PURPOSE
Human epidermal growth factor receptor 2 (HER2)–directed treatments improve outcomes for patients with HER2-positive metastatic breast cancer (MBC). Current identification of patients with HER2-positive disease relies on tumor tissue testing, which can be inaccurate because of tumor heterogeneity or tumor evolution. Circulating tumor cells (CTCs) are often present in patients with cancer. We hypothesized that HER2 assessment of CTCs in patients with HER2-negative breast cancer could identify a subset of patients with HER2-positive CTCs who could benefit from HER2-directed treatments.
METHODS
This was a single-arm, two-stage, phase II trial. Patients with HER2-negative progressive MBC with HER2-positive CTC (defined as HER2/CEP17 ratio ≥ 2.0 by fluorescence in situ hybridization), ≥ 1 prior chemotherapy regimen for MBC, and no prior vinorelbine received trastuzumab in combination with vinorelbine on days 1, 8, and 15 of a 21-day cycle. The primary end point was objective response rate.
RESULTS
From January 2013 to June 2014, we prospectively screened CTCs from patients with HER2-negative MBC. CTCs were detected in 201 of 311 patients (65%). The median number of CTCs was 10 (interquartile range, 3-57). Sixty-nine of 311 patients (22%) had HER2+ CTCs, with a median of three HER2+ CTCs (range 1-21). Twenty patients with HER2+ CTCs were treated on study. At data cutoff (January 13, 2017), no patients remained on study therapy. The objective response rate was 5% (95% CI, 0.1 to 24.9), with one of 20 patients experiencing a partial response. The clinical benefit rate was 20.0% (1 partial response and 3 stable diseases > 24 weeks, 95% CI, 5.7% to 43.7%). The median progression-free survival was 2.7 months.
CONCLUSION
CTC analysis of patients with HER2-negative MBC identifies a subset with HER2-amplified CTCs. However, clinical activity of an HER2-directed regimen in this population was low. The functional significance of HER2-positive CTCs remains uncertain.
BACKGROUND
Human epidermal growth factor receptor 2 (HER2) (HER-2/neu and erbB2) is a transmembrane tyrosine kinase with extensive homology to the epidermal growth factor receptor.1,2 HER2 protein overexpression and/or gene amplification occurs in approximately 20% of primary breast cancers. These cancers, designated as HER2-positive, are associated with diminished disease-free and overall survival.3 The use of trastuzumab, a monoclonal antibody directed against the HER2 extracellular domain, has improved both systemic control and overall survival in patients with metastatic breast cancer (MBC). Although there appears good concordance between the HER2 status of a patient's primary breast tumor and that of distant metastases, several studies have demonstrated that in a minority of patients (7%-26%) with HER2-negative primary breast cancers, biopsies of the metastatic tumor show evidence of HER2 overexpression4-6 and the majority of these patients' clinical care will be altered to include anti-HER2 therapy.7 Despite this evidence, it is likely that in many patients with clinically HER2-negative primary tumors, the HER2-positive status of their metastatic disease is never identified because repeat tumor biopsies are often not performed in this setting. Therefore, noninvasive tests to determine HER2 status could prove to be clinically useful.
CONTEXT
Key Objective
Human epidermal growth factor receptor 2 (HER2)–directed treatments improve outcomes for patients with HER2-positive metastatic breast cancer (MBC), but reliance on tissue-based assays to identify HER2-positive cancers may exclude patients who may benefit from HER2 therapy because of tumor heterogeneity or tumor evolution. To determine if analysis of circulating tumor cells (CTCs) could identify additional patients who may benefit from HER2 therapy, we conducted one of the first prospective clinical trials of an HER2 therapy (trastuzumab and vinorelbine) in patients with HER2+ CTCs and clinically HER2-negative MBC.
Knowledge Generated
We identified HER2+ CTCs in 22% of screened patients with MBC. The clinical benefit rate of trastuzumab and vinorelbine was 20% in this heavily pretreated population.
Relevance
CTC analysis of patients with HER2-negative MBC identifies a subset with HER2-amplified CTCs. Clinical activity of an HER2-directed regimen in this population was low. The functional and clinical significance of HER2-positive CTCs remains uncertain.
Methods to evaluate prognosis and response to therapy via measurement of microscopic tumor burden are of significant interest, and circulating tumor cells (CTCs) are one such tool. With the use of various technologies, it is possible to detect rare CTCs (one in one million to one in 100 million total blood cells) in women with advanced breast cancer, and CTCs correlate with both progression-free and overall survival in these patients.8-16 In addition to enumeration, CTCs can be molecularly characterized using immunofluorescence, RNA, or genomic methods to assess features of CTCs that may correlate with response or resistance to therapy. For example, evaluation of CTCs using immunofluorescence or fluorescence in situ hybridization (FISH) can detect the presence of HER2 overexpression and gene amplification. De Gregorio et al17 screened 1,123 patients with HER2-negative MBC for CTCs and found that of the 711 (63.3%) patients with at least one detectable CTC, 134 (11.9%) had at least one detectable HER2-positive CTC. Munzone et al showed that of 45 patients who did not overexpress HER2 on their primary tumors, eight (18%) acquired HER2 overexpression on CTCs with metastatic progression.18 Fehm et al4 found that 27% of patients with HER2-negative primary tumors acquired HER2 overexpression during the course of chemotherapy. Meng et al identified acquired HER2 gene amplification in CTCs in nine of 24 (37.5%) patients with primary tumor HER2-negative progressive breast cancer. Four of the nine patients were subsequently treated with a trastuzumab-containing regimen; 1 complete response (CR) and 2 partial responses (PRs) were observed.19 If HER2 overexpression and/or gene amplification can be acquired and detected on CTCs, it is important to understand if these patients would benefit from targeted, anti-HER2 therapy. To prospectively address this question, we conducted a single-arm phase II trial of the combination of trastuzumab and vinorelbine among patients with clinically HER2-negative MBC with HER2-amplified CTCs. Vinorelbine was chosen to pair with trastuzumab in this study as the combination is generally well-tolerated and has demonstrated synergy in HER2+ MBC with objective response rates (ORRs) in the range of 44%-86%.20 In addition, vinorelbine monotherapy is not typically used until late in the course of HER2-negative MBC, minimizing patients who would be excluded from this study because of prior vinorelbine use.
METHODS
CTC Sample Screening, Collection, and Analysis
To be eligible for CTC collection and screening, patients must have had clinically HER2-negative MBC, defined as an FISH ratio < 2.0 and/or immunohistochemistry (IHC) 0 or 1+ on a tissue sample (primary or metastatic). If the tumor was 2+ by IHC, it must have also been FISH-negative for the patient to be eligible. All other tumor biopsies from the patient must have been HER2-negative by the same criteria, if the results of those biopsies were available and they were evaluated for HER2 status. Patients must have received at least one prior chemotherapy regimen for MBC and could have received any number of biologic or hormonal regimens. CTC blood samples were collected in preservative tubes provided by Biocept Inc, with the goal of four tubes per patient. Samples were collected at screening, and for those patients who participated in the treatment phase of the study (see below), at the beginning of each treatment cycle, and at time of disease progression. The blood samples were transported and stored at room temperature (15-30°C) until processing. Initially, CTCs were collected and evaluated by FISH for HER2 copy number at the Dana-Farber Cancer Institute using a modification of the Veridex CellSearch Profile kit as previously described.21 After the first 11 patients were enrolled (cohort A), the Dana-Farber/Harvard Cancer Center Institutional Review Board required CTC analysis to be completed in a Clinical Laboratory Improvement Amendments–approved laboratory and subsequent CTC screening and HER2 FISH were performed by Biocept Inc (cohort B). Here, we report only on patients screened by Biocept Inc (cohort B). CTCs were assessed at Biocept Inc in a Clinical Laboratory Improvement Amendments–approved laboratory and were captured using an antibody cocktail directed to 10 tumor-associated cell surface antigens, further labeled with a biotin-conjugated secondary antibody, and captured in a proprietary streptavidin-derivatized microchannel, as previously described.22 FISH assays for HER2 and epidermal growth factor receptor using Abbott probes were performed in the microchannel and evaluated and scored by cytotechnicians using fluorescence microscopy at high power. CTCs were further evaluated in the microchannel for expression of cytokeratin using a mixture of anticytokeratin 7/17 (clone C-46), 18 (clone DA/7), 19 (clone A53-B/A2), and pan-cytokeratin (clone C-11) antibodies (BioLegend, San Diego, CA) labeled with AlexaFluor-488 (Life Technologies, Grand Island, NY) and CD-45 (labeled with Alexa-594) as previously described.22 Individual CTCs were considered amplified if the ratio of HER2 copy number to CEP17 copy number was ≥ 2.0. If a sample was FISH-positive by this definition, the patient's treating physician was notified that the patient was eligible for the treatment phase of the study.
Patients
Adult patients with progressive, HER2-negative MBC (see the above definition) with HER2-amplified CTCs were eligible for the treatment phase of the study (see the CTC Sample Screening, Collection, and Analysis). Other key inclusion criteria included the presence of measurable disease, Eastern Cooperative Oncology Group performance status of 0-2, and a cardiac ejection fraction of ≥ 50%. Participants must have received at least one prior treatment for MBC, and there was no limit on the number of previous therapy lines. Patients could not have previously received trastuzumab or vinorelbine. Patients were excluded if they had progressive or symptomatic brain metastases. All patients signed informed consent approved by the institutional review board and were enrolled from clinics within the Dana-Farber/Harvard Cancer Center.
Treatment Plan
This was a single-arm, two-stage phase II study designed to evaluate the efficacy of trastuzumab-vinorelbine combination therapy in patients with HER2-negative MBC (as defined on tumor tissue) and HER2 amplified CTCs. Intravenous trastuzumab (8 mg/kg for cycle 1 and 6 mg/kg each subsequent cycle) on day 1 and intravenous vinorelbine (25 mg/m2) on days 1, 8, and 15 were administered once daily on a 21-day cycle. Patients were evaluated every two cycles with computed tomography scans of the chest, abdomen, and pelvis.
Patients were to remain on study treatment until disease progression, unacceptable toxicity, discontinuation of therapy because of physician request, or withdrawal of consent. Treatment holds or discontinuation of trastuzumab was required for patients who experienced significant reduction in left ventricular ejection fraction. Treatment holds and/or dose reductions of vinorelbine were required for patients experiencing prolonged grade ≥ 2 hematologic, grade ≥ 2 hepatic, or any grade ≥ 3 toxicity.
Statistical Analysis
The primary objective was to assess ORR (to include [CR] + [PR]) to trastuzumab and vinorelbine. Evaluation was performed by local investigators according to RECIST 1.1 criteria. Simon's23 two-stage design was used. The null hypothesis that the true response rate is 20% was tested against a one-sided alternative. In the first stage, 17 patients were to be accrued. If there were four or fewer responses in these 17 patients, the study would be stopped. Otherwise, 18 additional patients would be accrued for a total of 35. The null hypothesis would be rejected if 11 or more responses were observed in 35 patients. If the true response rate was 20%, the probability of early termination was 0.76 and the probability that the regimen would be declared worthy of further study was 0.06. If the true response rate was 45%, the probability of early termination was 0.06 and the probability that the regimen would be declared worthy of further study was 0.92.
Secondary objectives included characterization of the safety and tolerability of the drug combination, assessment of progression-free survival (PFS), and clinical benefit rate (CR + PR + stable disease > 24 weeks). Additional end points included description of the number of CTCs and the CTCs' characteristics before and after therapy, and exploration of the correlation of these findings with response.
RESULTS
CTC Screening
A total of 311 patients with HER2-negative MBC were screened for the presence of HER2-amplified CTCs. CTCs were detected in 201 of 311 patients (65%) (Table 1). The median number of CTCs per patient was 10 (interquartile range, 3-57). Sixty-nine of the 311 patients (22%) had HER2+ CTCs, with a median number of three HER2+ CTCs (range 1-21). Of these, 36% (25 of 69) had cytokeratin (CK)+/HER2+ CTCs and 45% (31 of 69) of patients had only CK−/HER2+ CTCs. The remaining patients (13 of 9, 19%) had both CK+/HER2+ and CK−/HER2+ CTCs present.
TABLE 1.
Prevalence of HER2-Positive CTCs in Patients With Metastatic HER2-Negative Breast Cancer
There was no significant association between HER2+ CTC status and histopathologic characteristics of the patient's primary tumor (Table 2). This lack of association was observed when evaluating all HER2+ CTCs and within specific HER2+ CTC CK subgroups (data not shown).
TABLE 2.
Tumor Characteristics and CTC Status
We then explored the relationship between the presence of CTCs and overall survival, with a median follow-up of 72 months. As expected, patients with detectable CTCs experienced worse median overall survival compared with patients without detectable CTCs (14.55 months v 21.55 months, P = .00037; hazard ratio, 1.60; 95% CI, 1.23 to 2.08) (Fig 1A). Among the patients with detectable CTCs, there was no difference in overall survival between patients with HER2+ CTCs and those with HER2− CTCs (10.74 months v 14.95 months; P = .56; hazard ratio, 1.11; 95% CI, 0.81 to 1.50) (Fig 1B).
FIG 1.
OS by (A) total CTCs and (B) HER2 status for CTC-positive patients. CTC, circulating tumor cell; HER2, human epidermal growth factor receptor 2; OS, overall survival.
Patient Characteristics
A total of 20 women with HER2+ CTCs were enrolled in the treatment phase of the study between February 2013 and July 2014, and their baseline characteristics are shown in Table 3. The median age at the time of study entry was 54 (34-66). Thirteen (65%) patients had ER-positive disease, and seven (35%) had ER-negative disease. This was a relatively heavily pretreated population with a median of three prior lines of chemotherapy in the advanced disease setting.
TABLE 3.
Characteristics of Treated Patients
Efficacy
As of January 13, 2017, no patients remained on study treatment and the median follow-up time was 2.7 months (range 1-15). The study was stopped early under the Simon two-stage design. Reasons for discontinuation of study treatment are shown in Table 4. The median number of cycles received was four (range 2-21). One of 20 patients experienced a PR (ORR, 5%; 95% CI, 0.1 to 24.9).
TABLE 4.
Efficacy of Study Treatment and Reasons for Discontinuation of Treatment
The clinical benefit rate was 20.0% (1 PR and 3 stable diseases > 24 weeks, 95% CI, 5.7% to 43.7%) (Table 4). The median PFS was 2.7 months (lower 95% limit 2.6) (Data Supplement, online only).
Toxicity
Grade ≥ 3 adverse events for all patients in the treatment phase of the study are shown in the Data Supplement. The most common grade 3 event was neutropenia (20%), followed by anemia (10%). There were no ≥ 4 adverse events.
Serial CTCs
Total baseline CTC counts were available for all 20 patients (median 5.5, range 1-249). Twelve patients had both baseline and cycle 3 day 1 (C3D1) samples assessed. Signed-rank test was used to compare the baseline and C3D1 total CTCs and HER2-positive CTCs among the 12 patients. There was a significant decrease in total CTCs from baseline (median = 31) to the C3D1 (median 12.5) assessment (P = .03), but there was no difference in HER2-positive CTCs between baseline (median = 2) and C3D1 (median = 2) (P = .52) (Table 5). Of note, four of four CTCs detected at baseline were HER2+ in the one patient who experienced a PR.
TABLE 5.
On-Study CTCs
DISCUSSION
In this phase II, single-arm study, we screened patients with HER2-negative MBC for the presence of HER2-positive CTCs. To explore the functional significance of HER2-positive CTC, we evaluated the efficacy of the combination of trastuzumab and vinorelbine in these patients. Our study adds to the body of literature in breast cancer using liquid biopsy to attempt to identify markers predictive of treatment response. The prevalence of CTCs in our study, 65%, is consistent with other studies of MBC.6,8,9 Our finding of HER2-positive CTCs in 22% of patients with HER2-negative MBC is also consistent with what has been observed in previous studies.17-19,24
Although other studies have observed HER2-positive CTC in patients with HER2-negative MBC, to our knowledge, our trial, along with the previously published CirCe study,25 is the first to report on the prospective evaluation of HER2-directed therapy in such patients. In the current study, the ORR of 5% for the combination therapy was low and did not meet the prespecified threshold for further study. Although total CTCs decreased from baseline to C3D1 in the overall population, perhaps reflective of early treatment effect, we did not see meaningful changes in HER2-positive CTCs.
One explanation for the low clinical response rate could be the liberal threshold we used for declaring CTCs HER2-positive. In this study, a patient's sample was considered positive if the ratio of HER2 copy number to CEP17 copy number was ≥ 2.0. We did not specify the minimum number of CTCs needed to make the determination, and the median number of HER2-positive CTCs detected in this population was only three. We also did not require a minimum HER2 copy number, and confirmation of HER2 overexpression by IHC was not feasible in these CTC specimens. It is possible that a stricter definition of HER2-positive CTCs would identify a group of patients with HER2-negative primary disease who would benefit from HER2-directed regimens. A recent study by De Gregario et al17 used another method of CTC collection together with a more stringent cutoff of IHC staining of 3+ and found that 11.9% of screened patients had HER2-positive CTCs, approximately half of the prevalence we observed. Data from the DETECT III clinical trial (ClinicalTrials.gov identifier: NCT01619111) examining the efficacy of lapatinib in patients with HER2-negative MBC with HER2-positive CTCs are anticipated.26 In the CirCe trial, a prospective study of the efficacy of T-DM1 in patients with previously treated HER2-negative MBC and HER2-positive CTCs, one confirmed PR was observed of 11 patients treated.25
Our study used a CTC isolation method to select cells on the basis of multiple epithelial-specific cell surface markers with the goal of collecting both CK-positive and CK-negative CTCs.27 In MBC, and in other epithelial cancers, there is evidence that some tumor cells lose CK expression because of the process of epithelial mesenchymal transition (EMT), for example.28-30 In our study, the initial selection of CTC using epithelial-specific cell surface markers and the further identification of the cells via HER2 amplification help to confirm their malignant origin. Although the epithelial-specific markers and HER2 amplification make it highly likely these are tumor cells, it is certainly possible that these CK-negative cells may not act similar to classic HER2-amplified breast cancer cells in terms of response to therapy. If true, the significant presence of these CK− cells could have contributed to the low level of clinical activity seen in the study.
Additionally, the landscape of HER2-directed therapies has evolved significantly since we initiated our study, and a number of more effective treatment regimens are now commercially available or being tested in clinical trials. Some of these drugs may provide benefit for patients with tumors expressing lower levels of HER2. In particular, one promising drug, trastuzumab deruxtecan (DS8201a), has shown clinical activity in patients with HER2-negative MBC by conventional clinical criteria, but with low level (1+ or 2+) HER2 staining by IHC.31,32 It is possible that assessment of HER2-positive CTCs would be another, less invasive, way to identify patients who might benefit from this drug.
Another potential explanation for the lack of clinical activity seen in our study is that these patients were heavily pretreated, having received a median of three (range 1-6) lines of chemotherapy before enrollment, in addition to any hormonal therapy received. This may represent a group of patients with relatively treatment refractory disease, who might have benefited from the therapy had it been earlier in their course of treatment. Tumor heterogeneity may also play a role in the outcome of this study; HER2-positive CTCs found in patients with clinically HER2-negative disease could derive from cancers in which only a small subset of cells are HER2 amplified.
In summary, this study confirms previous reports indicating that HER2-positive CTCs are present in a subset of patients with HER2-negative MBC. Nonetheless, the clinical activity of an HER2-directed regimen in this patient population was very low and the functional significance of these HER2-positive CTCs remains uncertain.
Heather A. Parsons
Consulting or Advisory Role: Foundation Medicine
Research Funding: Puma Biotechnology (paid to institution)
William T. Barry
Employment: Rho
Gerburg M. Wulf
Stock and Other Ownership Interests: Selecta Biosciences
Research Funding: Merck, GlaxoSmithKline
Patents, Royalties, Other Intellectual Property: Pin1 as a marker for abnormal cell growth Patent number: 8129131
Steven J. Isakoff
Consulting or Advisory Role: AbbVie, OncoPep, Puma Biotechnology, Seattle Genetics, Novartis
Research Funding: Genentech, PharmaMar, AbbVie, OncoPep, Merck, AstraZeneca/MedImmune, Outcomes4Me
Ian E. Krop
Employment: AMAG Pharmaceuticals, Freeline Therapeutics
Leadership: AMAG Pharmaceuticals, Freeline Therapeutics
Stock and Other Ownership Interests: AMAG Pharmaceuticals, Freeline Therapeutics, Vertex
Honoraria: Genentech/Roche, AstraZeneca, Celltrion
Consulting or Advisory Role: Genentech/Roche, Seattle Genetics, Daiichi Sankyo, Macrogenics, Taiho Pharmaceutical, Context Therapeutics, Novartis, Merck, Ionis Pharmaceuticals, Bristol Myers Squibb, AstraZeneca
Research Funding: Genentech, Pfizer, Macrogenics
No other potential conflicts of interest were reported.
PRIOR PRESENTATION
Presented in part in poster format at the San Antonio Breast Cancer Symposium, December 9-13, 2014.
CLINICAL TRIAL INFORMATION
AUTHOR CONTRIBUTIONS
Conception and design: Erin R. Macrae, William T. Barry, Ian E. Krop
Provision of study materials or patients: Gerburg M. Wulf, Steven J. Isakoff
Collection and assembly of data: Heather A. Parsons, Erin R. Macrae, William T. Barry, Gerburg M. Wulf, Steven J. Isakoff, Ian E. Krop
Data analysis and interpretation: Heather A. Parsons, Erin R. Macrae, Hao Guo, Tianyu Li, Nabihah Tayob, Steven J. Isakoff, Ian E. Krop
Manuscript writing: All authors
Final approval of manuscript: All authors
Accountable for all aspects of the work: All authors
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
The following represents disclosure information provided by the authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.
Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).
Heather A. Parsons
Consulting or Advisory Role: Foundation Medicine
Research Funding: Puma Biotechnology (paid to institution)
William T. Barry
Employment: Rho
Gerburg M. Wulf
Stock and Other Ownership Interests: Selecta Biosciences
Research Funding: Merck, GlaxoSmithKline
Patents, Royalties, Other Intellectual Property: Pin1 as a marker for abnormal cell growth Patent number: 8129131
Steven J. Isakoff
Consulting or Advisory Role: AbbVie, OncoPep, Puma Biotechnology, Seattle Genetics, Novartis
Research Funding: Genentech, PharmaMar, AbbVie, OncoPep, Merck, AstraZeneca/MedImmune, Outcomes4Me
Ian E. Krop
Employment: AMAG Pharmaceuticals, Freeline Therapeutics
Leadership: AMAG Pharmaceuticals, Freeline Therapeutics
Stock and Other Ownership Interests: AMAG Pharmaceuticals, Freeline Therapeutics, Vertex
Honoraria: Genentech/Roche, AstraZeneca, Celltrion
Consulting or Advisory Role: Genentech/Roche, Seattle Genetics, Daiichi Sankyo, Macrogenics, Taiho Pharmaceutical, Context Therapeutics, Novartis, Merck, Ionis Pharmaceuticals, Bristol Myers Squibb, AstraZeneca
Research Funding: Genentech, Pfizer, Macrogenics
No other potential conflicts of interest were reported.
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