being activated33; this may account for these biologic differences. Identifying response differences in ERposHER-2/neupos DCIS to immune-based therapies may shed light on these differences in response in IBC as well.

This DC-based HER-2 vaccine therapy may prove a good adjunct for preventing both locoregional or distant recurrence, both of which are more frequent for the HER-2 and basal phenotypes.34, 35 Nonetheless, the emergence of HER-2/neuneg phenotypes after vaccination serves as a warning that a single target may not be sufficient to completely eliminate or prevent disease in all individuals. Because about 18% of the participants became triple-negative after being originally HER-2pos, these data provide evidence that at least some triple-negative breast cancers can be prevented through targeting and eliminating HER-2pos clones before breast cancer development. It also suggests that future experimental vaccine formulations must take other phenotypes (such as estrogen-dependent and triple-negative) into account to block potential escape variants. Refinements of this approach will therefore include pairing vaccine therapy with antiestrogen therapy such as tamoxifen to address hormone-dependent proliferating tumors, the inclusion of additional vaccine target antigens such as HER-1 (EGFR), HER-3, and survivin to block the emergence of escape variants with a triple-negative phenotype, and immune modulation of the primary site.

Clearly, new primary and secondary prevention strategies, especially for estrogen-independent breast cancers, are needed to reduce risk of recurrence and eventual deaths caused by these aggressive breast cancer phenotypes. As recently suggested, younger patients with triple-negative breast cancers may derive benefits in survival from contralateral prophylactic mastectomy.36 However, less radical alternative therapies would be preferable. Indeed, immune-based approaches targeting EGFR (HER-1), HER-2/neu, and HER-3 are of potential value because of the essential role these molecules play in the biology of both estrogen-dependent and estrogen-independent breast cancer phenotypes.37 Targeted immune therapies are currently becoming a mainstay for treatment of HER-2/neu–expressing invasive breast cancer, as the engineered monoclonal antibody trastuzumab (directed at HER-2) has demonstrated dramatic effects in diminishing distant recurrence and improving survival.38, 39 Targeting the HER-2/neu pathway in early breast cancer and DCIS using DC1 pulsed with HER-2/neu peptides clearly impacted these lesions, especially those with estrogen-independent HER-2/neupos DCIS. It is conceivable that these vaccines may reduce secondary recurrence and could be developed for primary prevention as well. Their combination with trastuzumab in patients with invasive breast cancer may also be worth pursuing, as a few of the patients in this trial were found to have some invasive disease large enough to warrant chemotherapy and trastuzumab, and completed this additional therapy after vaccination without increased morbidity. We are currently investigating DC1 vaccine therapy for HER-2/neupos IBC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

We thank Jeanne Schueller and Vickie Sallee for assistance in conducting this trial; the staff of the General Clinical Research Center; the Leukapheresis Unit at the Hospital of the University of Pennsylvania; and Robin Noel for graphic arts support.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

Supported by National Institutes of Health R01 CA096997, the Harrington Foundation, Pennies-in-action.org, and the Mistler Foundation.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES
  • 1
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