Abstract
Class III β-tubulin (βIII-tubulin) has been associated with tumor response to taxane-based therapies in breast cancer. However its role in the neoadjuvant setting has not been explored. We evaluated βIII-tubulin expression by immunohistochemistry in 44 patients, and found high expression associated with good pathologic response in estrogen receptor–negative (ER−) breast cancers. Our results give strong reason to consider βIII-tubulin as a predictive biomarker for neoadjuvant chemotherapy response.
Background
Expression of class III β–tubulin (βIII-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies including breast cancer. However its predictive value in a neoadjuvant setting in breast cancer remains unexplored. The objective of this explorative study was to determine whether βIII-tubulin expression in breast cancer correlated with pathologic characteristics and whether its expression was predictive of response to neoadjuvant chemotherapy.
Patients and Methods
We determined βIII-tubulin expression in 85 breast cancers, including 41 localized breast cancers treated with primary surgery and 44 treated with neoadjuvant chemotherapy before surgery. βIII-tubulin expression was evaluated by immunohistochemical methods and was correlated with pathologic characteristics and response to neoadjuvant chemotherapy using residual cancer burden (RCB) score.
Results
High βIII-tubulin expression was significantly associated with poorly differentiated high-grade breast cancers (P = .003) but not with tumor size, estrogen receptor (ER) status, or human epidermal growth factor receptor 2 (HER2)/neu overexpression. In ER− tumors treated with neoadjuvant chemotherapy, high βIII-tubulin expression was associated with a significantly greater likelihood of achieving a good pathologic response to chemotherapy as reflected by lower RCB scores (P = .021).
Conclusion
This study reveals differential βIII-tubulin expression in breast cancers of different histologic grades, hormone receptors, and HER2/neu status. It also suggests a potential role for βIII-tubulin as a predictive biomarker for response in neoadjuvant chemotherapy for ER− breast cancer, which has not been previously reported. These data provide a strong rationale for considering βIII-tubulin status and further validation of this marker in a large study.
Keywords: Breast cancer, Chemotherapy, Class III β-tubulin, Neoadjuvant, Predictive biomarker
Introduction
Microtubules are an essential cytoskeleton component that mediate key cellular events crucial to cell division. Tubulin heterodimers consisting of α- and β-tubulins are the basic structural components that constitute microtubules.1,2 There are multiple α- and β-tubulin isoforms with distinct patterns of distribution among different tissues. The antineoplastic effect of the taxanes is mainly related to their ability to bind the β-subunit of tubulin, thereby stabilizing microtubules and resulting in suppressed proliferation and cell death.2 Efforts have been made to find molecular markers capable of predicting the efficacy of taxanes that would lead to rational and biologically driven treatment selection.
βIII-tubulin is expressed normally in tissues of neuronal lineage and Sertoli cells of the testis.1 However, abnormal distribution and expression of βIII-tubulin have been demonstrated in a variety of human malignancies, including non–small-cell lung cancer and ovarian, gastric, pancreatic, prostate, and breast cancers.3–9 Altered tubulin isoform expression may relate to a more aggressive tumor phenotype and drug resistance.10,11 Many studies have identified βIII-tubulin as a prognostic marker of poor survival,4–8 whereas others have shown that βIII-tubulin expression may be associated with response to microtubule-interacting drugs.10 Furthermore, βIII-tubulin expression is associated with cell-based models of acquired paclitaxel resistance4,12 and also with resistance to DNA damaging drugs.13 Recently, it has been reported that βIII-tubulin expression may predict poor outcome in colorectal cancer, dependent on gender.14
To further determine the association of βIII-tubulin with pathologic characteristics of the tumor and chemotherapy response, we evaluated βIII-tubulin expression in patients with localized breast cancer who underwent primary surgical therapy and patients treated with neoadjuvant chemotherapy before surgery. Immunohistochemical staining was used for determining βIII-tubulin expression and a residual cancer burden (RCB) score was used for the pathologic response and clinical outcome analysis.
Patients and Methods
Tissue Collection
We retrospectively identified 44 patients with invasive breast cancer treated at our institution between 2006 and 2010 who received neoadjuvant chemotherapy and had sufficient tissue available for analysis. We also identified a cohort of 41 patients with operable breast cancer diagnosed during the same period who did not receive neoadjuvant chemotherapy.
Immunohistochemical Evaluation
βIII-tubulin (1:400, clone TUJ1; Covance, Princeton, NJ ), ER (1:50, clone 1D5; Dako North America Inc, Carpinteria, CA), progesterone receptor (PR) (1:400, clone 1A6, Dako), and human epidermal growth factor receptor 2 (HER2)/neu (Dako HercepTest, Dako) antibodies were used for immunohistochemical evaluation Four-micrometer sections were cut from formalin-fixed paraffin-embedded tissue blocks, heated at 60°C for 30 minutes, deparaffinized, rehydrated, and subjected to antigen retrieval by heating the slides in epitope retrieval buffer in a water bath at 95°C for 45 minutes. The slides were then incubated with either mouse monoclonal antibodies or rabbit polyclonal antibodies for 30 minutes at room temperature in a Dako autostainer. The immunoreactivity was detected using Dako EnVision methods, according to the manufacturers recommended procedures. Both the intensity and percentage of βIII-tubulin–positive cells were evaluated using the H-score system.15 Briefly, the number of positive cells was reported as a percentage (0%–100%) and the H-score was determined by multiplying the percentage of cells demonstrating each intensity (0–3).
Pathologic Evaluation
Tumor specimens were evaluated for tumor type, size, extent of disease, lymph node status, and histologic grade using the Nottingham combined histologic grading system. Immunohistochemical evaluation of ER, PR, and HER2/neu expression was classified according to the College of American Pathologists/American Society of Clinical Oncology guidelines.16,17 For patients treated with neoadjuvant chemotherapy, the pretreatment core biopsy and/or posttreatment excision specimens with residual tumor were used. Pathologic response to neoadjuvant chemotherapy was assessed by the American Joint Committee on Cancer staging and RCB score.18 This system uses the size of residual carcinoma, cellularity distributed over the tumor bed, number of lymph nodes with metastases, and size of the largest metastasis combined mathematically into a continuous index to define 4 RCB categories (0, I, II and III) associated with increasing risk of recurrence (http://www.mdanderson.org/breastcancer_RCB). An RCB score was graded by 2 pathologists (YW and SF) who were unaware of the βIII-tubulin expression at the time of analysis.
Statistical Analysis
Fisher exact tests (2-sided) were used to assess associations between βIII-tubulin expression, pathologic characteristics, and response to neoadjuvant chemotherapy.
Results
Patient Characteristics
In total, specimens from 85 patients with invasive breast cancers were part of this study. Forty-four patients received neoadjuvant chemotherapy and 41 patients underwent surgery without previous neoadjuvant chemotherapy during the same period at our institution. The characteristics of patients in the 2 groups are summarized in Table 1, including age, histologic grade, ER expression, HER2/ neu expression, and pathologic stage at surgery.
Table 1.
Clinical and Pathologic Characteristics
| Variable | Neoadjuvant Group | Operable Group | ||
|---|---|---|---|---|
| n | % | n | % | |
| Number of Patients | 44 | 41 | ||
| Age (y) | ||||
| Mean | 50 6 | 51.6 | ||
| Median | 48.5 | 49 | ||
| Range | 30–76 | 34–83 | ||
| Histologic Grade | ||||
| I | 0 | 0 | 7 | 17.1 |
| II | 13 | 29.5 | 13 | 31.7 |
| III | 31 | 70.5 | 21 | 51.2 |
| ER Status | ||||
| Positive | 20 | 45.5 | 22 | 45.5 |
| Negative | 24 | 54.5 | 19 | 54.5 |
| HER2/neu Status | ||||
| Positive | 8 | 18.2 | 11 | 26.8 |
| Negative | 36 | 81.8 | 30 | 73.2 |
| Pathologic Stage at Surgery | ||||
| T0 | 5 | 11.4 | 0 | 0 |
| T1 | 20 | 45.5 | 20 | 48.8 |
| T2 | 16 | 36.4 | 14 | 34.1 |
| T3 | 2 | 4.5 | 6 | 14.6 |
| T4 | 1 | 2.3 | 1 | 2.4 |
| Nx | 1 | 2.3 | 3 | 7.3 |
| N0 | 15 | 34.1 | 19 | 46.3 |
| N1 | 15 | 34.1 | 12 | 29.3 |
| N2 | 13 | 29.5 | 7 | 17.1 |
| Chemotherapy Used | ||||
| Taxane-containing | 33 | 75 | – | – |
| Other chemotherapy | 11 | 25 | – | – |
Abbreviations: ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2.
Correlation Between βIII-Tubulin Expression and Pathologic Characteristics
βIII-tubulin exhibited homogenous cytoplasmic staining when present as shown in Figure 1, but showed a wide range of intensity and percentage of expression in tumors (H-score range, 0–300). Using an H-score of 150 as a cutoff value, high βIII-tubulin expression was identified in 29 of the 85 patients (34%) in both groups, including 13 of 44 (30%) patients treated with neoadjuvant chemotherapy and 16 of 41 (39%) patients with operable disease who were not treated with neoadjuvant chemotherapy. High βIII-tubulin expression was observed in 0 of 7 (0%) well-differentiated (grade I) tumors, in 7 of 26 (26.9%) moderately differentiated (grade II) tumors, and in 22 of 52 (42.3%) poorly differentiated (grade III) tumors. Thus high βIII-tubulin expression was more commonly associated with grade III tumors compared with grade I and grade II tumors (2-sided Fisher’s exact test P = .003). No significant association was found between βIII-tubulin expression and tumor size, ER status, or HER2/neu overexpression.
Figure 1.
Immunohistochemical Staining Patterns of βIII-Tubulin in Invasive Breast Cancer. (A) Strong Diffuse βIII-Tubulin Staining; (B) Patchy Moderate Staining; (C) Weak Sparse Staining (arrow showing positive staining of nerve by βIII-tubulin); (D) Negative Staining
Correlation Between βIII-Tubulin Expression and Response to Neoadjuvant Chemotherapy
The correlation of βIII-tubulin expression by immunohistochemical methods and response to neoadjuvant chemotherapy are summarized in Table 2. When considering all patients treated with neoadjuvant chemotherapy or only those treated with taxane-containing therapy, high βIII-tubulin expression was not significantly associated with posttreatment RCB scores. However when cases were separately analyzed based on hormone receptor ER status, we found that in ER− tumors, high βIII-tubulin expression was significantly associated with lower posttreatment RCB scores (RCB 0 and I) in all patients treated with preoperative chemotherapy (P = .021) and in those treated with taxane-containing chemotherapy (P = .038). In contrast, βIII-tubulin expression did not predict the response in ER-positive (ER−) tumors.
Table 2.
βIII-Tubulin Expression and Correlation with Neoadjuvant Treatment Response
| Variable | Total | High βIII | Low βIII | Fisher Test P Value | |
|---|---|---|---|---|---|
| n | % | n | n | ||
| Overall | 44 | ||||
| RCB 0–I | 12 | 27 | 6 | 6 | .152 |
| RCB II–III | 32 | 73 | 8 | 24 | |
| ER− | 24 | ||||
| RCB 0–I | 8 | 33 | 5 | 3 | .021 |
| RCB II–III | 16 | 67 | 2 | 14 | |
| ER+ | 20 | ||||
| RCB 0–I | 4 | 20 | 1 | 3 | 1 |
| RCB II–III | 16 | 80 | 6 | 10 | |
| Taxane Treated | 33 | ||||
| RCB 0–I | 11 | 33 | 5 | 6 | .212 |
| RCB II–III | 22 | 67 | 4 | 18 | |
| ER− | 19 | ||||
| RCB 0-I | 7 | 37 | 4 | 3 | .038 |
| RCB II–III | 12 | 63 | 1 | 11 | |
| ER+ | 14 | ||||
| RCB 0–I | 4 | 29 | 1 | 3 | 1 |
| RCB II–III | 10 | 71 | 3 | 7 | |
Abbreviations: ER+ = estrogen receptor positive; ER− = estrogen receptor negative; RCB = residual cancer burden.
Discussion
High expression levels of βIII-tubulin have been demonstrated in a variety of human malignancies, including breast cancer. In our study, we found that high βIII-tubulin expression correlated with poor tumor grade but not with other clinical pathologic features. High βIII-tubulin expression in ER− disease was associated with a significantly greater likelihood of achieving a good pathologic response after preoperative neoadjuvant chemotherapy, including taxane-containing chemotherapy and other mainly AC (Adriamycin [doxorubicin] and cyclophosphamide)–based chemotherapy regimens. Such findings have not been reported previously. Cell-based studies have indicated that βIII-tubulin may regulate cancer cell response to DNA-damaging chemotherapy agents by modulating cell survival, death and tumorigenesis pathways.13 Others have suggested that immune signaling molecules and MAP2 are associated with response to neoadjuvant taxane treatment.19 Although the underlying molecular mechanism is still unclear, our study provides a rationale for evaluating βIII-tubulin expression as a predictive biomarker in ER− disease for chemotherapy to benefit patients undergoing neoadjuvant treatment and/or patients with operable breast cancer treated with adjuvant chemotherapy.
The relationship between βIII-tubulin expression and clinical outcomes has been studied extensively in various cancers.3–8 In general, these studies have associated high βIII-tubulin expression with poor clinical outcome and resistance to taxane-based chemotherapies in non–small-cell lung cancer and gastric, pancreatic, prostate, and ovarian cancers. However a few recent studies such as that of Aoki et al found that high βIII-tubulin expression was associated with a favorable prognosis in patients with clear cell carcinoma of the ovary.20 Seve et al reported better outcomes in patients with non–small-cell lung cancer with high βIII-tubulin expression, plus greater benefit from adjuvant chemotherapy.21
Several studies have also evaluated the relationship between βIII-tubulin expression and clinical outcome in advanced breast cancer. The comparisons among these studies, including ours, are listed in Table 3.9,22–25 They used the median expression level as the cutpoint to define high expression and two of these studies used the same TUJ1 antibody that was used by us. Bernard-Marty et al reported that βIII-tubulin expression was not associated with response to taxane therapy in patients with metastatic breast cancer.22 Others found βIII-tubulin over expression associated with taxane resistance23–25 using different methods, including RNA expression and immunohistochemical analysis with a different antibody clone. In contrast, Galmarini et al reported that high βIII-tubulin expression was associated with response to docetaxel in patients with locally advanced and metastatic breast cancer.9 These results correlated well with our findings. However, ours is the first study evaluating the relationship between βIII-tubulin expression and response to chemotherapy in a neoadjuvant setting. Evaluation of a tumor marker after surgery has confounding aspects. After a breast carcinoma is surgically removed, the next endpoint for a clinical trial is recurrence or death. These events may not occur for many years or even decades, and the outcome assessment of correlation with a marker can be complicated by other factors such as margin status, number of lymph nodes involved, presence of lymphovascular invasion, and tumor histologic grade and type, which have all proved to be important prognostic factors for determining disease recurrence and disease-free survival.
Table 3.
Comparison of Studies Evaluating βIII-Tubulin Expression in Breast Cancer
| Study | βIII-Tubulin Assessment Method | Definition of High βIII Expression | Number of Patients | Patient Population | ER−Disease | Endpoint | High βIII-Tubulin Correlated With Clinical Outcomes |
|---|---|---|---|---|---|---|---|
| Bernard-Marty et al22 | IHC clone TUJ1 | ≥ 50% | 34 | Metastatic 83% | Not stated | Time to progression | No |
| Paradiso et al23 | IHC clone SDL 3D10 | ≥ 50% | 70 | Metastatic 29% | 27% | Clinical response | Yes, associated with paclitaxel resistance |
| Tommasi et al24 | IHC clone SDL 3D10 | ≥ 50% | 92 | Advanced | 35.5% | Clinical response | Yes, associated with paclitaxel resistance |
| Hasegawa et al25 | mRNA RT-PCR | ≥ Median RNA expression level | 39 | Locally advanced (67%) | 56% | Clinical response | Yes, associated with docetaxel resistance |
| Galmarini et al9 | IHC clone TUJ1 | ≥ Median | 173 | Locally advanced | Not stated | Time to progression | Yes, associated with docetaxel sensitivity |
| Wang et al (Current Article) | IHC clone TUJ1 | ≥ Median | 44 | Operable or locally advanced | 55% | Pathologic response | Yes, associated with paclitaxel sensitivity |
Abbreviations: ER− = estrogen receptor negative; IHC = immunohistochemical; RT-PCR = reverse transcription polymerase chain reaction.
Our study demonstrated that although high βIII-tubulin expression was identified in both ER+ and ER− breast cancers, it did not predict response in ER+ cases. The molecular mechanism underlying this observation is unclear. Multiple complex signal transduction pathways can interact in the presence of ER. Studies in human cell lines and xenografts have shown that growth factor receptor signaling pathways, particularly those that converge on phosphatidylinositol 3-kinase, which is the most frequently altered pathway in breast cancer, can modulate ER and its transcription cofactors.26 In cell-based studies, researchers have suggested that the presence of a high level of ERα interferes with paclitaxel-induced cell death without affecting paclitaxel-induced microtubule bundling and mitotic arrest.27 Other researchers have demonstrated that high ER expression affected estrogen-regulated genes and enhanced cell motility, which was associated with resistance to paclitaxel therapy.28
Conclusion
Our study provides for the first time evidence that high βIII-tubulin expression is predictive of the response to preoperative neo-adjuvant chemotherapy in patients with ER− breast cancer. Validation in other larger data sets is needed.
Clinical Practice Points.
Expression of βIII-tubulin has been associated with tumor progression and response to taxane-based therapies in several human malignancies, including breast cancer. However the role of βIII-tubulin in a neoadjuvant setting has not been explored.
In this explorative study, we evaluated the role of βIII-tubulin expression in chemotherapy response and clinical outcome in advanced breast cancers, in a neoadjuvant setting.
This study reveals differential βIII-tubulin expression in breast cancers of different histologic grades, hormone receptor status, and HER2/neu status.
High βIII-tubulin expression was associated with high-grade tumors and good pathologic response in patients with ER− but not ER+ breast cancers.
These findings suggest that βIII-tubulin expression could be a potential predictive biomarker for response to neoadjuvant chemotherapy for ER− breast cancer, which has not been previously reported.
Our results provide a strong rationale for further evaluation of βIII-tubulin status in a large study.
Acknowledgments
This work was supported by National Cancer Institute Grant CA077263, the Breast Cancer Research Foundation, the National Foundation for Cancer Research, and the National Institutes of Health Grant K12CA132783-01A1.
Footnotes
Disclosure
The authors have stated that they have no conflicts of interest.
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