Purpose:

Doublets of everolimus with letrozole or trastuzumab have demonstrated activity against HER2-positive breast cancer, suggesting that the triple combination can have synergistic anticancer activity.

Patients and Methods:

This first-in-human dose-escalation study (NCT02152943) enrolled patients with hormone receptor− positive, HER2-positive (defined by amplification, overexpression, or mutation) treatment-refractory advanced cancers to receive escalating doses (3+3 design) of daily oral letrozole (days 1–21), daily oral everolimus (days 1–21), and intravenous trastuzumab (day 1) every 21 days to determine dose-limiting toxicities (DLT) and MTD or recommended phase II dose (RP2D).

Results:

A total of 32 patients with hormone receptor−positive, HER2-positive (amplification, n = 27; overexpression, n = 1; and mutation, n = 4) advanced breast cancer (n = 26) or other cancers (n = 6) were enrolled. The most frequent grade ≥3 adverse events included hyperglycemia (n = 4), anemia (n = 3), thrombocytopenia (n = 2), and mucositis (n = 2). DLTs included grade 3 mucositis and grade 4 neutropenia, and trastuzumab given as an 8 mg/kg loading dose on day 1 of cycle 1 followed by a 6 mg/kg maintenance dose on day 1 of subsequent cycles plus 10 mg everolimus daily and 2.5 mg letrozole daily every 21 days was declared as RP2D. Five patients with breast cancer (four with HER2 amplification and one with HER2 mutation) had partial responses. HER2 amplification in circulating cell-free DNA at baseline was associated with shorter progression-free and overall survival durations (P < 0.05).

Conclusions:

Everolimus, letrozole, and trastuzumab have a favorable safety profile and elicit encouraging signals of anticancer activity in patients with heavily pretreated hormone receptor- and HER2-positive advanced cancers.

This article is featured in Highlights of This Issue, p. 1215

Translational Relevance

The combination of everolimus, letrozole, and trastuzumab is well-tolerated and has encouraging activity against hormone receptor−positive, HER2-positive advanced cancers, especially breast cancer. HER2 amplification in circulating cell-free DNA at baseline is associated with shorter progression-free and overall survival.

Approximately 20% of breast cancers have overexpression or amplification of HER2, which can render tumors sensitive to therapeutic targeting with HER2 antibodies, HER2 antibody−drug conjugates, or tyrosine kinase inhibitors (1–8). Approximately half of patients with HER2-altered breast cancer, approximately 10% of all patients with breast cancer, have disease that also has hormone receptor expression (i.e., expression of estrogen receptor or progesterone receptor), and these patients have substantially worse clinical outcomes than those without HER2 alterations following hormone therapy (1). Furthermore, HER2-activating mutations occur in about 3% of breast cancers, and early clinical data suggest that HER2-targeting therapies have efficacy against these cancers (9, 10). In addition, HER2 overexpression, amplification, and/or activating mutations have been reported in other cancers that can respond to HER2-targeting therapies, including gastroesophageal, colorectal, lung, ovarian, and cervical cancers, and some of these cancers also express hormone receptors (8, 9, 11–13). Although targeting HER2 with mAbs, antibody–drug conjugates, or tyrosine kinase inhibitors has improved treatment outcomes in patients with breast and advanced gastric cancers, progression is almost inevitable in patients with metastatic disease, highlighting the unmet need for new therapies (1–8, 14–17).

The activation of the PI3K/AKT/mTOR pathway is a key mechanism of intrinsic and adaptive resistance to hormone therapy, as well as HER2-targeting agents (18–20). Specifically, the downstream activation of ribosomal protein S6 kinase beta-1 leads to the activation of estrogen receptor and the resultant transcription of estrogen receptor−responsive genes independent of estrogen ligand binding. Furthermore, estrogen receptors at the plasma membrane interact with tyrosine kinase receptors, thereby stimulating growth factor signaling (21–23). In the clinic, the combination of the mTOR inhibitor, everolimus, and the aromatase inhibitor, exemestane, has demonstrated superiority to hormone therapy with exemestane alone in postmenopausal patients with HER2-negative, hormone receptor−positive advanced breast cancer (24). In one study of patients with advanced HER2-positive, hormone receptor−positive breast cancer, the median progression-free survival (PFS) duration achieved with the combination of the aromatase inhibitor, anastrozole, and the HER2 antibody, trastuzumab (4.8 months), was significantly longer than that achieved with anastrozole alone (2.4 months; P = 0.0016; ref. 1). In addition, a randomized phase II study of HER2 antibody, trastuzumab, with antiestrogen, fulvestrant, and CDK4/6 inhibitor, abemaciclib, versus trastuzumab with abemaciclib or trastuzumab with chemotherapy in patients with advanced HER2-positive, hormone receptor−positive breast cancer demonstrated prolonged PFS for trastuzumab, fulvestrant, and abemaciclib arm (8.3 months vs. 5.7 months vs. 5.7 months, P = 0.05; ref. 25).

On the basis of these findings, we hypothesized that the combination of the mTOR inhibitor, everolimus, the aromatase inhibitor, letrozole, and the HER2 antibody, trastuzumab, can have synergistic activity and overcome resistance to hormone- and/or HER2-targeting therapies in patients with advanced or metastatic breast cancer or other solid tumors with hormone receptor expression and HER2 overexpression, amplification, or activating mutations. Therefore, we designed a dose-escalation phase I study to determine the MTD and/or recommended phase II dose (RP2D), as well as the dose-limiting toxicities (DLT), of this combination.

Study design and objectives

This was a single-center, open-label, nonrandomized, 3+3 dose-escalation phase I study (NCT02152943) to evaluate the safety, toxicity, and preliminary signals of anticancer activity of the combination of everolimus, letrozole, and trastuzumab in patients with hormone receptor−positive, HER2-positive advanced solid cancers. The study design allowed the expansion of the cohort at the MTD or RP2D for up to 12 additional patients with hormone receptor−positive, HER2-positive metastatic breast cancer. The full study protocol is available as Supplementary Data S1. In addition, patients were offered the opportunity to participate in a blood collection protocol (LAB10-0334) evaluating the dynamics of plasma-derived circulating cell-free DNA (cfDNA). Both protocols were approved by MD Anderson Cancer Center's (Houston, TX) Institutional Review Board and were conducted in accordance with the Declaration of Helsinki and Good Clinical Practice principles (26). All patients provided written informed consent before starting any study-related procedures.

Eligible patients had a histologically confirmed diagnosis of an advanced solid tumor with hormone receptor positivity (i.e., estrogen receptor positivity with or without progesterone receptor positivity), defined as >1% expression on IHC, and with HER2 positivity [defined in accordance with the American Society of Clinical Oncology/CAP 2013 guidelines as 3+ expression on IHC, or HER2 amplification detected by FISH (overall ratio ≥ 2 with an average HER2 copy number >4 signals/cell; overall ratio ≥ 2 with an average HER2 copy number <4; or average ratio < 2 with an average HER2 copy number ≥6 signals/cell) or by next-generation sequencing (NGS, ≥4 copies), or HER2 mutation detected by NGS]; had measurable or evaluable disease according to RECIST 1.1 (27); and had been off prior chemotherapy at least 4 weeks; off therapies with delayed toxicity (e.g., nitrosoureas, mitomycin-C, and liposomal doxorubicin) at least 6 weeks; off biologic/targeted therapies at least 4 weeks or five half-lives (whichever was shorter); and/or off hormone therapy at least 2 weeks. Patients who received palliative low-dose radiotherapy 1 week before treatment, provided that it was not given to only the targeted lesions, were eligible for the study. Patients had to have adequate organ function and an Eastern Cooperative Oncology Group performance status score of 0 or 1. Female patients had to be either premenopausal and receiving a gonadotropin-releasing hormone agonist or postmenopausal (defined as age ≥ 60 years or as age < 60 years with prior bilateral oophorectomy or at least 12 months of spontaneous amenorrhea and postmenopausal follicle-stimulating hormone and estradiol levels).

Patients received everolimus (2.5–10 mg orally daily), letrozole (2.5 mg orally daily), and trastuzumab (4–8 mg/kg loading dose and 2–6 mg/kg maintenance dose intravenously on day 1 of a 21-day cycle) until disease progression, unacceptable toxicity, or consent withdrawal. Safety was assessed using the NCI Common Terminology Criteria for Adverse Events v3. DLTs included any treatment-related grade 4 hematologic toxicity, grade 3 or 4 nonhematologic toxicity, and treatment delays due to adverse events that lasted more than 14 days during the first 21 days of therapy. Response to therapy was assessed every other cycle (i.e., every 6 weeks) using RECIST 1.1 (27).

Plasma collection and cfDNA mutation testing

Whole blood was collected from patients who consented to the optional laboratory collection protocol (LAB10-0334) outside of the main clinical study. Objectives were to test whether HER2 amplification can be detected in cfDNA and whether dynamic changes in cfDNA correlate with clinical outcomes. Samples were collected at baseline, during therapy, and at disease progression in ethylenediaminetetraacetic acid–containing tubes and centrifuged and spun twice within 2 hours to yield plasma. The QIAamp Circulating Nucleic Acid Kit (Qiagen) was used to isolate cfDNA according to the manufacturer's instructions.

Quantitation of cfDNA was done with Quant-iT PicoGreen dsDNA Reagent and Kits (catalog No. P7589, Invitrogen). Molecular testing of cfDNA was done with a QX200 Droplet Digital PCR System (Bio-Rad) using 8–16 ng (depending on availability) of unamplified cfDNA. A cutoff of 2.5 copies was used to detect the amplification of HER2 and other genes. In addition, mutation-specific probes were used to detect single-nucleotide variants in common oncogenes with a lowest limit of detection of 0.1% variant allele frequency. The results, presented as gene copy numbers for amplification and as variant allele frequencies for single-nucleotide variants, were compared with those of clinical molecular testing of the patients' archival tumor tissues.

Statistical analysis

Kruskal–Wallis one-way ANOVA was used to compare differences among independent groups of samples. The Fisher exact test was used to compare two categorical variables. PFS duration was defined as the time from the initiation of the investigational therapy to the date of disease progression or death from any cause. Overall survival (OS) duration was defined as the time from the initiation of the investigational therapy to the date of death or last follow-up. The Kaplan–Meier method was used to estimate PFS and OS, and a log-rank test was used to compare PFS or OS between subgroups. All tests were two-sided, and P < 0.05 was considered statistically significant. Statistical analyses were performed using the SPSS 25 Software program (SPSS).

Patient characteristics

Between November 2014 and February 2018, 37 patients were screened; of these, 32 met the eligibility criteria and were enrolled in the study (Fig. 1). The clinical characteristics of these 32 patients are depicted in Table 1. The median age was 55.5 years, and most patients were women (n = 31, 97%) and white (n = 21, 66%). Breast cancer was the most frequent tumor type (n = 26, 81%) followed by ovarian (n = 3, 9%), cervical (n = 1, 3%), endometrial (n = 1, 3%), and gastroesophageal junction cancer (n = 1, 3%). Patients received a median of five prior therapies. Four patients (13%) received prior therapy with everolimus. Prior lines of hormone therapies for metastatic disease included tamoxifen in eight patients (25%), letrozole in 11 patients (34%), anastrozole in 12 patients (38%), exemestane in six patients (19%), and fulvestrant in nine patients (28%). Prior lines of HER2-targeting agents in metastatic disease included trastuzumab in 25 patients (78%), trastuzumab emtansine in 20 patients (63%), lapatinib in 18 patients (56%), pertuzumab in 16 patients (50%), tucatinib in four patients (13%), neratinib in one patient (3%), ZW25 (HER2 antibody) in one patient (3%), and MM-302 (HER2 antibody conjugated to liposomal doxorubicin) in one patient (3%; Table 1; Supplementary Table S1).

Figure 1.

Patient enrollment and disposition. Between November 2014 and February 2018, 37 patients were screened; of these patients, 32 met the eligibility criteria and were enrolled in the study and received treatment with everolimus, letrozole, and trastuzumab.

Figure 1.

Patient enrollment and disposition. Between November 2014 and February 2018, 37 patients were screened; of these patients, 32 met the eligibility criteria and were enrolled in the study and received treatment with everolimus, letrozole, and trastuzumab.

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Table 1.

Patients' characteristics.

CharacteristicNo. of patients (%), N = 32
Median age (range), years 55.5 (31–78) 
Sex 
 Male 1 (3) 
 Female 31 (97) 
Ethnicity 
 White 21 (66) 
 Hispanic 6 (19) 
 African American 3 (9) 
 Asian 2 (6) 
ECOG performance status score 
 0 4 (12) 
 1 28 (88) 
No. of prior therapies for metastatic disease 
 Median (range) 5 (1–13) 
 ≤5 18 (56) 
 >5 14 (44) 
Prior hormone therapy for metastatic disease 25 (78) 
 Tamoxifen 8 (25) 
 Letrozole 11 (34) 
 Anastrozole 12 (38) 
 Exemestane 6 (19) 
 Fulvestrant 9 (28) 
Prior HER2-targeting therapy for metastatic disease 27 (84) 
 Trastuzumab 25 (78)a 
 Trastuzumab emtansine 20 (63) 
 Lapatinib 18 (56) 
 Pertuzumab 16 (50)b 
 Tucatinib 4 (13) 
 Neratinib 1 (3) 
 Other (ZW25, MM-302) 2 (6) 
Prior everolimus Cancer type 4 (13) 
 Breast 26 (81) 
 Ovarian 3 (9) 
 Otherc 3 (9) 
FFPE tissue 32 (100) 
HER2 amplification 27 (84) 
 HER overexpression 1 (3) 
HER2 mutationd 4 (12) 
Plasma cfDNA 14 (44) 
HER2 amplification 8 (25) 
CharacteristicNo. of patients (%), N = 32
Median age (range), years 55.5 (31–78) 
Sex 
 Male 1 (3) 
 Female 31 (97) 
Ethnicity 
 White 21 (66) 
 Hispanic 6 (19) 
 African American 3 (9) 
 Asian 2 (6) 
ECOG performance status score 
 0 4 (12) 
 1 28 (88) 
No. of prior therapies for metastatic disease 
 Median (range) 5 (1–13) 
 ≤5 18 (56) 
 >5 14 (44) 
Prior hormone therapy for metastatic disease 25 (78) 
 Tamoxifen 8 (25) 
 Letrozole 11 (34) 
 Anastrozole 12 (38) 
 Exemestane 6 (19) 
 Fulvestrant 9 (28) 
Prior HER2-targeting therapy for metastatic disease 27 (84) 
 Trastuzumab 25 (78)a 
 Trastuzumab emtansine 20 (63) 
 Lapatinib 18 (56) 
 Pertuzumab 16 (50)b 
 Tucatinib 4 (13) 
 Neratinib 1 (3) 
 Other (ZW25, MM-302) 2 (6) 
Prior everolimus Cancer type 4 (13) 
 Breast 26 (81) 
 Ovarian 3 (9) 
 Otherc 3 (9) 
FFPE tissue 32 (100) 
HER2 amplification 27 (84) 
 HER overexpression 1 (3) 
HER2 mutationd 4 (12) 
Plasma cfDNA 14 (44) 
HER2 amplification 8 (25) 

Note: All data are No. of patients (%) unless otherwise indicated.

Abbreviations: ECOG, Eastern Cooperative Oncology Group; FFPE, formalin-fixed paraffin-embedded.

aOne patient received trastuzumab only in the neoadjuvant setting, which is not captured in the table.

bOne patient received pertuzumab only in the adjuvant setting, which is not captured in the table.

cCervical cancer, endometrial cancer, and gastroesophageal junction cancer.

dHER2V777L, HER2P780_Y781insGSP, HER2Y772_A775dupYVMA, and HER2A775_G776insYVMA.

Safety

All 32 patients, who enrolled to six dose levels, were evaluable for toxicity. Two DLTs occurred in two patients: one patient had grade 3 mucositis at dose level 3 and one patient had grade 4 neutropenia at dose level 4 (Table 2). The MTD was not reached, and dose level 5 (intravenous trastuzumab at a loading dose of 8 mg/kg on day 1 of cycle 1 and a maintenance dose of 6 mg/kg on day 1 of each subsequent 21-day cycle; oral everolimus at a dose of 10 mg daily; and oral letrozole at a dose of 2.5 mg daily) was declared the RP2D.

Table 2.

Treatment dose levels and DLTs.

Dose levelTrastuzumab, mg/kg i.v. every 3 weeksEverolimus, mg p.o. dailyLetrozole, mg p.o. dailyNo. of patientsDLT
−1a LD: 4; MD: 2 2.50 2.50  
LD: 4; MD: 2 5.00 2.50  
LD: 6; MD: 4 5.00 2.50  
LD: 6; MD: 4 7.50 2.50 3b Grade 3 mucositis 
LD: 8; MD: 6 7.50 2.50 Grade 4 neutropenia 
LD: 8; MD: 6 10.00 2.50 11  
Dose levelTrastuzumab, mg/kg i.v. every 3 weeksEverolimus, mg p.o. dailyLetrozole, mg p.o. dailyNo. of patientsDLT
−1a LD: 4; MD: 2 2.50 2.50  
LD: 4; MD: 2 5.00 2.50  
LD: 6; MD: 4 5.00 2.50  
LD: 6; MD: 4 7.50 2.50 3b Grade 3 mucositis 
LD: 8; MD: 6 7.50 2.50 Grade 4 neutropenia 
LD: 8; MD: 6 10.00 2.50 11  

Abbreviations: i.v., intravenous; LD, loading dose; MD, maintenance dose; p.o., per oral.

aThe study allowed backfilling of the lower dose levels to obtain additional safety and/or correlative data.

bThe event was reclassified as grade 3 mucositis related to the study medication after dose level 4 had already been determined to be safe. Therefore, dose level 3 was not expanded to six patients.

Treatment-related adverse events are shown in Table 3. The most frequent grade 3 or 4 adverse events were hyperglycemia in four patients (13%), anemia in three patients (9%), thrombocytopenia in two patients (6%), mucositis in two patients, transaminitis in one patient (3%), headache in one patient, and neutropenia in one patient (Table 3). Four patients (13%) required dose reductions of everolimus because of grade 4 anemia with grade 4 gastrointestinal bleeding (one patient), symptomatic grade 2 hypertension (one patient), grade 3 transaminitis (one patient), and grade 3 mucositis (one patient). The most common grade 1 or 2 adverse events were mucositis in 15 patients (47%), hyperglycemia in nine patients (28%) fatigue in eight patients (25%), elevated triglycerides in eight patients (25%), and elevated cholesterol in seven patients (22%). In addition, two patients (6%) received G-CSF to treat or prevent neutropenia and one patient (3%) required blood transfusions for grade 4 gastrointestinal bleeding.

Table 3.

Treatment-related adverse events by Common Terminology Criteria for Adverse Events grade.

CTCAE grade
Adverse event1234Total
Mucositis 11 17 
Hyperglycemia 13 
Fatigue 
Hypertriglyceridemia 
Thrombocytopenia 
Anemia 
Hypercholesterolemia 
Rash 
Neuropathy 
Pain 
Headache 
Diarrhea 
Transaminitis 
Neutropenia 
Nausea 
Hypomagnesemia 
Creatinine 
Hypertension 
Infusion reaction 
Vomiting 
Constipation 
Vaginal bleeding 
Anorexia 
Hot flashes 
Elevated alkaline phosphatase 
Tremor 
Ear infection 
Leucopenia 
Lymphopenia 
Bronchitis 
CTCAE grade
Adverse event1234Total
Mucositis 11 17 
Hyperglycemia 13 
Fatigue 
Hypertriglyceridemia 
Thrombocytopenia 
Anemia 
Hypercholesterolemia 
Rash 
Neuropathy 
Pain 
Headache 
Diarrhea 
Transaminitis 
Neutropenia 
Nausea 
Hypomagnesemia 
Creatinine 
Hypertension 
Infusion reaction 
Vomiting 
Constipation 
Vaginal bleeding 
Anorexia 
Hot flashes 
Elevated alkaline phosphatase 
Tremor 
Ear infection 
Leucopenia 
Lymphopenia 
Bronchitis 

Abbreviation: CTCAE, Common Terminology Criteria for Adverse Events.

Efficacy

Of the 32 patients enrolled in the study, 31 (97%) had at least one follow-up imaging study for response assessment per RECIST 1.1 and were evaluable for response. The one patient who did not have a follow-up imaging study had clinical progression before the first restaging scan. Three patients had evaluable, but not measurable, disease.

Partial responses (PRs) occurred in five patients (16%), all of whom had breast cancer: four patients with HER2 amplification and one with HER2 mutation. These patients were heavily pretreated (3–9 prior therapies for metastatic disease) and their prior therapies are detailed in Supplementary Table S2. For these five patients, the maximum reductions in the sums of the diameters of their target lesions were −91%, −68%, −51%, −50%, and −32%, respectively (Fig. 2A), and their PFS durations were 16.1, 9.0, 22.2, 9.1, and 17.7 months, respectively (Fig. 2B).

Figure 2.

A, Waterfall plot depicting the best percentage changes in the sums of the diameters of target lesions per RECIST 1.1 in the 28 patients with measurable disease. Bar colors represent tumor types. Dashed lines mark changes from baseline of +20% and −30%. The patients' HER2 statuses and prior lines of aromatase inhibitors, trastuzumab, and/or everolimus for metastatic disease are noted above the plot, and their best responses per RECIST 1.1 are noted below the plot. B, Swimmer plot depicting the times to progression for all 32 patients included in the study. The patients' overall responses, HER2 statuses, and prior lines of aromatase inhibitors, trastuzumab, and/or everolimus for metastatic disease are noted to the left of the plot. GEJ, gastroesophageal junction; PD, progressive disease.

Figure 2.

A, Waterfall plot depicting the best percentage changes in the sums of the diameters of target lesions per RECIST 1.1 in the 28 patients with measurable disease. Bar colors represent tumor types. Dashed lines mark changes from baseline of +20% and −30%. The patients' HER2 statuses and prior lines of aromatase inhibitors, trastuzumab, and/or everolimus for metastatic disease are noted above the plot, and their best responses per RECIST 1.1 are noted below the plot. B, Swimmer plot depicting the times to progression for all 32 patients included in the study. The patients' overall responses, HER2 statuses, and prior lines of aromatase inhibitors, trastuzumab, and/or everolimus for metastatic disease are noted to the left of the plot. GEJ, gastroesophageal junction; PD, progressive disease.

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Twenty-three patients (72%) had stable disease (SD). The changes in the sums of the diameters of these patients' target lesions ranged from −23% to +19%, and the patients' PFS durations ranged from 1.4 to 21.0 months. Ten patients with SD for ≥6 months had PFS durations of 7.5–21.0 months and five patients with SD for ≥12 months had PFS durations of 12.3–21.0 months. Of note, three patients with breast cancer (one with HER2 amplification and two with HER2 mutations) who had evaluable, but not measurable, disease had SD for 9.0, 12.6, and 20.3 months, respectively.

PR rates did not differ between patients who received everolimus previously and those who did not [25% (1/4) vs. 14% (4/28); P = 0.51], patients who received aromatase inhibitors previously for metastatic disease and those who did not [17% (4/24) vs. 13% (1/8); P = 1.00], or patients who received trastuzumab previously for metastatic disease and those who did not [16% (4/25) vs. 14% (1/7); P = 1.00]. Similarly, PR rates did not differ between patients treated with the RP2D and those treated with lower doses [12% (2/17) vs. 20% (3/15); P = 0.65]. Finally, PR rates did not differ between patients with HER2 mutation and those with HER2 amplification or HER2 overexpression [25% (1/4) vs. 14% (4/28); P = 0.51], patients with PI3K mutations and those without PI3K mutations [17% (1/6) vs. 15% (4/26); P = 1.00], patients with ESR1 mutations and those without ESR1 mutations [50% (2/4) vs. 11% (3/28); P = 0.11], or patients with breast cancer and patients with other cancers [19% (5/26) vs. 0% (0/6); P = 0.56].

For all 32 patients, the median PFS duration was 4.3 months [95% confidence interval (CI), 0.0–9.5 months]. Median PFS durations did not differ between patients who received everolimus previously and those who did not (9.0 vs. 3.0 months; P = 0.24), patients who received trastuzumab previously for metastatic disease and those who did not (7.5 vs. 3.2 months; P = 0.96), patients treated with the RP2D and those treated with lower doses (3.2 vs. 9 months; P = 0.13), patients with HER2 mutations and those with HER2 amplification or HER2 overexpression (12.6 vs. 3.9 months; P = 0.19), patients with PI3K mutations and those without PI3K mutations (3.7 vs. 5.9 months; P = 0.73), or patients with ESR1 mutations and patients without ESR1 mutations (16.1 vs. 3.9 months; P = 0.19). There was a trend toward longer median PFS for patients treated with prior aromatase inhibitors for metastatic disease compared with those not treated with the agents (7.5 vs. 2.7 months; P = 0.06). Patients with breast cancer compared with those with other cancers had longer median PFS (7.5 vs. 2.7 months; P = 0.03; Fig. 3A).

Figure 3.

A, The median PFS duration of the 26 patients with breast cancer (7.5 months; 95% CI, 1.8–13.8 months; blue) was longer than that of the 6 patients with other cancers (2.7 months; 95% CI, 0.8–4.6 months; red; P = 0.03). B, The median OS duration of the 26 patients with breast cancer (20.7 months; 95% CI, 10.5–31.0 months; blue) was longer than that of the 6 patients with other cancers (4.9 months; 95% CI, 2.2–7.6 months; red; P = 0.001).

Figure 3.

A, The median PFS duration of the 26 patients with breast cancer (7.5 months; 95% CI, 1.8–13.8 months; blue) was longer than that of the 6 patients with other cancers (2.7 months; 95% CI, 0.8–4.6 months; red; P = 0.03). B, The median OS duration of the 26 patients with breast cancer (20.7 months; 95% CI, 10.5–31.0 months; blue) was longer than that of the 6 patients with other cancers (4.9 months; 95% CI, 2.2–7.6 months; red; P = 0.001).

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The median OS duration for all 32 patients was 13.9 months (95% CI, 2.9–24.9 months). The 26 patients with breast cancer had a longer median OS duration than the 6 patients with other cancers (20.7 vs. 4.9 months; P = 0.001; Fig. 3B).

Circulating cfDNA analysis

Fourteen patients (12 with breast cancer, one with cervical cancer, and one with endometrial cancer; all of whom had tumors with HER2 amplification) had plasma samples available for assessing HER2 amplification in circulating cfDNA. For eight of these patients (57%), HER2 amplification in cfDNA was detected in at least one plasma sample collected at baseline, during therapy, or at disease progression. The median numbers of HER2 copies collected at baseline (3.12; range, 1.61–9.94 copies), during therapy (1.80; range, 1.4–7.59 copies), and at disease progression (3.15; range, 1.56–11.00 copies) differed significantly (P = 0.01; Fig. 4A). Dynamic changes in the quantity of HER2 in cfDNA and other alterations corresponded with the clinical disease course and often predicted disease progression (Fig. 4B). Compared with patients without detectable HER2 amplification in cfDNA at baseline, those with HER2 amplification in cfDNA at baseline had a shorter median PFS duration (1.8 vs. 8.4 months; P = 0.049; Fig. 4C) and OS duration (5.2 vs. not reached; P = 0.021; Fig. 4D).

Figure 4.

A, The median numbers of HER2 copies in circulating cfDNA in plasma collected at baseline (median, 3.12; range, 1.61–9.94 copies), during therapy (median, 1.80; range, 1.4–7.59 copies), and at disease progression (median, 3.15; range, 1.56–11.00 copies) differed significantly (P = 0.01). B, Dynamic tracking of HER2 copies and other alterations in cfDNA isolated from serially collected plasma samples from a patient with breast cancer (top) and a patient with endometrial cancer (bottom). Gray bars depict the percentage changes in the sums of the largest diameters of the target lesions per RECIST 1.1. MAF, mean allelic frequency; CNV, copy number variation. C, Patients with HER2 amplification (≥2.5 HER2 copies) in cfDNA at baseline had a significantly shorter median PFS duration than patients without HER2 amplification in plasma cfDNA at baseline (1.8 vs. 8.4 months; P = 0.049). D, Patients with HER2 amplification in cfDNA at baseline had a significantly shorter median OS duration than patients without HER2 amplification in plasma cfDNA at baseline (5.2 vs. not reached; P = 0.021).

Figure 4.

A, The median numbers of HER2 copies in circulating cfDNA in plasma collected at baseline (median, 3.12; range, 1.61–9.94 copies), during therapy (median, 1.80; range, 1.4–7.59 copies), and at disease progression (median, 3.15; range, 1.56–11.00 copies) differed significantly (P = 0.01). B, Dynamic tracking of HER2 copies and other alterations in cfDNA isolated from serially collected plasma samples from a patient with breast cancer (top) and a patient with endometrial cancer (bottom). Gray bars depict the percentage changes in the sums of the largest diameters of the target lesions per RECIST 1.1. MAF, mean allelic frequency; CNV, copy number variation. C, Patients with HER2 amplification (≥2.5 HER2 copies) in cfDNA at baseline had a significantly shorter median PFS duration than patients without HER2 amplification in plasma cfDNA at baseline (1.8 vs. 8.4 months; P = 0.049). D, Patients with HER2 amplification in cfDNA at baseline had a significantly shorter median OS duration than patients without HER2 amplification in plasma cfDNA at baseline (5.2 vs. not reached; P = 0.021).

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The combination of everolimus, letrozole, and trastuzumab at the RP2D (in which all drugs are administered at their FDA-approved doses) demonstrated an acceptable safety profile and promising signals of anticancer activity in heavily pretreated patients with hormone receptor- and HER2-positive advanced cancers. DLTs included grade 3 mucositis and grade 4 neutropenia, and the most frequent treatment-related adverse events, mucositis, hyperglycemia, fatigue, and hypertriglyceridemia, were similar to those reported in previous studies in which everolimus was combined with hormone therapy or trastuzumab (1, 24). Of note, Rugo and colleagues demonstrated that the everolimus-related mucositis can be mitigated by the use of prophylactic dexamethasone mouthwash (28).

The combination had encouraging activity, eliciting a total of five durable PRs in patients with metastatic breast cancer and HER2 amplification or mutation. The overall response rate was 15%, and that among patients with breast cancer only was 19%. PRs were independent of prior exposure to everolimus or aromatase inhibitors or trastuzumab, which suggests that triple combination can overcome mechanisms of resistance; however, the small numbers of patients treated in our study preclude any definite conclusion. In addition, three patients with metastatic breast cancer and HER2 amplification or mutation who did not have measurable disease remained on therapy for at least 9 months. These results are similar to those of previous studies, which reported response rates of 9.5% for everolimus with exemestane in patients with HER2-negative and hormone receptor−positive metastatic breast cancer, and 20% for anastrozole and trastuzumab in patients with HER2-positive and hormone receptor−positive metastatic breast cancer, even though the patients in those studies appear to have been less heavily pretreated than those in this study (1, 24). In addition, our combination had encouraging activity, such as PR or prolonged SD (in patients without measurable disease), in patients with HER2 mutations. Our study can be also viewed in the broader context of efforts to develop triple nonchemotherapy combinations for HER2-positive and hormone receptor−positive disease. For instance, a randomized phase II study in patients with advanced HER2-positive, hormone receptor−positive breast cancer demonstrated prolonged PFS with triple combination of trastuzumab, with antiestrogen, fulvestrant, and CDK4/6 inhibitor, abemaciclib, when compared with trastuzumab and chemotherapy or trastuzumab and abemaciclib (8.3 vs. 5.7 vs. 5.7 months; P = 0.05; ref. 25). Also, a pivotal phase III study (NCT02947685) comparing standard anti-HER2 therapy (trastuzumab +/− pertuzumab) in combination with hormone therapy with or without palbociclib in patients with advanced HER2-positive, hormone receptor−positive breast cancer without evidence of disease progression after induction therapy is currently underway.

Our subgroup analysis revealed that 57% of patients with HER2 amplification in the tumor also had HER2 amplification in circulating cfDNA. The presence of HER2 amplification in cfDNA at baseline was associated with shorter PFS and OS durations. Our group and others have demonstrated that the quantity of circulating tumor DNA, as measured by the detection of single-nucleotide variants or methylation changes, is inversely correlated with PFS and OS (29–34). In this study, therefore, it was not entirely surprising that we observed similar outcomes when we quantified HER2 amplification in circulating tumor DNA. We also found that in some patients, the number of HER2 copies in cfDNA increased dynamically before progression. If this phenomenon is confirmed in larger studies, it could be useful for monitoring therapeutic efficacy.

Our study had several limitations. We enrolled relatively few patients with diverse tumor types. Patients were treated at multiple dose levels. Also, our study did not include analyses of additional factors, which could have impacted outcomes, such as pharmacokinetics and antidrug-antibody assessments. Subgroup analyses, including optional cfDNA testing, were retrospective and can be viewed as hypothesis generating at best. Despite these limitations, our study demonstrated that the combination of everolimus, letrozole, and trastuzumab has a favorable safety profile and elicits encouraging signals of anticancer activity in patients with heavily pretreated hormone receptor- and HER2-positive advanced cancers. Further investigation of the combination, especially in patients with breast cancer, is warranted.

J.J. Wheler reports leaving MD Anderson in 2015, joining Novartis, and has worked at Novartis from July 2015 until August 2018. D.D. Karp reports grants from Novartis and Genentech during the conduct of the study, and grants from National Center for Accelerating Translational Science (NCATS), Clinical Translational Sciences Award, NIH; The Jazz Connection Foundation, and National Academy of Recording Arts & Sciences. S.A. Piha-Paul reports other from AbbVie, Inc., ABM Therapeutics, Inc., Acepodia, Inc., Alkermes, Aminex Therapeutics, Amphivena Therapeutics Inc., BioMarin Pharmaceutical, Inc., Boehringer Ingelheim, Bristol Myers Squib, Cerulean Pharma, Inc., Chugai Pharmaceutical Co., Ltd., Curis, Inc., Daiichi Sanko, Eli Lilly, ENB Therapeutics, Five Prime Therapeutics, Gene Quantum, Genmab A/S, GlaxoSmithKline, Helix BioPharma Corp., Incyte Corp., Jacobio Pharmaceuticals Co., Ltd., Medimmune, LLC, Medivation, Inc., Merk Sharp and Dohme Corp., Novartis Pharmaceuticals, Pieris Pharmaceuticals, Inc., Pfizer, Principia Biopharma, Inc., Puma Biotechnology, Inc., Rapt Therapeutics, Inc., Seattle Genetics, Silverback Therapeutics, Taiho Oncology, Tesaro, Inc., TransThera Bio, and NCI/NIH P30CA016672, core grant (CCG Shared Resources) outside the submitted work. A.M. Tsimberidou reports grants from IMMATICS, OBI Pharmaceuticals, Parker Institute for Cancer Immunotherapy, EMD Serono, T-vardi, Karus, and Boston Biomedical, grants and other from Tempus, and other from Covance, Roche, and Genentech outside the submitted work. D.S. Hong reports research/grant funding from AbbVie, Adaptimmune, Aldi-Norte, Amgen, Astra-Zeneca, Bayer, BMS, Daiichi Sankyo, Eisai, Fate Therapeutics, Genentech, Genmab, Ignyta, Infinity, Kite, Kyowa, Lilly, LOXO, Merck, MedImmune, Mirati, miRNA, Molecular Templates, Mologen, NCI-CTEP, Novartis, Numab, Pfizer, Seattle Genetics, Takeda, Turning Point Therapeutics, and Verstatem, travel, accommodations, expenses from Bayer, LOXO, miRNA, Genmab, AACR, ASCO, and SITC, consulting or advisory role with Alpha Insights, Acuta, Amgen, Axiom, Adaptimmune, Baxter, Bayer, Boxer Capital, COG, ECOR1, Expert Connect, Genentech, GLG, Group H, Guidepoint, H.C. Wainwright, Infinity, Janssen, Merrimack, Medscape, NTRK Connect, Numab, Pfizer, Prime Oncology, Seattle Genetics, SlingShot, Takeda, Trieza Therapeutics, and WebMD, and other ownership interests with Molecular Match (advisor), OncoResponse (founder), and Presagia Inc (advisor). V. Subbiah reports grants from Novartis during the conduct of the study, research funding/grant support for clinical trials (to institution) from Novartis, Bayer, Berghealth, Incyte, Fujifilm, PharmaMar, D3, Pfizer, Multivir, Amgen, AbbVie, Alfa-Sigma, Agensys, Boston Biomedical, Idera Pharma, InhibRx, Exelixis, Blueprint medicines, Loxo oncology, Medimmune, Altum, Dragonfly therapeutics, Takeda, National Comprehensive Cancer Network, NCI-CTEP and UT MD Anderson Cancer Center, Turning point therapeutics, and Boston Pharmaceuticals, travel funding from Novartis, PharmaMar, ASCO, ESMO, Helsinn, and Incyte, and ad hoc advisory board relation with Helsinn, LOXO Oncology/Eli Lilly, R-Pharma US, INCYTE, QED pharma, Medimmune, Novartis, and Signant Health. F. Meric-Bernstam reports personal fees from DebioPharm, Pfizer Inc, Samsung Bioepis, Seattle Genetics Inc, Tyra Biosciences, Xencor, Zymeworks, Immunomedics, Inflection Biosciences, Mersana Therapeutics, Seattle Genetics, Silverback Therapeutics, Spectrum Pharmaceuticals, Zentalis, Chugai Biopharmaceuticals, Mayo Clinic, and Rutgers Cancer Institute of New Jersey, grants from Aileron Therapeutics, Inc, Bayer Healthcare Pharmaceutical, Calithera Biosciences, Curis Inc., CytomX Therapeutics Inc., Daiichi Sankyo, Debiopharm International, Guardant Health Inc., Millennium Pharmaceuticals, Novartis, and Taiho Pharmaceutical Co, grants and personal fees from Puma Biotechnology Inc, and other from Beth Israel Deaconess Medical Center outside the submitted work. F. Janku reports other from Novartis during the conduct of the study, and other from Genentech, BioMed Valley Discoveries, Astellas, Agios, Plexxikon, Piqur, Symphogen, Bristol-Myers Squibb, Asana, Synthorx, FujiFilm Pharmaceuticals, and Proximagen, personal fees and other from Deciphera, Ideaya Biosciences, Sotio, and Cardiff Oncology, and personal fees from Guardant Health, Illumina, IFM Therapeutics, Synlogic, PureTech Health, and ImmunoMet outside the submitted work. No disclosures were reported by the other authors.

A. Ballhausen: Data curation, formal analysis, writing-original draft, writing-review and editing. J.J. Wheler: Conceptualization, resources, supervision, funding acquisition, investigation, methodology, writing-review and editing, provision of patients. D.D. Karp: Investigation, writing-review and editing, provision of patients. S. A. Piha-Paul: Investigation, writing-review and editing, provision of patients. S. Fu: Investigation, writing-review and editing, provision of patients. S. Pant: Investigation, writing-review and editing, provision of patients. A.M. Tsimberidou: Investigation, writing-review and editing, provision of patients. D.S. Hong: Investigation, writing-review and editing, provision of patients. V. Subbiah: Investigation, writing-review and editing, provision of patients. V.R. Holley: Investigation, project administration, writing-review and editing. H.J. Huang: Investigation, writing-review and editing, cell-free DNA testing. A.M. Brewster: Writing-review and editing, provision of patients. K.B. Koenig: Writing-review and editing, provision of patients. N.K. Ibrahim: Writing-review and editing, provision of patients. F. Meric-Bernstam: Conceptualization, investigation, methodology, writing-review and editing, provision of patients. F. Janku: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, investigation, methodology, writing-original draft, project administration, writing-review and editing, provision of patients.

This study was funded by a research grant from Novartis (to F. Janku). The correlative studies were funded by the Sabin Family Foundation (to F. Janku). The study was also supported by MD Anderson Cancer Center Support grant (NIH/NCI P30 CA016672), Clinical Translational Science Award (NIH/HHS 1UL1 TR003167) and Cancer Prevention Research Institute of Texas (CPRIT) Precision Oncology Decision Support Core (RP150535), and Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy. Authors would like to acknowledge Joe Munch in MD Anderson's Research Medical Library for editing the article.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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