Oncodriver inhibition and CD4+ Th1 cytokines cooperate through Stat1 activation to induce tumor senescence and apoptosis in HER2+ and triple negative breast cancer: implications for combining immune and targeted therapies

In patients with HER2-expressing breast cancer many develop resistance to HER2 targeted therapies. We show that high and intermediate HER2-expressing cancer cell lines are driven toward apoptosis and tumor senescence when treated with either CD4+ Th1 cells, or Th1 cytokines TNF-α and IFN-γ, in a dose dependent manner. Depletion of HER2 activity by either siRNA or trastuzumab and pertuzumab, and subsequent treatment with either anti-HER2 Th1 cells or TNF-α and IFN-γ resulted in synergistic increased tumor senescence and apoptosis in cells both sensitive and cells resistant to trastuzumab which was inhibited by neutralizing anti-TNF-α and IFN-γ. Th1 cytokines induced minimal senescence or apoptosis in triple negative breast cancer cells (TNBC); however, inhibition of EGFR in combination with Th1 cytokines sensitized those cells causing both senescence and apoptosis. TNF-α and IFN-γ led to increased Stat1 phosphorylation through serine and tyrosine sites and a compensatory reduction in Stat3 activation. Single agent IFN-γ enhanced Stat1 phosphorylation on tyrosine 701 and similar effects were observed in combination with TNF-α and EGFR inhibition. These results demonstrate Th1 cytokines and antioncodriver blockade cooperate in causing tumor senescence and apoptosis in TNBC and HER2-expressing breast cancer, suggesting these combinations could be explored as non-cross-reactive therapy preventing recurrence in breast cancer.Fil: Rosemblit, Cinthia. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Datta, Jashodeep. University of Pennsylvania; Estados UnidosFil: Lowenfeld, Lea. University of Pennsylvania; Estados UnidosFil: Xu, Shuwen. University of Pennsylvania; Estados UnidosFil: Basu, Amrita. H. Lee Moffitt Cancer Center; Estados UnidosFil: Kodumudi, Krithika. H. Lee Moffitt Cancer Center; Estados UnidosFil: Wiener, Doris. H. Lee Moffitt Cancer Center; Estados UnidosFil: Czerniecki, Brian J.. H. Lee Moffitt Cancer Center; Estados Unidos. University of Pennsylvania; Estados Unido

Loss of Anti-HER-3 CD4+ T-Helper Type 1 Immunity Occurs in Breast Tumorigenesis and is Negatively Associated with Outcomes

We previously demonstrated a progressive loss of the anti-human epidermal growth factor receptor 2 (HER2) CD4+ T-helper type 1 (Th1) response during HER2 breast tumorigenesis. This loss is associated with residual disease following neoadjuvant therapy and increased risk of recurrence. In this study, we assessed the fate of anti-HER3 Th1 immunity during breast tumorigenesis. Peripheral blood from 131 subjects, including healthy donors (HDs), patients with benign breast disease (BD), ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC), was collected. Interferon (IFN)-γ immune responses to four HER3-derived major histocompatibility complex (MHC) class II promiscuous peptides were tested via enzyme-linked immunosorbent (ELISPOT) assays, and three immune response parameters were compared: anti-HER3 (i) responsivity, or proportion of subjects responding to at least one peptide; (ii) repertoire, or number of responding peptides; and (iii) cumulative response, or summed peptide response. A significant decline in anti-HER3 Th1 response was observed going from HDs to IBC patients; patients with triple-negative breast cancer (TNBC) demonstrated the lowest responses. HDs had significantly higher Th1 responses versus estrogen receptor (ER) IBC and TNBC patients across all three immune parameters; HER2 IBC patients displayed responses similar to HDs and BDs. Patients with recurrent breast cancer and residual disease following neoadjuvant therapy demonstrated significantly lower anti-HER3 Th1 immunity compared with patients without recurrence or with a pathologic complete response to neoadjuvant therapy. Anti-HER3 CD4+ Th1 responses decline during breast tumorigenesis, particularly in TNBC. Attempts to immunologically restore depressed responses in vulnerable subgroups may help mitigate recurrence

Optimizing dendritic cell-based approaches for cancer immunotherapy

Dendritic cells (DC) are professional antigen-presenting cells uniquely suited for cancer immunotherapy. They induce primary immune responses, potentiate the effector functions of previously primed T-lymphocytes, and orchestrate communication between innate and adaptive immunity. The remarkable diversity of cytokine activation regimens, DC maturation states, and antigen-loading strategies employed in current DC-based vaccine design reflect an evolving, but incomplete, understanding of optimal DC immunobiology. In the clinical realm, existing DC-based cancer immunotherapy efforts have yielded encouraging but inconsistent results. Despite recent U.S. Federal and Drug Administration (FDA) approval of DC-based sipuleucel-T for metastatic castration-resistant prostate cancer, clinically effective DC immunotherapy as monotherapy for a majority of tumors remains a distant goal. Recent work has identified strategies that may allow for more potent "next-generation" DC vaccines. Additionally, multimodality approaches incorporating DC-based immunotherapy may improve clinical outcomes

Abstract 1308: Depressed anti-HER2 CD4 Th1 responses correlate with residual disease following neoadjuvant therapy in HER2+ breast cancer patients and can be restored by dendritic cell vaccination

Abstract BACKGROUND: We have previously demonstrated a loss of anti-HER2 CD4 T-helper type 1 responses (Th1resp) in HER2+ invasive breast cancer (IBC) pts relative to healthy donors. Compared with pathologic complete response (pCR) after neoadjuvant trastuzumab/chemotherapy (T/C), residual disease at surgery (<pCR) portends a worse prognosis. We investigated differences in anti-HER2 Th1resp between pCR and <pCR pts following neoadjuvant T/C, and impact of HER2-pulsed dendritic cell (DC) vaccination in <pCR pts. METHOD: Th1resp were generated from PBMCs pulsed with 6 HER2 class II peptides by measuring IFN-γ production via ELISPOT, and compared between HER2+ IBC groups. Th1resp metrics were anti-HER2 responsivity, no. of reactive peptides (repertoire), and cumulative response across 6 peptides (spots/10^6 cells). Th1resp of <pCR IBC pts (n = 4) receiving adjuvant HER2-pulsed type 1-polarized DC (DC1) vaccination were analyzed pre-/post-immunization. RESULTS: The study comprised 85 pts. The diminished anti-HER2 Th1resp in treatment-naïve HER2+ IBC pts (n = 21) - assessed by responsivity, repertoire, or cumulative response - did not improve globally in T/C-treated IBC pts (n = 64). Within this T/C-treated cohort, neoadjuvant T/C receipt (60.9%) was associated with higher repertoire (1.5 vs 0.7; p = 0.04), but not responsivity or cumulative response, compared with adjuvant T/C. <pCR (n = 23) and pCR (n = 16) cohorts did not differ by age, menopausal status, race, BMI, comorbidity, or stage at diagnosis; however, pCR pts were more likely to have ER- tumors (69% vs 30%, p = 0.03). <pCR pts demonstrated dramatically lower anti-HER2 responsivity (30% vs 94%, p<0.001), repertoire (0.3 vs 3.3 peptides, p<0.001), and cumulative response (24.6 vs 148.2, p<0.001) compared with pCR pts. This disparity was not attributable to <pCR pts’ immune incompetence or increase in suppressive (Treg/MDSC) populations, but associated with shifts in IFNγ:IL10-producing Th phenotypes. Four pts in the residual disease cohort were recruited to our adjuvant HER2-pulsed DC1 vaccination trial, receiving 6 weekly injections followed by booster doses. Evaluable anti-HER2 Th1resp at 3 month post-vaccination (prior to 1st booster dose) indicated significantly restored repertoire (3.0 post vs 0.5 pre, p = 0.003) and cumulative response (152.5 vs 33.7; p = 0.03). CONCLUSION: Beyond the known association between ER status and pathologic response following neoadjuvant T/C in HER2+ breast cancer, we identify a novel correlation between <pCR and depressed anti-HER2 CD4 Th1resp. Even in these heavily pre-treated pts, this Th1 deficit is not “fixed” and can be restored by HER2-pulsed DC1 vaccination. Given the worse prognosis in <pCR pts, combinations of existing HER2-targeted therapies with strategies to boost anti-HER2 Th1 immunity may decrease the risk of recurrence. Note: This abstract was not presented at the meeting. Citation Format: Jashodeep Datta, Erik Berk, Shuwen Xu, Elizabeth Fitzpatrick, Lea Lowenfeld, Noah Goodman, David Lewis, Robert E. Roses, Angela DeMichele, Brian J. Czerniecki. Depressed anti-HER2 CD4 Th1 responses correlate with residual disease following neoadjuvant therapy in HER2+ breast cancer patients and can be restored by dendritic cell vaccination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1308. doi:10.1158/1538-7445.AM2015-130

Abstract 2489: HER2 peptide-specific immunogenicity correlates with pathologic response following HER2-pulsed dendritic cell vaccination for early breast cancer

Abstract Introduction: We have developed an autologous HER2-pulsed dendritic cell (DC) vaccine that induces robust T-cell responses in early breast cancer. We have also previously demonstrated anti-HER2 Th1 responses to select class II peptides are preferentially lost early in HER2pos breast tumorigenesis. We aimed to compare peptide-specific Th1 responses between pathologic complete (pCR) and incomplete responders (<pCR) following HER2-pulsed DC vaccination. Methods: Patients with HER2pos DCIS (n = 37) or stage I invasive breast cancer (IBC) (n = 10) received neoadjuvant HER2-pulsed DC vaccine prior to surgery. Specimens were examined for residual disease on pathology. CD4 Th1 responses to 6 HER2 Class II peptides (p42-56, p98-114, p328-345, p776-790, p927-941, p1166-1180) were measured using IFN-γ production by ELISPOT. Th1 response metrics were: (1) anti-HER2 responsivity, (2) response repertoire (i.e. no. of reactive peptides), and (3) cumulative response. Th1 responses post-vaccination were compared between pCR (n = 11) and <pCR (n = 36). Results: Forty-three of 47 (91.5%) vaccinated subjects mounted CD4 Th1 responses post-vaccination. Eleven of 47 (23.4%) achieved pCR; no significant difference in pCR rates were observed between IBC and DCIS patients (10% vs 27.0%; p = 0.41). pCR and <pCR patients did not differ by anti-HER2 responsivity (90.9% pCR vs 91.7% 0.05). We previously reported a significant loss of anti-HER2 Th1 responses to p42-56 and p927-941 very early in breast tumorigenesis (i.e. from healthy donors to DCIS ultimately to IBC). In this study, compared to <pCR, pCR patients demonstrated a significantly higher anti-HER2 responsivity (72.7% pCR vs 33.0% <pCR, p = 0.04) and response magnitude (354 vs 110; p = 0.04) to vaccination with p42-56; in addition, there was a trend toward higher response magnitude (318 pCR vs 168 <pCR, p = 0.06) to p927-941 vaccination. No differences were observed in the remaining four peptide responses between pCR and <pCR patients. Conclusion: HER2-pulsed DC vaccination induces pathologic responses in early breast cancer patients, supporting further clinical development. The key immunogenic HER2 class II peptides that appear to drive pCR following DC vaccination are incidentally lost very early in tumorigenesis, suggesting that immune restoration targeting these select peptides may enable control of early breast cancer. Citation Format: Megan E. Fracol, Jashodeep Datta, Shuwen Xu, Lea Lowenfeld, Elizabeth Fitzpatrick, Carolyn Mies, Paul J.L. Zhang, Robert E. Roses, Carla Fisher, Brian J. Czerniecki. HER2 peptide-specific immunogenicity correlates with pathologic response following HER2-pulsed dendritic cell vaccination for early breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2015-248

Multimodality Treatment of T4 Gastric Cancer in the United States: Utilization Trends and Impact on Survival

Background. National guidelines advocate use of multimodality therapy (MMT) for treatment of T4 gastric cancer (T4GC). Prior studies demonstrate poor compliance with these guidelines. We sought to assess treatment trends and association between different treatment approaches and overall survival (OS) in a large cohort of U.S. patients. Methods. Patients diagnosed with clinical T4 gastric adenocarcinoma were selected from the National Cancer Data Base (1998-2011). Temporal trends, risk factors associated with failure to receive treatment, and effect of treatments on OS were assessed. Results. Of 4369 patients with T4GC, only 15 % (n = 652) received MMT. Treatment with MMT increased over time, and was utilized in 25 % of patients after 2006. Older age, African American race, nonprivate insurance, proximal tumor location, and clinical node-negative disease were associated with failure to receive surgery; older age, female sex, poorly differentiated tumor grade, clinical node-negative disease, and prolonged postoperative length of stay were associated with failure to complete MMT in patients who underwent surgical resection. Median OS was longest in patients receiving MMT (19.2 months), and was similarly poor in patients undergoing surgical resection (9.0 months) or nonsurgical therapy (8.3 months; p < 0.001). Median OS was longer in patients receiving neoadjuvant therapy compared to patients receiving adjuvant therapy (27.8 vs. 16.6 months; p = 0.004). Conclusions. Treatment with neoadjuvant MMT is increasing and is associated with prolonged survival. Surgery alone and chemotherapy with or without radiotherapy without resection are associated with similarly poor outcomes. Appropriate treatment sequencing may facilitate delivery of MMT and improve outcomes in patients with T4GC

Multimodality Therapy Improves Survival in Resected Early Stage Gastric Cancer in the United States

National guidelines endorse adjuvant chemotherapy ± radiotherapy (C ± RT) for early-stage gastric cancer (ESGC). Compliance with these guidelines and the specific impact of adjuvant C ± RT on overall survival (OS) in ESGC have not been extensively explored. The National Cancer Data Base was queried for stage IB-II gastric adenocarcinoma patients undergoing gastrectomy (1998-2011). Multivariable modeling identified factors associated with adjuvant C ± RT receipt and compared risk-adjusted OS by treatment type (i.e., adjuvant therapy versus surgery alone). Of 23,461 ESGC patients (1998-2011), 79.4 % and 20.6 % received surgery alone and adjuvant C ± RT (chemoradiotherapy 17.7 %; chemotherapy alone 2.9 %), respectively. Predictors of adjuvant C ± RT receipt included age <67 years, pathologic nodal positivity, and adequate lymph node staging (LNS; ≥15 nodes examined; all p < 0.001). Survival analyses included 15,748 patients (1998-2006); median, 1-, and 5-year survival were 63.5 months, 86.0 %, and 27.0 % respectively. Omission of adjuvant C ± RT conferred an increased hazard of risk-adjusted mortality in the overall cohort, and stage IB and II subgroups (all p ≤ 0.001). The benefit of adjuvant C ± RT was most pronounced in stage II and node-positive patients-regardless of LNS adequacy (all p < 0.001)-and inadequately staged IB patients (p = 0.003). While associated with a trend toward improved OS in node-negative patients overall (p = 0.051), adjuvant C ± RT did not improve OS if surgical LNS was adequate in this subgroup (p = 0.960). Adoption of adjuvant C ± RT in ESGC remains incomplete nationally. Receipt of adjuvant therapy is associated with improved risk-adjusted survival relative to surgery alone; however, in adequately staged patients without lymph node metastasis, this benefit is less certain

Dendritic Cell Vaccination Enhances Immune Responses and Induces Regression of HER2(pos) DCIS Independent of Route: Results of Randomized Selection Design Trial

Purpose: Vaccination with HER2 peptide-pulsed DC1s stimulates a HER2-specific T-cell response. This randomized trial aimed to establish safety and evaluate immune and clinical responses to vaccination via intralesional (IL), intranodal (IN), or both intralesional and intranodal (ILN) injection. Experimental Design: Fifty-four HER2(pos) patients [42 pure ductal carcinoma in situ (DCIS), 12 early invasive breast cancer (IBC)] were enrolled in a neoadjuvant HER2 peptide-pulsed DC1 vaccine trial. Patients were randomized to IL (n = 19), IN (n = 19), or ILN (n = 16) injection. Immune responses were measured in peripheral blood and sentinel lymph nodes by ELISPOT or in vitro sensitization assay. Pathologic response was assessed in resected surgical specimens. Results: Vaccination by all injection routes was well tolerated. There was no significant difference in immune response rates by vaccination route (IL 84.2% vs. IN 89.5% vs. ILN 66.7%; P = 0.30). The pathologic complete response (pCR) rate was higher in DCIS patients compared with IBC patients (28.6% vs. 8.3%). DCIS patients who achieved pCR (n = 12) and who did not achieve pCR (n = 30) had similar peripheral blood anti-HER2 immune responses. All patients who achieved pCR had an anti-HER2 CD4 immune response in the sentinel lymph node, and the quantified response was higher by response repertoire (P = 0.03) and cumulative response (P = 0.04). Conclusions: Anti-HER2 DC1 vaccination is a safe and immunogenic treatment to induce tumor-specific T-cell responses in HER2(pos) patients; immune and clinical responses were similar independent of vaccination route. The immune response in the sentinel lymph nodes, rather than in the peripheral blood, may serve as an endpoint more reflective of antitumor activity. (C) 2016 AACR

Anti-HER2 CD4(+) T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

A progressive loss of circulating anti-human epidermal growth factor receptor-2/neu (HER2) CD4(+) T-helper type 1 (Th1) immune responses is observed in HER2(pos)-invasive breast cancer (IBC) patients relative to healthy controls. Pathologic complete response (pCR) following neoadjuvant trastuzumab and chemotherapy (T + C) is associated with decreased recurrence and improved prognosis. We examined differences in anti-HER2 Th1 responses between pCR and non-pCR patients to identify modifiable immune correlates to pathologic response following neoadjuvant T + C. Anti-HER2 Th1 responses in 87 HER2(pos)-IBC patients were examined using peripheral blood mononuclear cells pulsed with 6 HER2-derived class II peptides via IFN-γ ELISPOT. Th1 response metrics were anti-HER2 responsivity, repertoire (number of reactive peptides), and cumulative response across 6 peptides (spot-forming cells [SFC]/10(6) cells). Anti-HER2 Th1 responses of non-pCR patients (n = 4) receiving adjuvant HER2-pulsed type 1-polarized dendritic cell (DC1) vaccination were analyzed pre- and post-immunization. Depressed anti-HER2 Th1 responses observed in treatment-naïve HER2(pos)-IBC patients (n = 22) did not improve globally in T + C-treated HER2(pos)-IBC patients (n = 65). Compared with adjuvant T + C receipt, neoadjuvant T + C - utilized in 61.5 % - was associated with higher anti-HER2 Th1 repertoire (p = 0.048). While pCR (n = 16) and non-pCR (n = 24) patients did not differ substantially in demographic/clinical characteristics, pCR patients demonstrated dramatically higher anti-HER2 Th1 responsivity (94 % vs. 33 %, p = 0.0002), repertoire (3.3 vs. 0.3 peptides, p < 0.0001), and cumulative response (148.2 vs. 22.4 SFC/10(6), p < 0.0001) versus non-pCR patients. After controlling for potential confounders, anti-HER2 Th1 responsivity remained independently associated with pathologic response (odds ratio 8.82, p = 0.016). This IFN-γ(+) immune disparity was mediated by anti-HER2 CD4(+)T-bet(+)IFN-γ(+) (i.e., Th1) - not CD4(+)GATA-3(+)IFN-γ(+) (i.e., Th2) - phenotypes, and not attributable to non-pCR patients' immune incompetence, host-level T-cell anergy, or increased immunosuppressive populations. In recruited non-pCR patients, anti-HER2 Th1 repertoire (3.7 vs. 0.5, p = 0.014) and cumulative response (192.3 vs. 33.9 SFC/10(6), p = 0.014) improved significantly following HER2-pulsed DC1 vaccination. Anti-HER2 CD4(+) Th1 response is a novel immune correlate to pathologic response following neoadjuvant T + C. In non-pCR patients, depressed Th1 responses are not immunologically "fixed" and can be restored with HER2-directed Th1 immune interventions. In such high-risk patients, combining HER2-targeted therapies with strategies to boost anti-HER2 Th1 immunity may improve outcomes and mitigate recurrence

Addition of anti-estrogen therapy to anti-HER2 dendritic cell vaccination improves regional nodal immune response and pathologic complete response rate in patients with ERpos/HER2(pos) early breast cancer

HER2-directed therapies are less effective in patients with ERpos compared to ERneg breast cancer, possibly reflecting bidirectional activation between HER2 and estrogen signaling pathways. We investigated dual blockade using anti-HER2 vaccination and anti-estrogen therapy in HER2(pos)/ERpos early breast cancer patients. In pre-clinical studies of HER2(pos) breast cancer cell lines, ERpos cells were partially resistant to CD4(+) Th1 cytokine-induced metabolic suppression compared with ERneg cells. The addition of anti-estrogen treatment significantly enhanced cytokine sensitivity in ERpos, but not ERneg, cell lines. In two pooled phase-I clinical trials, patients with HER2(pos) early breast cancer were treated with neoadjuvant anti-HER2 dendritic cell vaccination; HER2(pos)/ERpos patients were treated with or without concurrent anti-estrogen therapy. The anti-HER2 Th1 immune response measured in the peripheral blood significantly increased following vaccination, but was similar across the three treatment groups (ERneg vaccination alone, ERpos vaccination alone, ERpos vaccination + anti-estrogen therapy). In the sentinel lymph nodes, however, the anti-HER2 Th1 immune response was significantly higher in ERpos patients treated with combination anti-HER2 vaccination plus anti-estrogen therapy compared to those treated with anti-HER2 vaccination alone. Similar rates of pathologic complete response (pCR) were observed in vaccinated ERneg patients and vaccinated ERpos patients treated with concurrent anti-estrogen therapy (31.4% vs. 28.6%); both were significantly higher than the pCR rate in vaccinated ERpos patients who did not receive anti-estrogen therapy (4.0%, p = 0.03). Since pCR portends long-term favorable outcomes, these results support additional clinical investigations using HER2-directed vaccines in combination with anti-estrogen treatments for ERpos/HER2(pos) DCIS and invasive breast cancer