Identification of predictive markers of the therapeutic effect of eribulin chemotherapy for locally advanced or metastatic breast cancer

The subjects included 52 patients with inoperable or metastasis/recurrent breast cancer who underwent chemotherapy using eribulin from August 2011 to June 2013 at our institute. Our previous reports have also used the same patient population and the present study, but it was the study of the significance of tumor-infiltrating lymphocytes [25]. The median follow-up time was 431 days (range, 50–650 days). The overall response rate (ORR), clinical benefit rate (CBR), disease control rate (DCR), overall survival (OS), time to treatment failure (TTF) and progression-free survival (PFS) were calculated regarding the efficacy of this regimen. Additionally, based on the immunohistochemical expression of ER, PgR, HER2 and Ki67, the tumors were categorized into immunophenotypes of luminal A (ER+ and/or PgR+, HER2-, Ki67-low), luminal B (ER+ and/or PgR+, HER2+) (ER+ and/or PgR+, HER2-, Ki67-high), HER2-enriched (ER-, PgR-, and HER2+), and TNBC (negative for ER, PgR and HER2).

Regarding the outline of the chemotherapy regimen, one course of treatment consisted of 21 days (three weeks). Eribulin mesylate (1.4 mg/m²) was intravenously administered on days 1 and 8, after which a withdrawal period was continued to day 21. This protocol was repeated until progressive disease (PD) was detected or a severe adverse event requiring the discontinuation of the scheduled chemotherapy was noted. The chemotherapy was administered on an outpatient basis in all cases. The antitumor effect was evaluated based on the criterion for therapeutic effects conforming to the RECIST criteria (Response Evaluation Criteria in Solid Tumors) version 1.1 [26, 27].

The morphology of the tumor, including the histological tissue type, nucleus grade, etc., was evaluated using conventional hematoxylin and eosin (HE) staining, and the expression levels of ER, PgR, HER2, Ki67, β-tubulin class III, GSTP1 and TLE3 were evaluated using immunostaining employing a needle biopsy specimen obtained prior to the start of chemotherapy with eribulin. The pathological diagnosis was made by several experienced pathologists specialized in cancer. This research conformed to the provisions of the Declaration of Helsinki in 1995. All patients were informed of the investigational nature of this study and provided their written informed consent. The study protocol was approved by the Ethics Committee of Osaka City University (#926).

TTF was evaluated on a daily basis and was set as the period from the date of treatment commencement to cancellation for any reason, including disease aggravation, treatment toxicity, and death. OS was evaluated on a daily basis and was set as the period from the date of treatment commencement to death. PFS was evaluated on a daily basis and was set as the period from the date of treatment commencement to the earlier of the date of death or confirmation of PD.

Immunohistochemical studies were performed as previously described [28, 29]. The tumor specimens were fixed in 10% formaldehyde solution and embedded in paraffin, after which they were cut into 4-μm-thick sections and mounted on glass slides. The slides were deparaffinized in xylene and heated for 20 min at 105 °C and 0.4 kg/m² using an autoclave in Target Retrieval Solution (Dako, Carpinteria, California, USA). The specimens were then incubated with 3% hydrogen peroxide in methanol for 15 min to block the endogenous peroxidase activity and were subsequently incubated with 10% normal goat or rabbit serum to block nonspecific reactions.

The cut-off value for ER and PgR positivity was ≥1% positive tumor cells with nuclear staining. HER2 expression was graded according to the accepted grading system as 0, 1+, 2+ or 3+. The following criteria were used for scoring: 0, no reactivity or membranous reactivity in less than 10% of cells; 1+, faint/barely perceptible membranous reactivity in 10% of cells or higher reactivity in only a part of the cell membrane; 2+, weak to moderate complete or basolateral membranous reactivity in 10% of tumor cells or higher and/or strong complete or basolateral membranous reactivity in 10% or higher in 30% or lower of tumor cells; 3+, strong complete or basolateral membranous reactivity in more than 30% of tumor cells. HER2 was considered to be positive if the grade of immunostaining was 3+, or a 2+ result showed gene amplification via fluorescent in situ hybridization (FISH). In the FISH analyses, each copy of the HER2 gene and its centromere 17 (CEP17) reference were counted. The interpretation followed the criteria of the ASCO/CAP guidelines for HER2 IHC classification for breast cancer, i.e., positive if the HER2/CEP17 ratio was higher than 2.0. A Ki67-labelling index of ≥14% was classified as positive. Only nuclear staining was considered distinct for TLE3. Cytoplasmic staining by β-tubulin class III and GTSP1 antibodies was observed in the cancer cells. The TLE3 and GSTP1 expression levels were semi-quantitatively analyzed according to the percentage of cells showing specific staining: 0, 0–10%; 1+, 10–30%; 2+, 30–70%; 3+, >70%. TLE3 expression was considered positive for scores of ≥2 and negative for scores of ≤1 (Fig. 1a ) [17, 18]. GSTP1 expression was considered positive for scores of ≥1 and negative for a score of 0 (Fig. 1c ) [23]. Tumor cells were acquired concerning the normally strong level of β-tubulin class III cytoplasmic staining within endothelial cells or nerves. Tumor cells that stained with at least equal intensity to the endothelial cells or nerves were considered to be positive. To determine the correlations with patient outcomes, the samples were scored as follows: (no staining), 1 (<50% positive cells) or 2 (≥50% positive cells) (Fig. 1b ) [20‐22].

The subjects included 52 patients who underwent chemotherapy using eribulin against inoperable or metastasis/recurrent breast cancer. The gender was female in all cases, with a median age of 63.5 ± 12.7 years. Regarding the line of administration (excluding adjuvant therapy), the average number of chemotherapeutic regimens that had been undertaken before eribulin administration was 2.4 ± 2.3, including 19 opportunities as first-line therapy. A total of 39 patients (75.0%) were suffering from visceral metastases at the administration, and the lesions in 14 cases were considered to be life-threatening. The site of metastasis included, in decreasing order: lung, 19 cases (36.5%); bone, 19 cases (36.5%); liver, 18 cases (34.6%) (Table 1).

The clinical effects of eribulin were as follows: ORR, 34.6% (18/52); CBR, 44.2% (23/52); DCR, 51.9% (27/52); median OS, 334 days; median TTF, 81 days; and median PFS, 275 days (Fig. 2a, b, c). The distribution of the intrinsic subtype classification was as follows: Luminal A, 12 cases (23.1%); Luminal B, 13 cases (15.0%); Luminal HER2, 2 cases (3.8%); HER2 enriched, 3 cases (5.8%) (non-TNBC 30 cases, 57.7%); and TNBC, 22 cases (42.3%). In investigation according to the intrinsic subtype, ORR was found to be 40.0% (12/30) in the non-TNBC cases and 27.3% (6/22) in the TNBC cases (Table 2).

TLE3, β-tubulin class III, and GSTP1 were expressed in 24 cases (46.2%), 21 cases (40.4%) and 24 cases (46.2%), respectively, among the 52 patients investigated. The expression of TLE3 was found significantly more frequently in the TNBC lesions than in the non-TNBC lesions (p = 0.030). However, no significant differences were found between the expression of either TLE3, β-tubulin class III or GSTP1 in the tumors and the clinicopathological background factors of the patients or tumors (Table 3 ). In a multivariate analysis including TLE3 and Ki67, no biomarkers useful for predicting the efficacy of eribulin in cases of MBC were found (Additional file 1).

TNBC, TLE3, β-tubulin class III and GSTP1 were expressed in 14 cases (63.6%), seven cases (31.8%) and 13 cases (59.1%), respectively, among 22 tumors showing characteristics of TNBC. When the clinicopathological background characteristics and expression of each factor were investigated, no factors are significantly affecting the expression levels of these three factors were identified (Table 4). However, patients with TLE3-negative tumors displayed significantly poorer outcomes in terms of PFS than patients with TLE3-positive tumors when the prognosis of patients with TNBC lesions was analyzed (p = 0.011, log-rank) (Fig. 3b) In contrast, no significant differences were found between TLE3-negative/positive patients when the prognosis of all patients (p = 0.433, log-rank) or of patients with non-TNBC lesions (p = 0.659, log-rank) was investigated (Fig. 3a, c). On the other hand, no significant differences were observed in β-tubulin class III or GSTP1 expression among the MBC, TNBC or non-TNBC groups (Fig. 4a, b, d, e, f), with the exception of β-tubulin class III expression in the non-TNBC group (p = 0.018, log-rank) (Fig. 4c).

Based on a univariate analysis of 22 TNBC cases, a better PFS correlated significantly with a positive TLE3 expression in the tumor (p = 0.025). A multivariate logistic regression analysis by Ki67 and TLE3 including 22 patients with TNBC also showed that a positive TLE3 expression significantly correlated with a better PFS (p = 0.037, Hazard ratio = 0.126, 95% CI = 0.018–0.885). Therefore, TLE3 expression in the tumor was identified to be an independent predictive marker of the therapeutic effect of eribulin chemotherapy among patients with TNBC lesions (Table 5).