Class III β-tubulin overexpression within the tumor microenvironment is a prognostic biomarker for poor overall survival in ovarian cancer patients treated with neoadjuvant carboplatin/paclitaxel

Abstract

Critics have suggested that neoadjuvant chemotherapy (NACT) followed by interval debulking may select for resistant clones or cancer stem cells when compared to primary cytoreduction. β-tubulins are chemotherapeutic targets of taxanes and epothilones. Class III β-tubulin overexpression has been linked to chemoresistance and hypoxia. Herein, we describe changes in class III β-tubulin in patients with advanced ovarian carcinoma in response to NACT, in relationship to clinical outcome, and between patients who underwent NACT versus primary debulking; we characterize in vitro chemosensitivity to paclitaxel/patupilone of cell lines established from this patient population, and class III β-tubulin expression following repeated exposure to paclitaxel. Using immunohistochemistry, we observed among 22 paired specimens obtained before/after NACT decreased expression of class III β-tubulin following therapy within stroma (p=0.07), but not tumor (p=0.63). Poor median overall survival was predicted by high levels of class III β-tubulin in both tumor (HR 3.66 [1.11,12.05], p=0.03) and stroma (HR 4.53 [1.28,16.1], p=0.02). Class III β-tubulin expression by quantitative-real-time-polymerase-chain-reaction was higher among patients who received NACT (n=12) compared to primary cytoreduction (n=14) (mean±SD fold-change: 491.2±115.9 vs. 224.1±55.66, p=0.037). In vitro subculture with paclitaxel resulted in class III β-tubulin upregulation, however, cell lines that overexpressed class III β-tubulin remained sensitive to patupilone. Overexpression of class III β-tubulin in patients dispositioned to NACT may thus identify an intrinsically aggressive phenotype, and predict poor overall survival and paclitaxel resistance. Decreases in stromal expression may represent normalization of the tumor microenvironment following therapy. Epothilones warrant study for patients who have received neoadjuvant carboplatin and paclitaxel.

Fig 1

Following treatment with NACT, a trend towards decreased strength of stromal (p = 0.07) but not tumoral staining (p = 0.76) was observed. Horizontal bars represent mean values

Fig 2

Following NACT, class III β-tubulin staining by immunohistochemistry was unchanged in tumor, but decreased in surrounding stroma. Representative slides are shown at original magnification of 100–200x and illustrate 3+ stromal staining before NACT (left) and 1+ stromal staining after NACT (middle); corresponding scores for tumor staining in this specimen are 3+ and 2+, respectively. Normal ovary does not stain for class III β-tubulin (right)

Fig 3

a Class III β-tubulin staining by both tumor (left) and stroma (right) predicts crude overall survival (OS). Median OS among patients with high [2+,3+] versus low [0,1+] expression by tumor was 707 days versus not reached (HR 3.66 [1.11,12.1], p=0.03). Similarly, median OS among patients with high [1+,2+,3+] versus low [0] expression by stroma was 649 versus 1494 days (HR 4.53 [1.28,16.1], p=0.02). b Decreased expression of class III β-tubulin expression following NACT in both tumor (ΔIHC score ≤ 1 vs ≥ 0: not reached vs 707 days, HR 2.116 [0.58, 7.69], p = 0.25) (left) and stroma (ΔIHC score ≤ −1 vs ≥ 0: 1494 vs 596 days, HR 2.73 [0.693, 10.72], p= 0.15) (right) appeared to confer survival benefit, though statistical significance was not reached

Fig 4

Patients dispositioned to NACT exhibit higher class III β-tubulin levels compared to those who undergo primary debulking. At time of cytoreduction, among 12 patients treated with NACT and 14 who underwent primary debulking class III β-tubulin expression values were 491.0 ± 115.7 and 224.1 ± 55.66, respectively

Fig 5

a Paclitaxel subculture of 10 cell lines results in upregulation of class III β-tubulin (from 985.8 ± 219 before treatment to 2306 ± 550 after treatment; p = 0.039). b Characteristics of cell lines exposed to paclitaxel subculture. c Ovarian serous cell lines that overexpress class III β-tubulin are exquisitely sensitive to patupilone; representative chemoresponsiveness to paclitaxel and patupilone among three different chemotherapy-naïve cell lines is shown in relationship to baseline class III β-tubulin expression. Molar concentration (x-axis) is shown against percentage viable cells (y-axis).