Tenner, Kathleen

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.

Purpose To develop a genomic signature that predicts benefit from trastuzumab in human epidermal growth factor receptor 2–positive breast cancer. Patients and Methods DASL technology was used to quantify mRNA in samples from 1,282 patients enrolled onto the Combination Chemotherapy With or Without Trastuzumab in Treating Women With Breast Cancer (North Central Cancer Treatment Group N9831 [NCCTG-N9831]) adjuvant trastuzumab trial. Cox proportional hazard ratios (HRs), adjusted for significant clinicopathologic risk factors, were used to determine the association of each gene with relapse-free survival (RFS) for 433 patients who received chemotherapy alone (arm A) and 849 patients who received chemotherapy plus trastuzumab (arms B and C). Network and pathway analyses were used to identify key biologic processes linked to RFS. The signature was built by using a voting scheme. Results Network and functional ontology analyses suggested that increased RFS was linked to a subset of immune function genes. A voting scheme model was used to define immune gene enrichment based on the expression of any nine or more of 14 immune function genes at or above the 0.40 quantile for the population. This model was used to identify immune gene–enriched tumors in arm A and arms B and C. Immune gene enrichment was linked to increased RFS in arms B and C (HR, 0.35; 95% CI, 0.22 to 0.55; P < .001), whereas arm B and C patients who did not exhibit immune gene enrichment did not benefit from trastuzumab (HR, 0.89; 95% CI, 0.62 to 1.28; P = .53). Enriched immune function gene expression as defined by our predictive signature was not associated with increased RFS in arm A (HR, 0.90; 95% CI, 0.60 to 1.37; P = .64). Conclusion Increased expression of a subset of immune function genes may provide a means of predicting benefit from adjuvant trastuzumab.

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 &lt; 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.

Abstract Purpose: This study investigated the association between tumor MYC protein expression and disease-free survival (DFS) of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the N9831 (Alliance) adjuvant HER2+ trastuzumab breast cancer trial. Experimental Design: This analysis included 1,736 patients randomized to Arms A, B, and C on N9831. Nuclear MYC protein expression was determined in tissue microarray sections containing three biopsies per patient or whole tissue sections using standard immunohistochemistry (clone 9E10). A tumor was considered positive for MYC protein overexpression (MYC+) if the nuclear 3+ staining percentage was more than 30%. Results: Five hundred and seventy-four (33%) tumors were MYC+. MYC+ was associated with hormone receptor positivity (χ2, P = 0.006), tumors 2 cm or more (χ2, P = 0.02), and a higher rate of nodal positivity (χ2, P &lt; 0.001). HRs for DFS (median follow-up: 6.1 years) for Arm C versus A were 0.52 (P = 0.006) and 0.65 (P = 0.006) for patients with MYC+ and MYC− tumors, respectively (Pinteraction = 0.40). For Arm B versus A, HRs for patients with MYC+ and MYC− tumors were 0.79 (P = 0.21) and 0.74 (P = 0.04), respectively (Pinteraction = 0.71). For Arm C versus B, HRs for patients with MYC+ and MYC− tumors were 0.56 (P = 0.02) and 0.89 (P = 0.49), respectively (Pinteraction = 0.17). Conclusions: Our data do not support an impact of tumor MYC protein expression on differential benefit from adjuvant trastuzumab. Clin Cancer Res; 19(20); 5798–807. ©2013 AACR.

BACKGROUNDIncreased soluble human epidermal growth factor receptor 2 (sHER2) is an indicator of a poor prognosis in HER2‐positive metastatic breast cancer. In this study, the authors evaluated levels of sHER2 during treatment and at the time of disease recurrence in the adjuvant North Central Cancer Treatment Group N9831 clinical trial.METHODSThe objectives were to describe sHER2 levels during treatment and at the time of recurrence in patients who were randomized to treatment arms A (standard chemotherapy), B (standard chemotherapy with sequential trastuzumab), and C (standard chemotherapy with concurrent trastuzumab). Baseline samples were available from 2318 patients, serial samples were available from 105 patients, and recurrence samples were available from 124 patients. The cutoff sHER2 value for the assay was 15 ng/mL. Statistical methods included repeated measures linear models, Wilcoxon rank‐sum tests, and Cox regression models.RESULTSThere were differences between groups in terms of age, menopausal status, and hormone receptor status. Within treatment arms A, B, and C, patients who had baseline sHER2 levels ≥15 ng/mL had worse disease‐free survival than patients who had baseline sHER2 levels <15 ng/mL (arm A: hazard ratio, 1.81; P = .0014; arm B: hazard ratio, 2.08; P = .0015; arm C: hazard ratio, 1.96; P = .01). Among the 124 patients who experienced disease recurrence, sHER2 levels increased from baseline to the time of recurrence in arms A and B but remained unchanged in arm C. Patients who had recurrence sHER2 levels ≥15 ng/mL had a shorter survival after recurrence with a 3‐year overall survival rate of 51% compared with 77% for those who had recurrence sHER2 levels <15 ng/mL (hazard ratio, 2.36; 95% confidence interval, 1.19‐4.70; P = .01).CONCLUSIONSIn patients with early stage, HER2‐positive breast cancer, a high baseline sHER2 level was identified as a prognostic marker associated with shorter disease‐free survival, and a high sHER2 level at recurrence was predictive of shorter survival. Cancer 2013;119:2675–2682. © 2013 American Cancer Society.

Abstract Purpose: To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). Experimental Design: Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to β2-microglobulin and calibrated to healthy blood using the 2–ΔΔCq algorithm; positivity was defined as 2 or more. Results: CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). Conclusions: CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients. Clin Cancer Res; 17(22); 7183–93. ©2011 AACR.

Abstract Background: In 2007, the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) recommended new guidelines to define HER2 positivity by immunohistochemistry (3+ IHC: uniform intense membrane staining of &gt;30% of invasive tumor cells) or fluorescent in situ hybridization (FISH+: HER2/CEP17 ratio &gt;2.2). The original criteria used for enrollment in the pivotal N9831 trial conducted from 2000-2005 were IHC&gt;10% (3+) or FISH ≥2.0.Purpose: To investigate the impact of the ASCO/CAP guidelines on patient (pt) eligibility and disease free survival (DFS) compared to the originally used FDA-approved definitions.Patients/Methods: This analysis included 2268 patients from the HER2+ N9831 adjuvant trastuzumab phase III trial. IHC was centrally performed at the Mayo Clinic using the DAKO HercepTest™ and was re-analyzed to determine the percent of tumor cells with 0, 1+, 2+, and 3+ staining intensities. DFS was compared between pts randomized to standard chemotherapy +/- concurrent trastuzumab (Arms A and C of the trial) within IHC/FISH subgroups using Cox proportional hazards regression stratified by hormone receptor and nodal status.Results: Based on 2268 pts, 83 pts (3.7%) were 3+ by FDA-approved guidelines but not 3+ by the ASCO/CAP guidelines. Among these pts, 77% (64/83) were FISH amplified centrally by ASCO/CAP guidelines. Only 1.5% (34/2268) of pts eligible under FDA-approved definitions from N9831 in this sample set would not meet the ASCO/CAP guidelines. In 1441 patients randomized to standard chemotherapy +/- concurrent trastuzumab, the exploratory DFS hazard ratio (HR) associated with trastuzumab in IHC/FISH subgroups were as follows: ASCO/CAP HER2+ HR=0.59 (95% CI: 0.47-0.75; p&lt;0.001); FDA-approved HER2+ HR=0.60 (95% CI: 0.47-0.75; p&lt;0.001); ASCO/CAP HER2 normal(not positive) HR=0.74 (95% CI: 0.35-1.57; p=0.43); and FDA-approved HER2 normal HR=0.53 (95% CI: 0.22-1.26; p=0.15).Conclusions: A small percentage (1.5%) of N9831 patients did not meet ASCO/CAP 2007 guidelines when applied retrospectively. Trastuzumab effect appeared similar for HER2+ patients regardless of ASCO/CAP or FDA-approved guidelines. Additional DFS analyses by IHC/FISH subgroups will be presented. We gratefully acknowledge support from the Breast Cancer Research Foundation, Genentech, and NIH CA25224 for this study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 701.

Abstract Background: Microtubules are crucial for spindle formation during mitosis and for cellular proliferation. The antineoplastic effect of paclitaxel is mainly related to its ability to bind the beta subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. Tubulins are expressed in human peripheral nerves and the binding of paclitaxel to tubulin may lead to neuropathy. Peripheral neuropathy is a common dose limiting toxicity of paclitaxel. We hypothesized that the occurrence of peripheral neuropathy may correlate with outcome (disease-free survival; DFS).Methods: This analysis sought to describe incidence of peripheral neuropathy following paclitaxel and its association to outcome (DFS) in patients who received paclitaxel (weekly x 12) in the adjuvant HER2+ intergroup trial N9831. Only eligible pts who initiated paclitaxel and did not have peripheral neuropathy at initiation of paclitaxel that were randomized to arms A (955 pts; chemotherapy alone) and C (889 pts; chemotherapy plus concurrent trastuzumab) of N9831 were included. Cox regression analysis stratified by ER/PR status and nodal status was used to compare DFS within arm between patients with and without peripheral neuropathy.Results: Out of 1844 eligible pts, 379 developed neuropathy (20.5%). For pts in arm A, those who developed neuropathy had better DFS than pts who did not (3 yr DFS: 86.2% vs 81.8%; HR 0.65; p=0.01), despite lower doses of paclitaxel in the pts with neuropathy. Grade of neuropathy did not appear to impact DFS. No statistical difference was noted for pts treated in the trastuzumab-containing arm (3 yr DFS: 92.8% vs 91.1% for pts with neuropathy vs not; HR 0.79; p=0.34). There were no differences in paclitaxel dose intensity between arms A and C.Conclusion: Patients with early stage HER2+ breast cancer who received adjuvant paclitaxel-containing chemotherapy in arm A and developed peripheral neuropathy had a better DFS than pts who did not develop neuropathy. This effect was possibly abrogated by the use of trastuzumab in Arm C. This side effect may represent effective bindings of paclitaxel to the target tubulin, lack of point mutations in tubulin at the paclitaxel binding site and/or lack of selective overexpression of β-III tubulin. This is a hypothesis generating study and additional analysis needs to be conducted from other large taxane-based trials.Partial support from Genentech and the Breast Cancer Research Foundation Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2100.

Purpose We examined associations between tumor characteristics (human epidermal growth factor receptor 2 [HER2] protein expression, HER2 gene and chromosome 17 copy number, hormone receptor status) and disease-free survival (DFS) of patients in the N9831 adjuvant trastuzumab trial. Patients and Methods All patients (N = 1,888) underwent chemotherapy with doxorubicin and cyclophosphamide, followed by weekly paclitaxel with or without concurrent trastuzumab. HER2 status was determined by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) at a central laboratory, Mayo Clinic, Rochester, MN. Patients with conflicting local positive HER2 expression results but normal central laboratory testing were included in the analyses (n = 103). Results Patients with HER2-positive tumors (IHC 3+, FISH HER2/centromere 17 ratio ≥ 2.0, or both) benefited from trastuzumab, with hazard ratios (HRs) of 0.46, 0.49, and 0.45, respectively (all P < .0001). Patients with HER2-amplified tumors with polysomic (p17) or normal (n17) chromosome 17 copy number also benefited from trastuzumab, with HRs of 0.52 and 0.37, respectively (P < .006). Patients who received chemotherapy alone and had HER2-amplified and p17 tumors had a longer DFS than those who had n17 (78% v 68%; P = .04), irrespective of hormone receptor status or tumor grade. Patients with HER2-normal tumors by central testing (n = 103) seemed to benefit from trastuzumab, but the difference was not statistically significant (HR, 0.51; P = .14). Patients with hormone receptor–positive or –negative tumors benefited from the addition of trastuzumab, with HRs of 0.42 (P = .005) and 0.60 (P = .0001), respectively. Conclusion These results confirm that IHC or FISH HER2 testing is appropriate for patient selection for adjuvant trastuzumab therapy. Trastuzumab benefit seemed independent of HER2/centromere 17 ratio and chromosome 17 copy number.