Ann Surg Oncol DOI 10.1245/s10434-015-4600-6
ORIGINAL ARTICLE – BREAST ONCOLOGY
Circulating Tumor Cells After Neoadjuvant Chemotherapy in Stage I–III Triple-Negative Breast Cancer Carolyn Hall, PhD, Mandar Karhade, MBBS, MPH, Barbara Laubacher, BSN, Amber Anderson, BS, Henry Kuerer, MD, PhD, Sarah DeSynder, MD, and Anthony Lucci, MD Department of Surgical Oncology, Unit 444, The University of Texas MD Anderson Cancer Center, Houston, TX
ABSTRACT Background. Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen and progesterone receptor expression and HER-2 gene amplification. Circulating tumor cells (CTCs) can be identified in 25 % of nonmetastatic breast cancer patients, and the identification of C1 CTC predicts outcome. This study was designed to determine whether CTCs present after neoadjuvant chemotherapy (NACT) predicted worse outcome in nonmetastatic TNBC patients. Methods. CTCs were assessed in 57 TNBC patients with nonmetastatic TNBC after the completion of NACT. CTCs (per 7.5 ml blood) were identified using the Cell SearchÒ System (Janssen). Log-rank test and Cox regression analysis were applied to establish the association of CTCs with relapse-free (RFS) and overall survival (OS). Results. Median follow-up was 30 months, and mean age was 53 years. Fifty-four patients (95 %) had [2-cm tumors, 42 (84 %) were nuclear grade 3, and 42 (74 %) had positive axillary lymph nodes. One or more CTC was identified in 30 % of patients. CTC presence was not associated with primary tumor size, high grade, or lymph node positivity. Multivariate analysis demonstrated that detection of C1 CTC predicted decreased RFS (log-rank P = 0.03, HR 5.25, 95 % CI 1.34–20.56) and OS (log-rank P = 0.03, HR 7.04, 95 % CI 1.26–39.35). Conclusions. One or more CTCs present after NACT predicted relapse and survival in nonmetastatic TNBC patients. This information would be helpful in future clinical trial design of adjuvant treatments for TNBC patients who are at risk for relapse after completing NACT.
Ó Society of Surgical Oncology 2015 First Received: 26 March 2015 A. Lucci, MD e-mail:
[email protected]
Breast cancer is a heterogeneous disease that is classified and staged using clinical and pathologic features, including tumor size, histological grade, estrogen (ER) and progesterone (PR) hormone receptor status, human epidermal growth factor receptor 2 (HER2/neu) amplification, and axillary lymph node (ALN) involvement. ER and/or PR are expressed in the majority of primary breast cancers, whereas HER2/neu is amplified in approximately 20 % of tumors. On the other hand, 10–20 % of primary breast cancers lack ER/ PR and HER2/neu expression and thus are classified as triplenegative breast cancer (TNBC).1 Effective management of TNBC patients remains clinically challenging because of the absence of ER/PR and HER2/neu, and the aggressive nature of TNBC. TNBC patients often are treated with a combination of sequential chemotherapy, surgery, and radiation. A study of 1782 breast cancer patients by Liedtke et al. demonstrated that TNBC patients respond favorably to neoadjuvant chemotherapy (NACT) and are more likely to achieve pathologic complete response (pCR, defined as no residual invasive cancer in the excised tumor and lymph nodes) following completion of NACT than non-TNBC patients (22 vs. 11 %, respectively).2 Although pCR is a powerful predictor of outcome, TNBC patients with residual disease following NACT experience significantly shorter relapse-free (RFS) and overall survival (OS) than non-TNBC patients with residual disease after NACT.2,3 The poorer outcome observed for TNBC patients with post-NACT residual disease might be explained by the prognostic features of TNBC (higher nuclear grade, increased incidence of visceral/cerebral metastases) or because non-TNBC patients may derive benefit from both chemotherapy and endocrine/anti-HER2/neu therapies.2,4,5 Interestingly, the reported recurrence and death rates are higher for TNBC compared with non-TNBC only in the first 3 years after diagnosis.2,3,6 NACT has been increasingly used in the management of nonmetastatic patients to increase the number of patients
C. Hall et al.
who will be candidates for breast conservation and to provide early information regarding systemic treatment response.7 The rationale for NACT administration in nonmetastatic breast cancer is determined by prognostic indicators, including primary tumor size, ALN metastasis, and primary tumor characteristics, such as high-grade, ER/ PR negativity, nonlobular invasive histology, and high Ki67 staining.7,8 Response to NACT is routinely assessed at surgical resection, and the information obtained is used to assess the likelihood of breast cancer recurrence and survival. Randomized trials have demonstrated that pCR predicted improved RFS and OS in TNBC patients.9–12 However, published studies utilizing standard chemotherapy regimens have not demonstrated significant differences in RFS or OS when these modalities were administered either as NACT or as postoperative (adjuvant) therapies.13,14 During the past decade, clinical researchers have actively pursued studies to identify and determine the prognostic significance of occult micrometastatic cells within the blood (circulating tumor cells, CTCs) of nonmetastatic breast cancer patients. CTCs are subpopulations of cells within heterogeneous primary tumors that have acquired motility and invasive capabilities that facilitate dissemination into the bloodstream. After invading the bloodstream, they can travel to distant sites, sometimes remaining undetected in a quiescent state for an extended period of time, before they establish distant metastases in bone, lung, liver, or brain.15 Although the mechanisms mediating micrometastatic dissemination to the bloodstream are unknown, CTCs are rare (as few as 1 CTC/106 hematopoietic cells) cells that remain undetected by serum marker assays (carcinoma antigen 15.3, carcinoembryonic antigen) and standard high-resolution imaging modalities currently employed to stage patients.16 They are heterogeneous populations of cells with varying biomarker expression, viability, dormancy, and metastatic capabilities.15 The Cell Search SystemÒ (Janssen Diagnostics, Raritan, NJ) can detect as few as one CTC per 7.5 mL of peripheral blood and to date remains the only U.S. FDA-approved methodology for CTC detection in metastatic breast cancer patients. Recent studies have demonstrated the predictive significance of CTCs in nonmetastatic patients. Results from the French REMAGUS02 trial, the German SUCCESS-A trial, Franken et al., van Dalum et al., and our group, indicate that identification of one or more CTC predicts both RFS and OS in nonmetastatic breast cancer patients.17–21 However, no data have been published regarding the prognostic significance of CTC identification in nonmetastatic TNBC patients after NACT. We hypothesized that presence of CTCs following NACT would predict worse RFS and OS in nonmetastatic TNBC patients, irrespective of ALN status or other primary tumor characteristics. If CTC presence following NACT
were to contribute to the currently available prognostic information, it would be beneficial to identify nonmetastatic TNBC patients at high risk for disease progression who might derive benefit from additional adjuvant therapies or inclusion in clinical trials of novel therapies for high-risk patients. MATERIALS AND METHODS Patients This study included 57 stage I-III TNBC patients undergoing surgery for their primary tumor between February 2005 and February 2014. All eligible TNBC patients with nonmetastatic breast cancer who had completed NACT were offered enrollment by the participating surgeons (from 2005 to 2010: Dr. Lucci, and from 2010 to 2014: Drs. Lucci, Kuerer, and DeSnyder) at The University of Texas MD Anderson Cancer Center. The institutional review board at The University at Texas MD Anderson Cancer Center approved this prospective study (04-0698; PI: A.L.), which included CTC assessment on samples taken before initial surgery for the primary breast cancer. We obtained informed, written consent from all patients prior to collecting blood. Enrollment was strictly voluntary, and patient results were blinded from investigators by use of a random number system as the unique patient identifier. Patients with bilateral breast cancer or any other malignancy within 5 years of diagnosis of the current cancer were ineligible. Staging and Classification The primary TNM staging [primary tumor (T), regional nodes (N), distant metastases (M)] and tumor grade was designated according to the criteria set by the American Joint Commission on Cancer (AJCC) and Black’s nuclear grading system, respectively.22,23 Clinical stage was defined as TNM stage determined at the time of first diagnostic procedure confirming the invasive component of the tumor. Tumor sections were immunostained for ER, PR, and HER2 using previously published procedures.24 Primary breast tumors that expressed nuclear staining in C10 % of tumor cells were regarded as positive for ER and/or PR expression. Immunostaining results for HER2 were scored as positive when[10 % of the tumor cells had membranous staining or when fluorescence in situ hybridization (FISH) for HER2 gene amplification using the Abbott PathVysion HER2 DNA probe kit (Abbott Laboratories, Abbott Park, IL) HER2/CEP17 ratio was [2.2. TNBC was defined by absence of primary tumor ER, PR expression, and HER-2 immunostaining and/or gene amplification.
Circulating Tumor Cells in TNBC TABLE 1 Patient demographics Variables
Overall cohort Number of subjects
Total subjects
57
Age (mean) (years)
53 (range 50.2–55.2)
Median follow-up (months)
30 (range 25–36)
1 or more circulating tumor cell Percentage
Patients positive (%)
Patients negative (%)
17 (30)
40 (70)
Tumor size \2 cm (T1)
3/57
5
0/17 (0)
3/40 (8)
2–5 cm (T2)
17/57
30
4/17 (24)
13/40 (33)
[5 cm (T3) (T4)
9/57 28/57
16 49
3/17 (18) 10/17 (59)
6/40 (15) 18/40 (45)
Pathologic nodal status Node negative
15/57
26
5/17 (29)
10/40 (25)
1–3 lymph nodes
19/57
33
3/17 (18)
16/40 (40)
[3 lymph nodes
23/57
40
9/17 (53)
14/40 (35) 1/34 (3)
Histologic tumor grade Low grade (Grade 1)
1/50
2
0/16 (0)
Intermediate grade (Grade 2)
7/50
14
1/16 (6)
6/34 (18)
High grade (Grade 3)
42/50
84
15/16 (94)
27/34 (79)
Missing
7/57
12
1/17 (6)
6/15 (15)
No
27/43
63
8/13 (62)
19/30 (63)
Yes
16/43
37
5/13 (39)
11/30 (37)
Missing
14/57
25
4/17 (24)
10/40 (25)
Lymphovascular invasion
Pathologic complete response No
40/55
73
12/17 (71)
28/38 (74)
Yes
15/55
27
5/17 (29)
10/38 (26)
Missing*
2/57
4
0/17 (0)
2/40 (5)
Premenopausal
17/57
30
4/17 (23)
13/40 (32)
Postmenopausal
40/57
70
13/17 (77)
27/40 (68)
Menopausal status
Isolation, Staining, and Enumeration of Circulating Tumor Cells Peripheral blood (7.5 mL) was collected at the time of primary tumor surgery (but prior to any surgical manipulation of the primary tumor). CTC status was determined using the CellSearch SystemÒ (Janssen Diagnostics, LLC) within 72 h of blood collection. This semiautomated technology enriches blood samples for cells expressing the epithelial-cell-adhesion molecule with antibody-coated magnetic beads, labels the nuclei of these enriched cells with fluorescent dye 4,2diamidino-2-phenylindole dihydrochloride, and stains enriched cells using a combination of CK 8,18,19 and CD45 fluorescent antibodies. A semiautomated fluorescence-based microscope system was employed to identify CTCs: nucleated cells positive for CK and negative for CD45, as described previously.25 A qualified laboratory technician blinded to patient data reviewed all results.
Statistical Analyses REMARK biomarker guidelines for reporting were utilized.26 We used v2 or Fisher exact tests to test associations between presence of CTCs and primary tumor characteristics. Fisher’s exact test was applied when expected value in one or more cells was less than five. Endpoints were characterized using STEEP criteria with RFS and OS as the primary endpoints.27 RFS and OS were defined as time elapsed between date of diagnosis and either the date of clinical disease progression, death, or the last follow-up. Log-rank test was used to detect significant differences between groups. The Cox proportional hazards regression model was used to determine hazard ratios for RFS and OS. P values were two-tailed, and values\0.05 were considered statistically significant. Kaplan–Meier curves were derived using STATA/IC 11.2 (StataCorp, College Station, TX) for comparison of groups defined by different counts of CTCs.
C. Hall et al. TABLE 2 Cox regression analyses of survival associated with presence of CTCs Relapse-free survival
Primary tumor [5 cm
Univariate
Multivariate
Hazard ratio
95 % CI
Cox P value
Hazard ratio
95 % CI
Cox P value
4.10
0.93–18.09
0.03
2.14
0.21–22.34
0.52
Pathologic node-negative vs. 1–3 lymph nodes
1.45
0.24–8.68
0.68
0.71
0.10–4.79
0.72
[3 lymph nodes
5.55
1.2–25.14
0.03
2.23
0.35–14.34
0.40
Histologic grade 3 Lymphovascular invasion
2.67 2.63
0.35–20.35 0.83–8.31
0.27 0.10
7.73 4.24
0.72–83.35 1.09–16.51
0.09 0.04
CTC C1
2.91
1.09–7.78
0.04
5.25
1.34–20.56
0.02
Overall survival
Hazard ratio
95 % CI
Cox P value
Hazard ratio
95 % CI
Cox P value
Primary tumor [5 cm
3.88
0.48–31.54
0.21
–
–
Pathologic node-negative vs. 1–3 lymph nodes
0.46
0.04–5.10
0.53
0.34
0.03–3.96
0.39
[3 lymph nodes
2.04
0.40–10.54
0.24
1.66
0.27–10.00
0.58
Histologic grade 3 Lymphovascular invasion
1.25 1.22
0.15–10.18 0.27–5.44
0.83 0.80
2.02 1.86
0.16–24.07 0.34–10.14
0.58 0.47
CTC C1
3.99
0.95–16.74
0.06
7.04
1.26–39.35
0.03
RESULTS Patient Characteristics A total of 57 patients were enrolled for this study, and their demographic data are reported in Table 1. The mean age was 53 years, and median follow-up was 30 months. Three patients (5 %) were T1, 26 patients (46 %) were T2/ T3, and 28 patients (49 %) had T4 tumors. Fifteen patients (26 %) had node-negative disease, 19 patients (33 %) had 1–3 positive lymph nodes, and 23 patients (40 %) had more than 3 positive lymph nodes on surgical pathologic assessment. Forty-two of 50 patients (84 %) had grade 3 tumors. Sixteen patients (28 %) relapsed, and eight (14 %) died. Circulating Tumor Cell Presence
patients (47 %) with one or more CTC relapsed compared with 8 of 40 patients (20 %) with no CTCs. Two of three patients with two or more CTCs relapsed, but additional events would be needed to perform RFS analyses on patients with multiple CTCs. Circulating Tumor Cells and Overall Survival As shown in Table 2, univariate (log-rank P = 0.03; HR 3.99; 95 % CI 0.95–16.74) and multivariate analysis (HR 7.04; 95 % CI 1.26–39.35; P = 0.03; Fig. 1b) demonstrated that one or more CTC predicted decreased RFS compared with patients with no CTCs. Five of 17 (29 %) patients with one or more CTC died compared with 3 of 40 patients (8 %) with no CTCs. One of three patients with two or more CTCs died, but again, a higher number of events would be needed to perform OS analyses on patients with multiple CTCs.
Forty patients (70 %) had no CTCs, whereas 17 patients (30 %) had one or more CTCs identified. Two or more CTCs were identified in three patients (5 %), and two patients (4 %) had three or more CTCs.
Circulating Tumor Cells and Primary Tumor Characteristics
Circulating Tumor Cells and Relapse-Free Survival
We identified no significant correlation between CTC presence and tumor size (C5 vs. \5 cm; P = 0.23), or primary tumor grade (P = 0.41)
Univariate analysis demonstrated one or more CTC predicted decreased RFS compared with patients with no CTCs (log-rank P = 0.03; hazard ratio [HR] 2.91; 95 % confidence interval [CI] 1.09–7.78; Table 2). These findings were confirmed with multivariate analysis (HR 5.25; 95 % CI 1.34–20.56; P = 0.02; Fig. 1a). Eight of 17
Circulating Tumor Cells and Axillary Lymph Node Status We identified no significant correlation between CTC presence and pathologic ALN status (LN negative vs.
Circulating Tumor Cells in TNBC
A
0.50
0.75
1.00
Kaplan-Meier Relapse-Free survival
0.00
0.25
FIG. 1 Kaplan–Meier survival estimates of probabilities of RFS and OS according to CTCs following NACT in nonmetastatic triple-negative breast cancer. a Probability of RFS in patients with CTC count C1 (HR 2.91; 95 % CI 1.09–7.78; log-rank P = 0.03). b Probability of OS in patients with CTC count C1 (HR 3.99; 95 % CI 0.95–16.74; log-rank P = 0.03)
Log-rank P=0.03 0
10
20
30
40
21 6
0 0
analysis time Number at risk ctc1 = 0 ctc1 = 1
40 17
33 14
29 8 ctc1 = 0
B
ctc1 = 1
0.00
0.25
0.50
0.75
1.00
Kaplan-Meier Overall survival
Log-rank P=0.03 0
10
20
30
40
24 10
0 0
analysis time Number at risk ctc1 = 0 ctc1 = 1
40 17
34 16
30 12 ctc1 = 0
positive, P = 0.75). This lack of significant association persisted after stratifying lymph node-positive patients into one to three, or more than three positive lymph nodes (P = 0.17). DISCUSSION To our knowledge, this is the first study demonstrating that the identification of one or more CTCs independently predicted both RFS and OS in nonmetastatic TNBC patients after NACT. In this study, one or more CTCs was identified in 30 % of our patient cohort, confirming the hypothesis that CTCs can be identified in a significant
ctc1 = 1
number of TNBC patients even after NACT has been completed. These results are in agreement with several published reports demonstrating that CTCs can be identified in 18–49 % of patients using immunocytochemical methods following neoadjuvant and adjuvant treatments.17,18,28,29 In all of these studies, the identification of CTCs posttherapy predicted worse outcome. In agreement with Kim et al., standard prognostic indicators, such as primary tumor size and high grade, did not predict outcome in TNBC patients.14 Tumor size and grade were not significantly associated with CTC presence, nor did they predict outcome in our study. In addition, lymph node positivity was not associated with CTC identification
C. Hall et al.
in our TNBC patient cohort. Yet identification of one or more CTCs after NACT independently predicted both relapse and death. Interestingly, pCR was not associated with CTC identification, yet none of the patients who achieved pCR had relapsed at last follow-up. These data suggest that CTC dissemination can occur independently of lymphatic spread and of primary tumor therapy response. Larger studies will be needed to establish firmly that CTC assessment provides independent prognostic information that could be used to identify TNBC patients who are at increased risk for disease progression following NACT. Currently, CTC assessment is not utilized routinely in the clinic for staging or to guide management decisions for nonmetastatic breast cancer patients. However, data are rapidly emerging regarding their prognostic significance. The REMAGUS 02 and SUCCESS-A trials—Franken et al., van Dalum et al., and Lucci et al.—have demonstrated that presence of one or more CTC was an independent predictor of both RFS and OS rates in stage I– III breast cancer patients.17–21 In all of these studies, HRs associated with RFS and OS and presence of one or more CTC were greater than those observed for primary tumor size and grade and often were as prognostically powerful as presence of ALN metastasis. This raises the question of whether it might be advantageous at some time in the future to utilize information on CTC presence either in combination with, or in place of information on ALN status, especially in patients presenting with clinically negative lymph nodes. Within the entire cohort of TNBC patients studied at our institution, we found no differences (P = 0.54) in CTC identification rates between TNBC patients who were chemonaı¨ve, and in those who received NACT before CTC analysis.30 This finding is not surprising as CTCs have been reported to be dormant and express multidrug resistance proteins (MRPs).31–33 Therapies directed against biomarkers expressed in primary tumors also might be ineffective against CTCs. Recent reports indicate that HER2/neu-positive CTCs can be identified in a significant number of patients with HER2/neu-negative primary tumors.34–39 Because therapy is usually based solely on primary tumor characteristics, these patients are not typically offered anti-HER2/neu therapies. Preliminary data also suggest that many patients with ER-positive tumors have ER-negative CTCs; therefore, it is unlikely antiestrogen treatments directed against primary tumor ER expression would eradicate ER-negative CTCs.40 Given the aggressive nature and limited targeted therapy options available for TNBC patients, CTC profiling could expedite the development of novel therapeutic agents that benefit patients who are at high risk for disease recurrence. In the present study, the presence of one or more CTC following NACT independently predicted RFS and OS for
TNBC patients, irrespective of the presenting clinical stage of the disease. One limitation of our data regarding increased relapse risk with increasing CTC numbers remains due to small numbers of patients who had multiple CTCs in this study. Larger clinical studies will be needed to confirm our findings and to document the increased likelihood of relapse when higher numbers of CTCs are identified. Because in current clinical practice the majority of TNBC patients do not receive any additional therapy after completion of NACT, there is a clear need to identify highrisk patients who might benefit from additional adjuvant therapies. The data from this study emphasize the importance of CTC assessment as a means of identifying patients at high-risk and offer a valuable research tool for future clinical studies investigating development of novel therapy options for nonmetastatic TNBC patients. ACKNOWLEDGMENT This work was supported by The Society of Surgical Oncology Clinical Investigator Award (A Lucci), The Morgan Welch Inflammatory Breast Cancer Program, and The Institute for Personalized Therapy at U.T. M.D. Anderson Cancer Center, the State of Texas Rare and Aggressive Breast Cancer Research Program, and philanthropic funds for which we thank our many generous donors. DISCLOSURES Dr. Lucci served as a consultant at Janssen Diagnostics in December, 2014.
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