Norton, Nadine

Abstract Importance: 20-25% of patients with early stage HER2-positive breast cancer develop tumor relapse after adjuvant trastuzumab. Identification of such patients is a key goal for clinical management decisions. Objective: To assess molecular heterogeneity among early stage HER2-positive patients using the Prosigna™ algorithm, to define intrinsic subtypes, and to determine the clinical significance of such heterogeneity. Design: The NanoString® platform was used to measure the abundance of the PAM50 subtype signature transcripts. Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna™ algorithm to define intrinsic subtype and risk scores. Subtypes were evaluated for recurrence-free survival following chemotherapy with or without trastuzumab. Setting: Samples were obtained from a multi-center randomized phase III trial of chemotherapy versus chemotherapy plus trastuzumab. Participants: All tumors were centrally evaluated for HER2 positivity, defined as IHC 3+ and/or FISH >2.0; 1392 patients were evaluated for molecular subtype. Intervention(s): Patients received adjuvant chemotherapy (doxorubicin plus cyclophosphamide followed by paclitaxel) (n=484) or chemotherapy plus trastuzumab (n=908). Main Outcome Measure(s): The primary outcome was recurrence-free survival as a function of subtype and treatment. Results: Patients with HER2-positive tumors with HER2-enriched features comprised about 70% of the sample cohort, and these individuals received significant benefit from adjuvant trastuzumab (HR=0.68, 95%CI: 0.52, 0.89, p=0.005), as did the relatively fewer patients (291/1392) with Luminal-type tumors (HR=0.52, 95%CI: 0.32, 0.85, p=0.01). The sample cohort contained a small number of patients with tumors having Basal-like features (97/1392), and the data suggest that these individuals may have received less benefit from trastuzumab, beyond that received from chemotherapy alone (HR=1.06, 95%CI:0.53,2.13, p=0.87). Conclusions: The majority of HER2-positive tumors are classified as HER2-enriched or Luminal using the Prosigna algorithm, and patients with such tumors benefit from adjuvant trastuzumab. About 10% of HER2-positive tumors exhibit Basal-like genomic features, and such tumors appear to recur at fairly similar frequency irrespective of treatment with chemotherapy or chemotherapy plus trastuzumab. Patients with HER2-positive/Basal-like tumors may represent a cohort that should be considered for enrollment in trials to evaluate emerging novel HER2-targeted agents, other targeted therapies, or combinations of both approaches. Support provided in part by CA129949 and CA15083. Citation Format: Perez EA, Ballman KV, Mashadi-Hossein A, Tenner KS, Kachergus JM, Norton N, Necela BM, Carr JM, Ferree S, Perou CM, Cheang MCU, Thompson EA. Intrinsic subtype and therapeutic response among early stage HER2-positive breast tumors from the North Central cancer treatment group (Alliance) N9831 trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-04.

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 Introduction: Invasive lobular carcinoma (ILC) comprises approximately ∼10-20% of breast cancers (BC) and appears to have a distinct biology from invasive ductal carcinoma (IDC). ILC is known to have a higher incidence of multifocal (MF) disease when compared to IDC (Biglia N, EJSO, 2013) and in general, MF BC has been associated with an increased rate of regional lymph node metastases (Cabioglu N, JACS, 2009). Treatment decisions are most often defined by the largest of the MF tumors without targeting potentially relevant biologic heterogeneity of the disease. We conducted a genomic analysis of MF ILC to help address this knowledge void. Methods: We characterized gene expression in multiple foci from 11 patients (pts) with MF ILC (all ER+ and HER2-, mean number of foci = 2.62) and 13 benign breast tissues. RNA was extracted from 3×1.5mm cores from each foci and gene expression was measured in 730 known cancer genes using the NanoString PanCancer pathways panel. Differential gene expression analyses were performed between 1) MF tumors and benign tissue (BT) accounting for pairing of pts and 2) different foci of MF tumors from the same ILC pt by ANOVA. The association of clinical covariates (age, number of tumors, grade and stage) in the top genes that were differentially expressed between multiple foci was assessed by linear mixed models accounting for pt and tumor. Results: A total of 20 genes (including MET and EGFR, top 5 genes: SPRY2, SOX17, FIGF, PDGFA, IL6) were differentially expressed (p<0.05) between malignant foci from the same pt, after correction by false discovery rate (FDR). EGFR expression was also negatively associated with the number of positive lymph nodes (N1a vs N0 absolute fold change = 0.51, p = 0.043). We observed an excess of genes that were down-regulated in MF ILC compared to BT. 277 genes were differentially expressed at p<0.05 after correction by FDR. The top 5 differentially expressed genes (by fold change) between ILC and BT were COL11A1, COL1A1, WIF1, FIGF and PROM1. CDH1, a known marker of ILC, was down-regulated in ILC compared to benign (absolute fold change = 2.54, p = 6.02×10−6), ranked only 39/277 genes by fold change. MET and EGFR ranked 29 and 42/277 genes (p = 5.99×10−9 and 3.32×10−8 respectively). Conclusion: We observed significant heterogeneity for 20/730 genes between individual foci of MF ILC within the same pt suggesting that MF synchronous ILC lesions may have different biologic behavior. This in turn may have important prognostic implications with potential therapeutic utility. Our analyses also shows significant down-regulation of gene expression between ILC tumors and BT for many genes other than CDH1 suggesting that additional biomarkers can be used to further characterize ILC Study funded in part by Mayo Clinic Cancer Focus Research Team, the 26.2 with Donna Foundation and the Serene M. and Frances C. Durling endowment. Citation Format: Pooja Advani, Nadine Norton, Daniel J. Serie, Xochiquetzal Geiger, Julia E. Crook, Aubrey E. Thompson, Edith A. Perez, Alvaro Moreno Aspitia. Characterization of individual foci of multifocal invasive lobular breast cancer using gene expression profiling. [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 4761. doi:10.1158/1538-7445.AM2015-4761

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.