Pegram, Mark D.

Abstract The addition of trastuzumab to chemotherapy extends survival among patients with HER2+ breast cancer. Prior work showed that trastuzumab and chemotherapy augments HER2 extracellular domain (ECD)-specific antibodies. The current study investigated whether combination therapy induced immune responses beyond HER2-ECD and, importantly, whether those immune responses were associated with survival. Pretreatment and posttreatment sera were obtained from 48 women with metastatic HER2+ breast cancer on NCCTG (now Alliance for Clinical Trials in Oncology) studies, N0337 and N983252. IgG to HER2 intracellular domain (ICD), HER2-ECD, p53, IGFBP2, CEA, and tetanus toxoid were examined. Sera from 25 age-matched controls and 26 surgically resected HER2+ patients were also examined. Prior to therapy, some patients with metastatic disease had elevated antibodies to IGFBP2, p53, HER2-ICD, HER2-ECD, and CEA, but not to tetanus toxin, relative to controls and surgically resected patients. Treatment augmented antibody responses to HER2-ICD in 69% of metastatic patients, which was highly associated with improved progression-free survival (PFS; HR = 0.5, P = 0.0042) and overall survival (OS; HR = 0.7, P = 0.038). Augmented antibody responses to HER2-ICD also correlated (P = 0.03) with increased antibody responses to CEA, IGFBP2, and p53, indicating that treatment induces epitope spreading. Paradoxically, patients who already had high preexisting immunity to HER2-ICD did not respond to therapy with increased antibodies to HER2-ICD and demonstrated poorer PFS (HR = 1.6, P < 0.0001) and OS (HR = 1.4, P = 0.0006). Overall, the findings further demonstrate the importance of the adaptive immune system in the efficacy of trastuzumab-containing regimens. Cancer Res; 76(13); 3702–10. ©2016 AACR.

Abstract Background: In the adjuvant setting, HER2 breast cancer patients treated with trastuzumab along with chemotherapy have superior survival compared to patients treated with chemotherapy alone (Perez EA, et al. J Clin Oncol. 2013 31:2115-22). Functional evidence suggests a role for the FC gamma receptor genes in antibody response. Mice deficient in the FC gamma receptor genes show significantly reduced antitumor effect. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab and a polymorphism in FCGR2B (I232T, T allele) is associated with impaired negative regulatory activity. Association of FC gamma receptor polymorphisms with clinical response among trastuzumab-treated patients is equivocal with both positive (Musolino A, et al. J Clin Oncol. 2008 26:1789-96) and negative associations (Hurvitz SA, et al. Clin Cancer Res. 2012 18:3478-86), a lack of sufficiently powered clinical samples and a lack of cohorts with clearly demonstrated survival benefit to trastuzumab containing regimens. Methods: Genomic DNA was analyzed for FcyRIIIa, FcyRIIa, and FcyRIIb polymorphisms (FCGR3A V158F, FCGR2A H131R and FCGR2B I232T) using Taqman SNP real-time PCR assays (Applied Biosystems) in 1,336 patients from the N9831 clinical trial of adjuvant trastuzumab in HER2+ breast cancer. 1325 patients were evaluable. Patients in Arm A (N=419) received chemotherapy only. Patients in Arms B (N=469) and C (N= 437) were treated with chemotherapy and trastuzumab (sequentially in Arm B and concurrently in Arm C). We performed Kaplan-Meier analyses of disease free survival (DFS) by arm and genotype for each polymorphism. Results: There were only minor differences in demographics and tumor features when comparing the subset with DNA to other N9831 participants who did not provide DNA. Patients who provided DNA in Arms B and C demonstrated superior DFS relative to patients in Arm A who provided DNA (p<0.001). Genotype distributions in the N9831 population for FCGR2A and FCGR3A polymorphisms were in Hardy-Weinberg equilibrium, p>0.05 and FCGR2B, p>0.01. In contrast to previous reports in the metastatic setting, we did not find that trastuzumab-treated patients who had the FCGR3A 158 V and/or FCGR2A 131 H genotypes demonstrated superior DFS to those without those genotypes. Similarly, we did not see an effect of FCGR2B I232T with the exception that I/I patients (p<0.001) apparently derived more benefit, in terms of DFS, from trastuzumab than did T carriers (p=0.81). However, 5-year DFS was not different between these two groups, I/I and T carriers within Arms B/C. Conclusions: Our analysis failed to reveal strong associations between FCGR genotypes and outcome in patients with HER2+ breast cancer treated with trastuzumab. While the present data do not preclude a functional role for FC gamma receptors in the clinical activity of trastuzumab, the results suggest that these naturally occurring polymorphisms may have minor (if any) impact in the adjuvant setting, which is consistent with previous findings by Hurvitz and colleagues. Citation Format: Mark Pegram, Rebecca M. Olson, Nadine Norton, Kathleen Tenner, Karla L. Ballman, Raphael Clynes, Keith L. Knutson, Edith A. Perez. Analysis of the associations of FC gamma receptor polymorphisms (FCGR3A, FCGR2A, and FCGR2B) with outcome in HER2+ breast cancer patients treated with trastuzumab in NCCTG (Alliance) trial N9831. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B123.

Abstract Patients with HER2+ breast cancer treated with trastuzumab and chemotherapy have superior survival compared with patients treated with chemotherapy alone. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab, and a polymorphism in FCGR2B (I232T) is associated with impaired regulatory activity. The association of these polymorphisms with clinical response among trastuzumab-treated patients is equivocal, with both positive and negative associations. We performed genotyping analysis on the FCGR3A V158F, FCGR2A R131H, and FCGR2B I232T polymorphisms in 1,325 patients from the N9831 clinical trial. Patients in arm A (N = 419) received chemotherapy only. Patients in arms B (N = 469) and C (N = 437) were treated with chemotherapy and trastuzumab (sequentially in arm B and concurrently in arm C). Using log-rank test and Cox proportional hazard models, we compared disease-free survival (DFS) among genotypic groups within pooled arms B/C. We found no differences in DFS between trastuzumab-treated patients who had the FCGR3A 158 V/V and/or FCGR2A 131 H/H high-affinity genotypes and patients without those genotypes. Furthermore, there was no significant interaction between FCGR3A and FCGR2A and treatment. However, there was a difference in DFS for FCGR2B I232T, with I/I patients deriving benefit from trastuzumab (P < 0.001), compared with the T carriers who did not (P = 0.81). The interaction between FCGR2B genotype and treatment was statistically significant (P = 0.03). Our analysis did not reveal an association between FcγR high-affinity genotypes and outcomes. However, it seems that the FCGR2B inhibitory gene may be predictive of adjuvant trastuzumab benefit. Cancer Immunol Res; 2(10); 962–9. ©2014 AACR.

AbstractPurpose: Passive immunotherapy with antitumor antibodies has the potential to induce active tumor immunity via the opsonic enhancement of immunogenicity of tumor antigen. We have assessed whether immune sensitization to the HER-2/neu tumor antigen occurs during treatment with the anti-HER-2/neu monoclonal antibody trastuzumab.Experimental Design: Twenty-seven patients treated with trastuzumab and chemotherapy were assessed for the induction of HER-2/neu–specific immunity. Sera and peripheral blood mononuclear cells obtained before and after trastuzumab therapy were compared for the presence of anti-HER-2/neu endogenous Igλ antibodies and HER-2/neu–specific CD4 responses by ELISA and enzyme-linked immunospot, respectively.Results: Anti-HER-2/neu antibodies were detectable in 8 of 27 (29%) patients before trastuzumab treatment and in 15 of 27 (56%) patients during trastuzumab treatment. In the overall study population, anti-HER-2/neu humoral responses significantly increased during therapy (P < 0.001) and were not associated with development of an anti-idiotypic response. In 10 evaluable individuals, 6 showed augmented HER-2/neu–specific CD4 T-cell responses during therapy. Of the 22 individuals treated for metastatic disease, those patients showing objective clinical responses exhibited more frequent (P = 0.004) and larger (P = 0.006) treatment-associated anti-HER-2/neu humoral responses.Conclusion: Humoral immune sensitization occurs during treatment with chemotherapy and trastuzumab. Further studies are warranted to investigate whether augmented anti-HER-2/neu humoral and cellular immunity contributes mechanistically to clinical outcome.

Abstract Lapatinib (GW572016) is a selective inhibitor of both epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases. Here, we explore the therapeutic potential of lapatinib by testing its effect on tumor cell growth in a panel of 31 characterized human breast cancer cell lines, including trastuzumab-conditioned HER-2-positive cell lines. We further characterize its activity in combination with trastuzumab and analyze whether EGFR and HER-2 expression or changes induced in the activation of EGFR, HER-2, Raf, AKT, or extracellular signal-regulated kinase (ERK) are markers of drug activity. We report that concentration-dependent antiproliferative effects of lapatinib were seen in all breast cancer cell lines tested but varied significantly between individual cell lines with up to 1,000-fold difference in the IC50s (range, 0.010-18.6 μmol/L). Response to lapatinib was significantly correlated with HER-2 expression and its ability to inhibit HER-2, Raf, AKT, and ERK phosphorylation. Long-term in vivo lapatinib studies were conducted with human breast cancer xenografts in athymic mice. Treatment over 77 days resulted in a sustained and significant reduction in xenograft volume compared with untreated controls. For the combination of lapatinib plus trastuzumab, synergistic drug interactions were observed in four different HER-2-overexpressing cell lines. Moreover, lapatinib retained significant in vitro activity against cell lines selected for long-term outgrowth (>9 months) in trastuzumab-containing (100 μg/mL) culture medium. These observations provide a clear biological rationale to test lapatinib as a single agent or in combination with trastuzumab in HER-2-overexpressing breast cancer and in patients with clinical resistance to trastuzumab. (Cancer Res 2006; 66(3): 1630-9)