Flow cytometry MFI and % positive cells data for fluorescently labelled trastuzumab and pertuzumab in SKBR3, HCC1954, T47D and MCF-7 treated with TKIs

Additional supporting information for methods outlined in main manuscript

Flow cytometry control scatter plots for trastuzumab-488 and pertuzumab-550

MFI comparison at T0 and T6 for T47D/MCF-7

Densitometry analysis for Figure 6D/E

Background: ExteNET showed that neratinib, an irreversible pan-HER tyrosine ki-nase inhibitor, given for 1 year after trastuzumab-based therapy significantly improved inva-sive disease-free survival in women with early-stage HER2-positive breast cancer. We report the final analysis of overall survival in ExteNET.Methods: In this international, randomised, double-blind, placebo-controlled, phase 3 trial, women aged 18 years or older with stage 1-3c (amended to stage 2-3c) HER2-positive breast cancer who had completed neoadjuvant and adjuvant chemotherapy plus trastuzumab were eligible. Patients were randomly assigned to oral neratinib 240 mg/day or placebo for 1 year. Randomisation was stratified according to hormone receptor (HR) status (HR-positive vs. HR-negative), nodal status (0, 1-3 or 4 thorn ), and trastuzumab regimen (sequentially vs. concur-rently with chemotherapy). Overall survival was analysed by intention to treat. ExteNET is registered (Clinicaltrials.gov: NCT00878709) and is complete.Results: Between July 9, 2009, and October 24, 2011, 2840 women received neratinib (n = 1420) or placebo (n = 1420). After a median follow-up of 8.1 (IQR, 7.0-8.8) years, 127 patients (8.9%) in the neratinib group and 137 patients (9.6%) in the placebo group in the intention-to-treat population had died. Eight-year overall survival rates were 90.1% (95% CI 88.3-91.6) with neratinib and 90.2% (95% CI 88.4-91.7) with placebo (stratified haz-ard ratio 0.95; 95% CI 0.75-1.21; p = 0.6914).Conclusions: Overall survival in the extended adjuvant setting was comparable for neratinib and placebo after a median follow-up of 8.1 years in women with early-stage HER2-positive breast cancer.(c) 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contains supplementary tables and methods

Densitometry analysis for Figure 6B

Rituximab ADCC negative control

All ADCC ratios from Figure 3 and trastuzumab/pertuzumab ADCC comparison

Neratinib (Nerlynx®) is an irreversible pan-HER inhibitor that significantly improves invasive disease-free survival (iDFS) vs placebo as extended adjuvant therapy in HER2-positive (HER2+) early-stage breast cancer (eBC) after trastuzumab-based therapy. The phase III ExteNET trial (NCT00878709) showed an absolute iDFS benefit of 2.5% and distant disease-free survival benefit of 1.7% with neratinib after 5 years’ follow-up. The brain is a common first site of metastasis after HER2-directed adjuvant therapy for eBC. To date, no HER2-directed therapies have prevented central nervous system (CNS) metastases in this setting. Women with HER2+ eBC who had completed adjuvant therapy (± neoadjuvant therapy) with trastuzumab + chemotherapy were randomized to oral neratinib 240 mg/d or placebo for 1y. OS was analyzed after 248 events (powered for the ITT population). CNS outcomes: cumulative incidence of CNS recurrences (time from randomization to CNS recurrence as first distant recurrence); CNS disease-free survival (CNS-DFS, time from randomization to any CNS recurrence or death from any cause). Cut-off dates: 5y (Mar 2017); OS (Jul 2019). 2840 patients were randomized (1420 per group). After a median follow-up of 8.1y, 8y OS rates were 90.1% (95% CI 88.3‒91.6) for neratinib and 90.2% (95% CI 88.4‒91.7) for placebo (absolute difference –0.1%; stratified HR=0.95; 95% CI 0.75‒1.21; p=0.6914). At 5y, cumulative incidence of CNS recurrences was 1.3% (95% CI 0.8–2.1) with neratinib and 1.8% (95% CI 1.2–2.7) with placebo, and CNS-DFS rates were 97.5% (95% CI 96.4–98.3) and 96.4% (95% CI 95.2–97.4), respectively (HR=0.73; 95% CI 0.45–1.17). No new safety signals were reported. There were fewer deaths with neratinib than placebo in ExteNET, but the results did not reach statistical significance. Long-term CNS outcomes at 5y were also improved with neratinib. Neratinib is the first HER2-directed agent to show a trend towards improved CNS outcomes in HER2+ eBC, providing further support for current NCCN guidelines that recommend neratinib-based therapy for brain metastases from HER2-positive breast cancer.

Progression-free survival by subgroup in (A) cohort 1 and (B) cohort 2 HR based on unstratified Cox regression model with <1 favoring enzalutamide arm. Abbreviations: ECOG PS, Eastern Cooperative Oncology Group Performance Status; HR, hazard ratio.

ExteNET, an international, randomized, placebo-controlled phase III trial, showed that extended adjuvant neratinib given for 12 months after trastuzumab-based adjuvant therapy significantly improved 2-year (HR=0.50, p=0.003) and 5-year (HR=0.58, p=0.002) invasive disease-free survival (iDFS) in early-stage HER2+ and hormone-receptor positive (HR+) breast cancer. We explored the impact of treatment duration on outcomes in the ITT population and in the subgroup of patients (pts) with HR+ disease who started neratinib <1 year after prior trastuzumab (EU label population). Pts with early-stage HER2+ breast cancer received oral neratinib 240 mg/day or placebo for 12 months (or until disease recurrence or unacceptable toxicity) after trastuzumab-based adjuvant therapy. Pts who received neratinib for ≤3 or ≥11 months (including those who had recurrence prior to 11 months) were compared with the ITT placebo group. iDFS (primary endpoint) and secondary endpoints (DCIS, DDFS, time to distant recurrence, and 5-year CNS recurrence) were analyzed using Kaplan-Meier methods and Cox proportional-hazards models adjusted for prognostic factors. Data cut-off: March 1, 2017. There were 2840 pts in the ITT population and 1334 patients who were HR+, <1-year. The table shows iDFS findings for HR+, <1-year pts and stratified by treatment duration (≤3, ≥11 months). Greater benefit was seen in pts who stayed on treatment for ≥11 months when compared to pts who received ≤3 months of treatment. Similar findings were seen for secondary endpoints.TableDurationN5-year iDFS rate, %NeratinibPlaceboNeratinibPlaceboDifferenceHR (95% CI)EU label population67066490.885.7+ 5.1%0.58 (0.41-0.82)≤3 months20166485.985.7+ 0.2%0.88 (0.51-1.42)≥11 months40266493.185.7+ 7.4%0.44 (0.28-0.68) Open table in a new tab These exploratory data suggest that pts who received a longer duration of treatment with neratinib (≥11 months) derived greater benefit compared to those who stopped treatment early (≤3 months). Patients who receive recommended duration of treatment with neratinib of 12 months may have improved outcomes when compared to patients who discontinue early (within 3 months).

Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that inhibits PI3K/Akt and MAPK signaling pathways after HER2 receptor activation. The ExteNET study showed that neratinib significantly improved 5-year invasive disease-free survival (iDFS) in women who completed trastuzumab-based adjuvant therapy for early breast cancer (EBC). We assessed the prognostic and predictive significance of PIK3CA alterations in patients in ExteNET. Participants were women aged ≥ 18 years (≥ 20 years in Japan) with stage 1–3c (modified to stage 2–3c in February 2010) operable breast cancer, who had completed (neo)adjuvant chemotherapy plus trastuzumab ≤ 2 years before randomization, with no evidence of disease recurrence or metastatic disease at study entry. Patients were randomized to oral neratinib 240 mg/day or placebo for 1 year. Formalin-fixed, paraffin-embedded primary tumor specimens underwent polymerase chain reaction (PCR) PIK3CA testing for two hotspot mutations in exon 9, one hot-spot mutation in exon 20, and fluorescence in situ hybridization (FISH) analysis for PIK3CA amplification. The primary endpoint (iDFS) was tested with log-rank test and hazard ratios (HRs) estimated using Cox proportional-hazards models. Among the intent-to-treat population (n = 2840), tumor specimens were available for PCR testing (991 patients) and PIK3CA FISH (702 patients). Overall, 262 samples were PIK3CA altered: 201 were mutated (77%), 52 (20%) were amplified, and 9 (3%) were mutated and amplified. iDFS was non-significantly worse in placebo-treated patients with altered vs wild-type PIK3CA (HR 1.34; 95% CI 0.72–2.50; P = 0.357). Neratinib’s effect over placebo was significant in patients with PIK3CA-altered tumors (HR 0.41; 95% CI 0.17–0.90, P = 0.028) but not PIK3CA wild-type tumors (HR 0.72; 95% CI 0.36–1.41; P = 0.34). The interaction test was non-significant (P = 0.309). Although there was a greater absolute risk reduction associated with neratinib treatment of patients with PIK3CA-altered tumors in ExteNET, current data do not support PIK3CA alteration as a predictive biomarker of response to neratinib in HER2-positive EBC. ClinicalTrials.gov , NCT00878709 . Trial registered April 9, 2009.

Background: We report longitudinal health-related quality-of-life (HRQoL) data from the international, randomized, double-blind, placebo-controlled phase III ExteNET study, which demonstrated an invasive disease-free survival benefit of extended adjuvant therapy with neratinib over placebo in human epidermal growth factor receptor-2-positive early-stage breast cancer. Patients and methods: Women (N = 2840) with early-stage HER2-positive breast cancer who had completed trastuzumab-based adjuvant therapy were randomly assigned to neratinib 240 mg/day or placebo for 12 months. HRQoL was an exploratory end point. Patients completed the Functional Assessment of Cancer Therapy-Breast (FACT-B) and EuroQol 5-Dimensions (EQ-5D) questionnaires at baseline and months 1, 3, 6, 9, and 12. Changes from baseline were compared using analysis of covariance with no imputation for missing values. Sensitivity analyses used alternative methods. Changes in HRQoL scores were regarded as clinically meaningful if they exceeded previously reported important differences (IDs). Results: Of the 2840 patients (intention-to-treat population), 2407 patients were evaluable for FACT-B (neratinib, N = 1171; placebo, N = 1236) and 2427 patients for EQ-5D (neratinib, N = 1186; placebo, N = 1241). Questionnaire completion rates exceeded 85%. Neratinib was associated with a decrease in global HRQoL scores at month 1 compared with placebo (adjusted mean differences: FACT-B total, -2.9 points; EQ-5D index, -0.02), after which between-group differences diminished at later time-points. Except for the FACT-B physical well-being (PWB) subscale at month 1; all between-group differences were less than reported IDs. The FACT-B breast cancer-specific subscale showed small improvements with neratinib at months 3-9, but all were less than IDs. Sensitivity analyses exploring missing data did not change the results. Conclusions: Extended adjuvant neratinib was associated with a transient, reversible decrease in HRQoL during the first month of treatment, possibly linked to treatment-related diarrhea. With the exception of the PWB subscale at month 1, all neratinib-related HRQoL changes did not reach clinically meaningful thresholds.

AbstractPurpose: Addition of alpelisib to fulvestrant significantly extended progression-free survival in PIK3CA-mutant, hormone receptor–positive (HR+) advanced/metastatic breast cancer in the phase III SOLAR-1 study. The combination of alpelisib and letrozole also had promising activity in phase I studies of HR+ advanced/metastatic breast cancer. NEO-ORB aimed to determine whether addition of alpelisib to letrozole could increase response rates in the neoadjuvant setting. Patients and Methods: Postmenopausal women with HR+, human epidermal growth factor receptor 2-negative, T1c-T3 breast cancer were assigned to the PIK3CA-wild-type or PIK3CA-mutant cohort according to their tumor PIK3CA status, and randomized (1:1) to 2.5 mg/day letrozole with 300 mg/day alpelisib or placebo for 24 weeks. Primary endpoints were objective response rate (ORR) and pathologic complete response (pCR) rate for both PIK3CA cohorts. Results: In total, 257 patients were assigned to letrozole plus alpelisib (131 patients) or placebo (126 patients). Grade ≥3 adverse events (≥5% of patients) in the alpelisib arm were hyperglycemia (27%), rash (12%), and maculo-papular rash (8%). The primary objective was not met; ORR in the alpelisib versus placebo arm was 43% versus 45% and 63% versus 61% in the PIK3CA-mutant and wild-type cohorts, respectively. pCR rates were low in all groups. Decreases in Ki-67 were similar across treatment arms and cohorts. In PIK3CA-mutant tumors, alpelisib plus letrozole treatment induced a greater decrease in phosphorylated AKT versus placebo plus letrozole. Conclusions: In contrast to initial results in advanced/metastatic disease, addition of alpelisib to 24-week neoadjuvant letrozole treatment did not improve response in patients with HR+ early breast cancer.

Abstract Background: The international, randomized, placebo-controlled phase III ExteNET trial showed that 1 year (yr) of neratinib 240 mg/day after trastuzumab-based adjuvant therapy significantly improved invasive disease-free survival (iDFS) in 2840 patients with early-stage HER2+ breast cancer at 2 yr (hazard ratio 0.67; 95% CI 0.50–0.91; p=0.009) [Chan 2016] and 5 yr (hazard ratio 0.73; 95% CI 0.57-0.92; p=0.008) [Martin 2017]. A prespecified subgroup analysis by hormone receptor (HR) status suggested enhanced efficacy with neratinib in patients with HR+ (2-yr hazard ratio 0.51; 95% CI 0.33–0.77) vs. HR– tumors (2-yr hazard ratio 0.93; 95% CI 0.60–1.43). The efficacy of neratinib was also greater in patients who initiated treatment within 1 yr of prior trastuzumab compared with those who started neratinib later. The European Medicines Agency's Committee for Medicinal Products for Human Use recently recommended neratinib for use in patients with HR+ tumors who initiate treatment within 1 yr of completing trastuzumab-based adjuvant therapy. Subgroup analyses from ExteNET examining iDFS benefits in this patient population are presented here. Methods: Patients with early-stage HER2+ breast cancer who completed trastuzumab-based (neo)adjuvant therapy were assigned to oral neratinib 240 mg/day or placebo for 1 yr. Randomization was stratified by HR status (determined locally before trial entry), nodal status, and trastuzumab regimen. Endocrine therapy was allowed in patients with HR+ disease. The primary endpoint, iDFS, was tested by 2-sided log-rank test and hazard ratios (95% CI) were estimated using Cox proportional hazards models. Kaplan-Meier methods were used to estimate iDFS rates. Secondary endpoints were DFS-DCIS, time to distant recurrence, distant DFS, and CNS recurrences. The primary analysis was conducted at 2 yr, and a sensitivity analysis conducted at 5 yr. Clinicaltrials.gov:NCT00878709. Results: Of the 2840 patients (neratinib, n=1420; placebo, n=1420), 1631 (57%) had HR+ disease (neratinib, n=816; placebo, n=815). Most (93%) HR+ patients were receiving endocrine therapy at baseline. 1334 of 1631 (82%) patients with HR+ tumors were randomized to start neratinib within 1 yr of last trastuzumab dose (neratinib, n=670; placebo, n=664). iDFS benefits from neratinib in this population are shown in the table. Secondary endpoints were also improved with neratinib vs. placebo in this population. Safety data in this subset will be presented at the meeting. Table. iDFS in patients with an interval between last trastuzumab dose and randomization of ≤1 yr HR+ population (N=1334)ITT population (N=2297) Hazard ratiob Hazard ratiob Δ, %a(95% CI)P-valueΔ, %a(95% CI)P-value2-yr analysisc+4.50.490.002+2.90.630.006 (0.30–0.78) (0.45–0.88) 5-yr analysisd+5.10.580.002+3.20.700.006 (0.41–0.82) (0.54–0.90) aDifference in iDFS rates between neratinib vs. placebo; bNeratinib vs. placebo; cData cut-off: July 2014; dData cut-off: March 2017 Conclusions: Neratinib may have enhanced and sustained efficacy in patients with HR+ disease who initiate treatment within 1 yr of trastuzumab-based adjuvant therapy. Citation Format: Gnant M, Martin M, Holmes F-A, Jackisch C, Chia SK, Iwata H, Moy B, Martinez N, Mansi J, Morales S, Ruiz-Borrego M, von Minckwitz G, Buyse M, Delaloge S, Bhandari M, Murias Rosales A, Galeano T, Fujita T, Luczak A, Barrios CH, Saura C, Rugo HS, Chien J, Johnston SR, Spencer M, Xu F, Barnett B, Chan A, Ejlertsen B. Efficacy of neratinib in hormone receptor-positive patients who initiated treatment within 1 year of completing trastuzumab-based adjuvant therapy in HER2+ early-stage breast cancer: Subgroup analyses from the phase III ExteNET trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-13-01.