Clinical value of next-generation sequencing in guiding decisions regarding endocrine therapy for advanced HR-positive/HER-2-negative breast cancer

Dan Lyu ,Binliang Liu,2 ,Bo Lan ,Xiaoying Sun ,Lixi Li ,Jingtong Zhai ,Haili Qian,Fei Ma

1Department of Medical Oncology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China;2Department of Breast Cancer Medical Oncology,Hunan Cancer Hospital/the Affiliated Cancer Hospital of Xiangya School of Medicine,Central South University,Changsha 410013,China;3 Department of Medical Oncology,Cancer Hospital of Huanxing Chaoyang District,Beijing 100122,China;4 State Key Laboratory of Molecular Oncology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China

Abstract Objective:The mechanism of acquired gene mutation plays a major role in resistance to endocrine therapy in hormone receptor (HR)-positive advanced breast cancer.Circulating tumor DNA (ctDNA) has been allowed for the assessment of the genomic profiles of patients with advanced cancer.We performed this study to search for molecular markers of endocrine therapy efficacy and to explore the clinical value of ctDNA to guide precise endocrine therapy for HR-positive/human epidermal growth factor receptor-2 (HER-2)-negative metastatic breast cancer patients.Methods:In this open-label,multicohort,prospective study,patients were assigned to four parallel cohorts and matched according to mutations identified in ctDNA: 1) activation of the phosphatidylinositol-3-kinase(PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway preferred mTOR inhibitor combined with endocrine therapy;2) estrogen receptor 1 (ESR1) mutation preferred fulvestrant;3) HER-2 mutations preferred pyrotinib;and 4) no actionable mutations received treatment according to the clinical situation.In all cohorts,patients were divided into compliance group and violation group.The primary outcome measure was progression-free survival (PFS),and the secondary outcome measure was overall survival (OS).Results:In all cohorts,the combined median PFS was 4.9 months,and median PFS for the compliance and violation groups was 6.0 and 3.0 months,respectively [P=0.022,hazard ratio (HR)=0.57].Multivariate Cox regression model showed the risk of disease progression was lower in compliance group than in violation group(P=0.023,HR=0.55).Among the patients with HER-2 mutations,the median PFS was 11.1 months in the compliance group and 2.2 months in the violation group (P=0.011,HR=0.20).There was no significant difference in the median PFS between patients who did and did not comply with the treatment protocol in patients with activation of the PI3K/AKT/mTOR or ESR1 mutation.Conclusions:The results suggest that ctDNA may help to guide the optimal endocrine therapy strategy for metastatic breast cancer patients and to achieve a better PFS.Next-generation sequencing (NGS) detection could aid in distinguishing patients with HER-2 mutation and developing new treatment strategies.

Keywords: ctDNA;next-generation sequencing;breast cancer;endocrine therapy

Introduction

Breast cancer,one of the most common malignant cancer types,is the leading cause of cancer-related death in women worldwide (1,2).Nearly 70% of breast cancer patients express the estrogen receptor (ER) (3),and endocrine therapy can significantly reduce the recurrence rate and prolong the overall survival (OS) of these patients.Endocrine therapy resistance is a major challenge for patients with hormone receptor (HR)-positive metastatic breast cancer (4,5).

Activation of the phosphatidylinositol-3-kinase(PI3K)/AKT/mammalian target of rapamycin (mTOR)signaling pathway is associated with endocrine resistance,and multiple clinical trials have consistently confirmed that the mTOR inhibitor everolimus (6) or rapamycin(sirolimus) (7) was able to reverse endocrine resistance in patients with abnormal activation of the PI3K/AKT/mTOR signaling pathway and to significantly prolong the progression-free survival (PFS) of patients.Patients with estrogen receptor 1 (ESR1) mutations receiving aromatase inhibitor (AI) treatment were found to be more likely to develop endocrine resistance (8).For patients with ESR1 mutations,treatment with fulvestrant may achieve greater survival benefits (9,10).Approximately 2%-5% of breast cancer patients carry human epidermal growth factor receptor-2 (HER-2) somatic mutations (11-14).It is reported that most HER-2 somatic mutations occur in HER-2-negative breast cancer (15).Recently,research found that some patients with HER-2 mutation may also respond to anti-HER-2 therapy (16,17).In our previous study,we revealed that HER-2 somatic mutations are common in metastatic breast cancer patients,and HER-2 amplification-negative patients with HER-2 somatic mutations may benefit from pyrotinib (18).Among patients with HR-positive/HER-2-negative patients,it is unclear whether patients with HER-2 somatic mutations will benefit from endocrine therapy combined with anti-HER-2 treatment.

With the continuous evolution of tumors caused by antitumor therapy,repeated biopsy is needed to guide the treatment of metastatic breast cancer because the mechanism of resistance is not very obvious in the analysis of primary tumors.However,for many patients,continuous biopsy of recurrent metastatic lesions carries the risks of trauma due to its invasiveness,making it difficult to be widely used in clinical practice (19).Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA)presents a noninvasive way of analyzing tumor genetics and has become an important method for monitoring drug resistance and predicting efficacy (20,21).Referring to previous umbrella trials,we present a phase II trial that explored NGS-guided targeted therapy for refractory metastatic HR-positive/HER-2-negative patients(Figure 1).We aimed to search for molecular markers of endocrine therapy efficacy and explore the clinical value of ctDNA to guide precise endocrine therapy in patients with HR-positive/HER-2-negative metastatic breast cancer.

Figure 1 Clinical profile.HR,hormone receptor;ctDNA,circulating tumor DNA;NGS,next-generation sequencing;PI3K,phosphatidylinositol-3-kinase;mTOR,mammalian target of rapamycin;ESR1,estrogen receptor 1;HER-2,human epidermal growth factor receptor-2.

Materials and methods

Study design and participants

We conducted an open-label,single-center,multicohort,prospective study in patients with metastatic HRpositive/HER-2-negative breast cancer.This study was registered at ClinicalTrials.gov (NCT03786575).The main inclusion criteria were patients willing to voluntarily sign an Institutional Review Board-approved written informed consent document;at least 18 years of age;with histologically or cytologically confirmed HR-positive/HER-2-negative patients with advanced breast cancer;measurable diseases using the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1);an Eastern Cooperative Oncology Group performance status (ECOG PS) 0-2;a life expectancy ＞12 weeks;and no visceral crisis.Patients who relapsed during or after adjuvant endocrine therapy or who progressed after completing at least one previous line of treatment for advanced breast cancer were candidates for inclusion. Exclusion criteria included pregnant and/or lactating females;a history of mental disorders;severe infection or active gastrointestinal ulcers;severe liver disease (such as cirrhosis);kidney disease;respiratory disease or uncontrolled diabetes; or participation in other clinical trials within one month.

All metastatic breast cancer patients who volunteered for NGS were enrolled,and all blood samples were obtained from patients before therapy.Enrollment was based on blood-based NGS assay results,and the patients were assigned to four parallel treatment cohorts matched to mutations identified in ctDNA: 1) Cohort A included patients with abnormal activation of the PI3K/AKT/mTOR signaling pathway,preferred mTOR inhibitor (10 mg of everolimus or 2 mg of rapamycin monotherapy orally) combined with physician-determined endocrine therapy;2) Cohort B included patients with ESR1 mutation who had not received fulvestrant treatment previously.This cohort received 500 mg of fulvestrant intramuscularly (standard dose);3) Cohort C included patients with any of HER-2 mutations,preferred 400 mg of pyrotinib monotherapy orally combined with/without physician-determined endocrine therapy;and 4) Cohort D included patients with no significant gene mutations,received physician-determined treatment plan according to their clinical situation.If more than one gene mutation was identified,the priority of entry was cohort C,cohort A and cohort B.The primary outcome measure was PFS,and the secondary outcome measure was OS.

All participants voluntarily provided written informed consent to participate.This study was approved by the Regulatory and Ethics Committees of National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College.Once a patient was enrolled,treatment was administered until tumor progression occurred,there was intolerable toxicity,or the participant requested withdrawal from treatment for personal reasons.During treatment,dose adjustment was allowed for patients who experience treatment-related toxicity.At baseline,patients received computed tomography (CT) or magnetic resonance imaging (MRI)scans,and patients with bone metastases received wholebody bone emission computed tomography (ECT)scanning.CT or MRI scans were repeated every 8 weeks until 32 weeks after the beginning of treatment and then repeated every 12 weeks thereafter.RECIST 1.1 criteria were used to evaluate the tumor response.The study flowchart is shown inFigure 1.

Sample collection and DNA extraction

A volume of 10 mL of peripheral blood was collected from each patient.Peripheral blood samples were collected in Streck tubes (Streck,Omaha,NE,USA) and centrifuged within 72 h to separate the plasma from the peripheral blood cells.

Circulating DNA was isolated from plasma by the QIAamp Circulating Nucleic Acid Kit (Qiagen) and extracted by the DNeasy Blood and Tissue Kit.An Agilent 2100 Bioanalyzer and the DNA HS Kit (Agilent Technologies,Santa Clara,CA,USA) were used to analyze the size distribution of the cell-free DNA (cfDNA).

Target capture and NGS

The steps for library preparation,hybridization capture,and 1021 gene panel sequencing for whole-genome ctDNA were performed as previously described (22,23).cfDNA sequencing libraries were prepared using the KAPA DNA Library Preparation Kit (Kapa Biosystems,Wilmington,MA,USA),and sequencing libraries were prepared for gDNA using the protocols recommended in the Illumina TruSeq DNA Library Preparation Kit (Illumina,San Diego,CA,USA).DNA sequencing was conducted with the HiSeq 3000 Sequencing System (Illumina,San Diego,CA,USA) with 2×101-bp paired-end reads.

One-step NGS of 14 gene panel libraries were constructed using multiplex polymerase chain reaction(PCR) methods (reaction conditions are described inSupplementary Table S1,S2),DNA sequencing was carried out with the Ion Proton system (Thermo Fisher Scientific).A panel of 14 genes was assayed in the present study,includingPIK3CA,ESR1,HER-2,AKT1andPTEN(Supplementary Table S3).

Table S1 PCR amplification reaction system

Table S2 PCR amplification reaction conditions

Table S3 Forteen genes included in the panel

Table S3 (continued)

Table S3 (continued)

Table S3 (continued)

Table S3 (continued)

Statistical analysis

Descriptive statistics was used to summarize clinicopathological characteristics.Chi-square test or Fisher’s exact test was used to determine whether there were differences between the compliance and violation groups in baseline factors.PFS was estimated by the Kaplan-Meier method,and the log-rank test was used to compare PFS between the treatment groups.We used Cox regression analysis to determine whether there was a difference in PFS between the compliance and violation groups.Hazard ratios (HRs) were estimated using the Cox proportional hazard model.The median follow-up time was calculated using the reverse Kaplan-Meier method.P＜0.05 was considered statistically significant.All of the statistical analyses were performed using the SPSS Statistics (Version 22.0;IBM Corp.,New York,USA) or GraphPad Prism 8.0(GraphPad Software,Inc.,LaJolla,CA,USA).

Results

Patient cohort and sample collection

Between December 1,2018 and July 21,2021,a total of 113 patients underwent NGS detection of ctDNA.Seventeen patients were excluded because they started treatment without waiting for the test report.Seven patients were excluded due to loss to follow-up.Five patients were not enrolled because of incomplete baseline information.Finally,84 patients were enrolled in the study.Thirty-one patients underwent 1021 gene panel wholegenome ctDNA sequencing,and 53 patients underwent 14 gene panel one-step sequencing. The baseline characteristics of the 84 treated patients according to compliance are shown inTable 1,and according to mutations identified in ctDNA are shown inTable 2.

Table 1 Baseline characteristics of compliance and violation groups

Table 2 Baseline characteristics of patients treated according to mutations identified in ctDNA

Thirty-two patients were treated with other regimens due to comorbid diseases,drug availability,rapid disease progression,poor economic conditions and patients’request for chemotherapy and interventional therapy.The median age was 50.0 years,62 (74%) patients had baseline visceral metastases,and 22 (26%) patients had no visceral metastases.Patients had received a median of two previous lines of endocrine therapy and a median of two previous lines of chemotherapy.The median follow-up time was 30.8 [95% confidence interval (95% CI),14.1-47.6]months.The outcome of death occurred in three patients,and the secondary endpoint was not reached.

Treatment efficacy and survival analysis of umbrella trial

Among patients included in cohorts A-D,the combined median PFS was 4.9 (95% CI,3.7-6.4) months,and the median PFS in the compliance group was longer than that in the violation group (6.0vs.3.0 months,P=0.022,HR=0.57,95% CI,0.33-1.01) (Figure 2).After adjusting molecular subtype,lines of chemotherapy,lines of endocrine treatment and visceral metastasis in multivariate Cox regression model,we found the risk of disease progression was lower in the compliance group than in the violation group (P=0.023,HR=0.55,95% CI,0.33-0.92).In patients who adhered to the treatment regimen,one patient’s efficacy evaluation cannot be obtained,nineteen(31%) patients had progressive disease (PD),thirty-three(54%) patients had stable disease (SD),seven (11%)patients experienced a partial response (PR),and one patient experienced a complete response (CR).The objective response rate (ORR,CR+PR) was 13%.The disease-control rate (DCR,CR+PR+SD) was 67%.In the violation group,the best efficacy of three patients was PR,that of nine patients was SD,and that of eleven patients was PD.In the violation group,the ORR and DCR were 13% and 52%,respectively,which were lower than those in the compliance group (Figure 3).

Thirty-two patients were included in cohort A,and the median PFS of these patients was 5.2 months.In cohort A,15 patients received a treatment regimen of everolimus/sirolimus combined with endocrine therapy,and 17 patients received other treatment regimens for various reasons.The median PFS in the compliance and violation groups was 5.2 months and 3.5 months,respectively.There was no significant difference between the median PFS of the two groups (P=0.505,HR=0.80,95% CI,0.40-1.59)(Figure 4A).Eleven patients were included in cohort B,with a median PFS of 5.4 months,of whom 9 patients received an endocrine treatment regimen containing fulvestrant,and 2 patients did not adhere to the treatment regimen.The median PFS of the compliance group and violation group were 5.4 months and 5.3 months,respectively,with no significant difference observed(P=0.733,HR=0.77,95% CI,0.14-4.15) (Figure 4B).Eight patients were included in cohort C,of whom 4 patients followed the treatment regimen containing pyrotinib,and 4 patients violated the regimen.Among cohort C,the median PFS was 11.1 months in the compliance group and 2.2 months in the violation group (P=0.011,HR=0.20,95%CI,0.03-1.22) (Figure 4C).In the remaining 33 patients,targeted drug-related gene mutations were not detected,so they were assigned to cohort D to follow the treatment plan formulated by the doctor.The median PFS of patients in cohort D was 6.0 months.

Figure 2 Kaplan-Meier curves for median PFS of compliance group and violation group (HR=0.57,95% CI,0.33-1.01;P=0.022).mPFS,median progression-free survival;HR,hazard ratio;95% CI,95% confidence interval.

Figure 3 Efficacy evaluation of patients in treatment cohorts according to mutations identified in ctDNA.ctDNA,circulating tumor DNA;SD,stable disease;PD,progressive disease;PR,partial response;CR,complete response.

Figure 4 Kaplan-Meier curves for median PFS in the compliance and violation groups in patients with PI3K/AKT/mTOR mutation(HR=0.80,95% CI,0.40-1.59;P=0.505) (A),ERS1 mutation (HR=0.77,95% CI,0.14-4.15;P=0.733) (B) and HER-2 mutation (HR=0.20,95% CI,0.03-1.22;P=0.011) (C).PI3K,phosphatidylinositol-3-kinase;mTOR,mammalian target of rapamycin;ESR1,estrogen receptor 1;HER-2,human epidermal growth factor receptor-2;PFS,progression-free survival;mPFS,median PFS;HR,hazard ratio;95% CI,95%confidence interval.

Discussion

Breast cancer recurrence,metastasis and treatment resistance may be related tode novoactionable mutations.Most studies in the field of ctDNA have only focused on monitoring treatment response (24-26).Our previous studies revealed that the molecular tumor burden index(mTBI) of ctDNA could be used as a potential evaluation criterion for antitumor therapy response in patients with breast cancer (27).At present,the main source of identifying mutations in patients with metastatic breast cancer is tumor biopsy.Previous studies have shown that the use of plasma to detect mutations has good consistencycompared to matching tumor biopsies (28).Reliable and comprehensive real-time genomic ctDNA detection is of great importance to conduct targeted and precise treatment(29).Recently,several prospective clinical studies have used NGS detection to guide clinical strategies,but these have all been pan-cancer studies and included only a limited number of breast cancer cases (30-32).A recent trial demonstrated that genomically guided therapy did not improve outcomes compared with the physician’s treatment choice in triple-negative breast cancer (TNBC) (33).It focused on adjuvant therapy for residual early-stage TNBC patients (33).In contrast,in the present study,we investigated the application of blood-based NGS detection of ctDNA to search for molecular markers which could be valuable in developing endocrine therapy strategies for HR-positive/HER-2-negative patients with metastatic breast cancer.

Our results reveal the clinical relevance of endocrine therapy for HR-positive/HER-2-negative metastatic breast cancer activation mutations based on NGS findings.In our study,the median PFS of the patients who followed the scheduled treatment plan determined by ctDNA testing was significantly (3.0 months) longer than that of the patients who did not follow the scheduled treatment plan.This indicates that a targeted therapeutic strategy guided by NGS detection for refractory metastatic HRpositive/HER-2-negative breast cancer may be able to achieve a more favorable PFS.This finding has also been confirmed in studies on metastatic gastric cancer,colorectal cancer and non-small-cell lung cancer,which revealed that ctDNA could guide treatment management to improve clinical outcomes (34-36).In addition,we also observed that breast cancer patients with HER-2 mutations identified by ctDNA testing could achieve an 11.1-month median PFS benefit by receiving a treatment regimen containing pyrotinib.In contrast,the median PFS of patients who did not receive a treatment regimen containing pyrotinib was only 2.2 months.This finding is similar to that of previous studies.In a previous phase II study,metastatic HER-2 amplification-negative,mutationpositive breast cancer received pyrotinib treatment,achieving a 40% objective remission rate and 60% clinical benefit rate (18).In the FUTURE study,two assessable patients in the cohort of HER-2-mutant TNBC achieved a PR after two cycles of pyrotinib with capecitabine therapy(37).In the same way,patients with an HER-2 mutation identified by ctDNA testing who were given neratinib had a 45% clinical benefit rate (38).The findings of this study suggest that NGS detection may provide new treatment options for HR-positive/HER-2-negative metastatic breast cancer patients with HER-2 mutation.

The therascreen PIK3CA RGQ polymerase chain reaction assay was recently approved by the US Food and Drug Administration (FDA) as a companion diagnostic assay to detect PIK3CA mutations in breast cancer,which highlights the role of liquid biopsy in treatment and in guiding the application of alpelisib in HR-positive/HER-2-negative breast cancer (39,40).Nevertheless,alpelisib is currently unavailable for breast cancer survivors in China.Everolimus and sirolimus,which are also mTOR inhibitors,are currently mainly used in the field of immunosuppression after kidney transplantation and have certain application value in the field of cancer (7,41).In our study,cohort A was used to test the hypothesis that HRpositive/HER-2-negative metastatic breast cancer with PI3K/AKT/mTOR mutation may benefit from mTOR inhibitors,but patients who followed the treatment regimen and received everolimus or rapamycin did not benefit significantly in terms of PFS compared with patients who violated the treatment regimen.A previous study discovered that the detection of ESR1 mutations through ctDNA analysis in metastatic breast cancer predicted a lack of benefit from AI therapy;it was found that these patients may benefit from fulvestrant (42).However,in our study,patients with ESR1 mutations who adhered to the regimen and received fulvestrant did not achieve a PFS benefit compared to the patients who did not adhere to the treatment regimen.We speculate that these inconsistencies may be due to the small sample size,and some patients in the violation group received chemotherapy.A larger sample size may produce different results,so we plan to carry out research with a larger sample size in the future.

The present study was a prospective study based on an umbrella experiment concept designed to identify the emergence of actionable mutations through blood-based NGS detection to guide endocrine therapy for HRpositive/HER-2-negative metastatic breast cancer so that more precise treatment can be provided.Based on the results of NGS detection,patients could benefit from receiving an endocrine therapy regimen in terms of PFS.In particular,among patients with HER-2 mutation,treatment with pyrotinib was able to significantly prolong the median PFS.This indicates that ctDNA testing by NGS is a promising strategy to clinically guide endocrine therapy selection.

Several limitations in the use of blood-based NGS detection to guide clinical endocrine therapy for HRpositive/HER-2-negative metastatic breast cancer need to be acknowledged.One is that the detection of ctDNA by NGS detection depends on the tumor cells flowing into the blood,so patients with less tumor abscission and those with a lower tumor burden may not be sufficiently evaluated by this method.The false negative rate of ctDNA based on blood testing is still higher than that of tissue testing.Another limitation of the current analysis is that the number of patients in each cohort was relatively small.Multi-center studies are necessary to expand the study population and further validate our conclusions in the future.In addition,due to the limited follow-up time,the secondary endpoint was not reached,so OS could not be analyzed.Third,all patients were from one center,which limits the generalizability of these findings.

Conclusions

In summary,ctDNA testing is a promising strategy to guide patient management and targeted therapy selection for refractory metastatic HR-positive/HER-2-negative breast cancer,which can help to achieve more favorable PFS outcomes.The study also confirmed that patients with HER-2 mutation can be identified by NGS detection,which could aid in the development of new treatment strategies.The findings from our prospective study are expected to further promote the realization of precision treatment for HR-positive/HER-2-negative metastatic breast cancer survivors.

Acknowledgements

This work was supported by grant from the CAMS Innovation Fund for Medical Sciences (CIFMS,No.2021-I2M-1-014).

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.