Transducin-Like Enhancer of Split 3 (TLE3) Expression Is Associated with Taxane Sensitivity in Nonserous Ovarian Carcinoma in a Three-Cohort Study

Epithelial ovarian carcinoma is the most lethal gynecologic cancer, with a 5-year survival rate of 30% to 50%. Epithelial ovarian cancers are generally classified according to their histology and molecular characteristics (1). Histologic and molecular characterization of ovarian carcinoma has revealed that the different histotypes of ovarian cancer represent distinct diseases with distinct sites of origin, pathogenesis, and responses to therapy. High-grade serous carcinomas are characterized by mutations in TP53, whereas endometrioid, clear cell, low-grade serous, and mucinous carcinomas are characterized by mutations in genes including KRAS, BRAF, PTEN, PIK3CA, CTNNB1, ARID1A, and/or PPP2R1A (1, 2).

The poor survival rate in women with ovarian cancer reflects late diagnosis (due to lack of adequate screening options) and poor response to chemotherapy. The standard treatment regimen following debulking surgery includes a combination of platinum-based and taxane-based chemotherapy, which shows improved response rates, progression-free survival, and overall survival in ovarian cancer patients compared with platinum-based chemotherapy alone (3). Although a majority of women initially respond to treatment, up to 75% eventually relapse with chemoresistant disease (4). Chemoresistance in ovarian cancer develops in response to alterations in intracellular drug concentration (increased sequestration, increased efflux), DNA repair/cell-cycle regulation and/or intracellular signaling (5). In taxane-treated tumors, resistance may also result from alterations in microtubule structure (6). Whole-genome sequencing studies of primary high-grade serous carcinomas (93% of whom were treated with taxane-containing therapy) and matched tumor cells from recurrent ascites samples demonstrate a higher mutational burden in the recurrent samples compared with the primary tumors (7). One of the genes found to be altered in the recurrent samples following treatment is ABCB1, which codes for MDR1 (multidrug-resistant protein), which is involved in efflux of taxanes and other chemotherapeutic agents (8). As treatment with taxanes may lead to adverse side effects, including axonal damage and neuropathy (9), biomarkers that can predict response to taxane treatment are imperative to maximize benefit to chemosensitive patients while limiting adverse side effects in patients with chemoresistant disease.

Transducin-like enhancer of split 3 (TLE3) is a homolog of the Drosophila Groucho protein and a transcriptional co-repressor of β-catenin in the Wnt pathway (10). Somatic mutations in CTNNB1 and other components of the Wnt pathway are commonly present in endometrioid ovarian cancer (11). TLE3 expression has been demonstrated to be de-regulated in subsets of various cancers, including colorectal, prostate, non–small cell lung and appendiceal cancers, and angiosarcomas (12–15). TLE3 has been more extensively investigated as a biomarker of chemotherapy response in breast cancer. In one study, TLE3 expression correlated with improved outcomes in triple-negative breast cancer patients treated with eribulin, an agent that, like taxane, inhibits microtubule dynamics (16). In addition, TLE3 was identified as a potential biomarker of taxane response in triple-negative breast cancer; TLE3 expression was associated with progression-free survival in taxane-treated patients (17). In a follow-up study, we demonstrated that high TLE3 expression is associated with a favorable response in nonserous ovarian cancers treated with a taxane-containing regimen compared with a platinum-based agent alone, reflecting the predominance of mutations in the Wnt pathway in nonserous ovarian cancers (18). The small numbers of nonserous ovarian cancers in that study limited our power to analyze associations between TLE3 expression and recurrence in these tumors; we undertook the current study in three additional clinical cohorts with increased numbers of nonserous tumors to confirm our previous findings and to explore the association between taxane treatment and response in specific subtypes of nonserous tumors.

Ovarian cancer cohorts, including paraffin tissue blocks and anonymized clinical data (treatment and outcome data) from the Australian Ovarian Cancer Study (AOCS), Memorial Sloan Kettering Cancer Center (MSKCC) and the Gynecological Oncology Biobank at Westmead (Westmead) were used in this study. Institutional review board approval was obtained for the use of patient blocks and examination of clinical data at each respective institute (AOCS: HREC 01/60; MSKCC: protocol 06-107; Westmead: HREC 92/10/4.13). A total of 1,059 patients were analyzed, of whom 1,041 had invasive carcinoma. Of those patients with invasive carcinoma, 633 (496 AOCS, 29 MSKCC, and 108 Westmead) had undergone primary treatment with a taxane-containing regimen. Progression-free survival was determined by CA-125 criteria (19), imaging, and clinical assessment.

The AOCS cohort consisted of 15 tissue microarray (TMA) blocks containing one to eight 0.6-mm cores sampled from representative paraffin blocks from each patient. The three Westmead TMAs each contained one to three samples from representative paraffin blocks of each patient's tumor, whereas the three MSKCC TMAs each contained two to 15 samples from representative paraffin blocks of each patient's tumor. Staining methods and scoring criteria are as described earlier (17, 18). Briefly, slides were de-paraffinized by submersing in xylene 3 × 10 minutes and rehydrated by rinsing 3x in 100% ethanol and 2x in 95% ethanol. Antigen retrieval was performed by boiling in a microwave for 11 minutes in 10 μmol/L buffered citrate (pH 6.0). Slides were blocked in 0.03% hydrogen peroxide and stained using antibody diluted to appropriate titer in Dako Diluent (DakoCytomation, Glostrup, Denmark) for 1 hour at room temperature. Breast cancer cases and tumor-derived cell lines were tested as positive and negative controls. Secondary antibody was applied for 1 hour and staining was visualized using the DakoCytomation Envision staining kit according to the manufacturer's instructions. Cores were manually scored by a pathologist (R. Murali), and considered positive if greater than 30% of nuclei stained regardless of staining intensity. Replicate scores for a single case were compressed by assuming the maximum score (for 1–3 replicate cores, or by using the rounded average score for ≥4 replicate cores).

The correlation between TLE3 expression (TLE3⁺ or TLE3⁻) and outcome was analyzed using S-plus software (Tibco Software Inc.). Kaplan–Meier and log-rank analyses were used to analyze the association between expression of TLE3 and progression-free survival or overall survival in invasive cases. χ², Log-rank, and Kruskal–Wallis analyses were used to analyze differences between the cohorts (as indicated in Table 1). Cox proportional hazards analysis was performed to investigate the association between TLE3 expression and outcome grouped by grade or stage. Interactions between TLE3 expression and taxane treatment were examined by multivariate analysis in which TLE3 expression, taxane treatment, and the interaction term were assessed simultaneously by Cox proportional hazards. All P values are two-sided.

Table 1 describes the clinical characteristics for the patient cohorts analyzed in this study, which significantly differed between the AOCS, the MSKCC, and the Westmead cohorts. In the combined study (62.7%) of patients were treated with a regimen containing a taxane, ranging from 34% (MSKCC) to 65% (AOCS; Table 1). Across all cohorts, 465/886 (53%) of patients expressed TLE3 (58% AOCS, 49% MSKCC, 40% Westmead). Expression of TLE3 was slightly higher in the nonserous histotypes compared with serous cases (58.0% TLE3⁺ in nonserous, 50.3% serous, χ² P = 0.04). Within patients of known treatment used for the analyses in this study, there was no significant difference in TLE3 expression (52.9% TLE3⁺ in nonserous, 51.2% serous, χ² P = 0.75). Fig. 1 presents immunohistochemical expression of TLE3 in different tumors types.

Table 2 and Fig. 2 describe the associations between TLE3 expression (TLE3⁺ or TLE3⁻) and progression-free survival (3- and 5-year) in patient subsets defined by treatment (with or without taxane), institutional cohort and tumor histology. Our original study assessed 5-year progression-free survival (18); however, as the median recurrence rate was high, outcome was assessed at 3 and 5 years for the nonserous cases. Across all cohorts, TLE3 expression was significantly associated with reduced recurrence within 3 years in taxane-treated patients with nonserous tumors [n = 160; HR, 0.56; 95% confidence interval (CI), 0.33–0.95; P = 0.03), whereas there was no significant relationship with recurrence in patients not treated with a taxane (n = 40; HR, 0.94; 95% CI, 0.33–2.71; P = 0.91; Table 2; Fig. 2A and B). At 5 years, the association between TLE3 expression and recurrence in the taxane-treated nonserous cancer patients was no longer statistically significant (HR, 0.68; 95% CI, 0.42–1.1; P = 0.12). The association between TLE3 expression and outcome was independent of the contributing institution. Within the AOCS cohort, TLE3 expression was significantly associated with recurrence within 3 years in the nonserous cancers treated with taxane (n = 96, HR = 0.46; 95 CI, 0.23–0.91; P = 0.02). An interaction test to assess the apparent differential response to therapy based on TLE3 expression (TLE3:taxane) was not significant.

Similar results were found upon examination of associations between TLE3 expression and overall survival (Table 2). Across all cohorts, TLE3 expression was significantly associated with improved survival in taxane-treated patients with nonserous tumors (n = 160; HR, 0.53; 95% CI, 0.29–0.99; P = 0.04), whereas there was no significant relationship with overall survival at 5 years in patients not treated with taxane (n = 40; HR, 1.37; 95% CI, 0.44–4.25; P = 0.59; Table 2; Fig. 2C and D). We also performed Cox proportional hazards analysis in the taxane-treated, nonserous cases to investigate the association between TLE3 expression and outcome grouped by grade (low or high) or stage (early or late; Supplementary Tables S1 and S2). When broken down by stage or grade, TLE3 expression was only significantly associated with 3-year PFS in the high-grade group (P = 0.03), possibly due to the decreased size in the subsets. TLE3 was independent of grade when examined in a Cox model including stage (n = 110; HR, 0.52; 95% CI, 0.27–0.99; P = 0.05); however, TLE3 failed to remain significant when assessed in combination with stage.

Among the nonserous carcinomas, a significant association between TLE3 expression and outcome was found only in patients with clear cell carcinoma treated with a taxane, for both 3-year progression-free survival and 5-year overall survival (Table 3; Fig. 3A and B). In the other nonserous tumors, no significant association was observed (Fig. 3C and D). The interaction between taxane and TLE3 was significant at 5-year overall survival (n = 79, P = 0.003) among clear cell carcinoma cases. The interaction between taxane and TLE3 at 3-year progression-free survival in clear cell carcinoma was stronger than in all nonserous tumors, but did not reach significance (P = 0.13). In all nonserous tumors, associations of TLE3 with survival were not independent of stage; when examined in a Cox model, including stage, the effect size in the clear cell carcinomas was larger and there was a trend toward significance for improved 5-year overall survival (n = 63; HR, 0.38; 95% CI, 0.14–1.07, P = 0.07).

TLE3 is a member of the TLE family, and acts as a transcriptional repressor of β-catenin in the Wnt pathway. The Wnt pathway regulates cellular processes that promote tumor progression, including differentiation, cell polarity, and cytoskeletal remodeling, the target of taxane agents (20, 21). Taxanes exert their mechanism of action by interacting with the microtubule structure within the cytoskeleton; thus, it is reasonable to hypothesize that TLE3, as a regulator of the Wnt pathway, may serve as a biomarker of response to agents that disrupt downstream processes of this pathway.

In a previous study, we found an association between TLE3 expression and favorable outcome in a small cohort of nonserous ovarian cancer cases after treatment with taxane (18). As our original study was underpowered to parse out differences between histologic subtypes, in this study we analyzed additional biospecimens to determine whether the association with response to taxane treatment was more prevalent in any specific nonserous subtype. Because nonserous subtypes make up a minority of overall ovarian cancer cases (∼30%), we obtained TMAs from 3 different institutions in an effort to validate our previous finding and to strengthen our ability to examine associations of TLE3 expression and taxane sensitivity in individual nonserous subtypes. We examined a total of 1,041 cases and found that TLE3 was expressed in 52% of tumors, compared with 30% in our previous study (Table 1). This difference in the proportion of cases expressing TLE3 could reflect the enrichment for nonserous histotypes used in our current study (in particular, mucinous cases are significantly over-represented in the group of TLE3⁺ tumors). Studies have demonstrated that mutations in the Wnt pathway are frequently present in nonserous histotypes of ovarian cancer (11, 22), suggesting that TLE3 expression may also be altered in this subset of tumors. In the combined cohorts, and in AOCS alone (the cohort with the largest sample size), expression of TLE3 was associated with improved progression-free survival at 3 years specifically in taxane-treated, nonserous cases (Table 2). Furthermore, TLE3 expression was associated with favorable overall survival only in taxane-treated, nonserous cases (Table 2). Additional analysis demonstrated that clear cell tumors specifically drive this association (Table 3), warranting additional molecular studies in this tumor type.

A number of studies have investigated TLE3 as a biomarker of taxane sensitivity in other cancer types, with conflicting results. Kulkarni and colleagues (17) first identified an association between TLE3 expression and reduced recurrence and improved disease-free interval in cancer patients treated with a taxane-based regimen. This finding was validated in a cohort of tumors from women with triple negative breast cancer who had been treated with a taxane-based regimen, suggesting that TLE3 does not merely serve as a surrogate biomarker for ER or HER2 expression. Although early studies have supported the role of TLE3 as a marker of taxane response, the findings of other studies are contradictory. In a study of TLE3 expression in angiosarcoma, TLE3 expression was not associated with taxane sensitivity (12). More recently, Bartlett and colleagues (23) prospectively examined TLE3 expression in a large NCIC Clinical Trial Group breast cancer cohort which had been randomized to two taxane-based treatment arms and a single non–taxane-based treatment arm. In this study, 83% of breast cancers expressed TLE3, compared with 58% in the original breast cancer study. The authors found no evidence that TLE3 expression was associated with taxane response in women diagnosed with breast cancer, contrary to earlier findings. These differences may be due to the heterogeneity of the breast cancer cohort used in this study (24).

These conflicting findings indicate that there is still a great need for identification and validation of biomarkers of chemotherapy response, particularly in cancers with poor outcomes and high rates of recurrence (such as ovarian cancer and triple negative breast cancer). The inability to validate response-associated biomarkers may be due to (i) insufficient sample numbers; (ii) incomplete stratification of histologic subtypes, and (iii) variability in biomarker staining and quantification.

In summary, we have demonstrated an association between high TLE3 expression and a favorable response to taxane-containing chemotherapy regimens in a large multi-institutional cohort of patients with nonserous (especially clear cell) ovarian carcinoma. Further studies are needed to explore in detail the molecular mechanisms underlying this apparent chemosensitivity. In patients with nonserous ovarian carcinoma being considered for systemic therapy, TLE3 expression may serve as a simple and useful method of identifying patients who are likely to benefit from taxane-based regimens.

A. deFazio reports receiving commercial research funding from AstraZeneca.No potential conflicts of interest were disclosed by the other authors.

This material should not be interpreted as representing the viewpoint of the U.S. Department of Health and Human Services, the NIH, or the National Cancer Institute.

Conception and design: G. Samimi

Development of methodology: B.Z. Ring, G. Samimi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Murali, R.A. Soslow, D.D.L. Bowtell, S. Fereday, A. deFazio, N. Traficante, C.J. Kennedy, A. Brand, R. Sharma, Paul Harnett, G. Samimi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B.Z. Ring, R. Murali, S. Fereday, A. deFazio, R. Sharma, G. Samimi

Writing, review, and/or revision of the manuscript: B.Z. Ring, R. Murali, R.A. Soslow, D.D.L. Bowtell, S. Fereday, A. deFazio, C.J. Kennedy, A. Brand, R. Sharma, Paul Harnett, G. Samimi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Fereday

AOCS: The AOCS gratefully acknowledges the cooperation of the participating institutions in Australia, and also acknowledges the contribution of the study nurses, research assistants, and all clinical and scientific collaborators. A complete list of the AOCS Study Group can be found at www.aocstudy.org. AOCS was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729, The Cancer Council Victoria, Queensland Cancer Fund, The Cancer Council New South Wales, The Cancer Council South Australia, The Cancer Foundation of Western Australia, The Cancer Council Tasmania and the National Health and Medical Research Council of Australia (NHMRC; ID400413, ID400281). The AOCS gratefully acknowledges additional support from Ovarian Cancer Australia and the Peter MacCallum Foundation. MSKCC: This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. Westmead: We acknowledge the Gynaecological Oncology Biobank at Westmead (GynBiobank), a member of the Australasian Biospecimen Network-Oncology group, which was funded by the National Health and Medical Research Council Enabling Grants ID 310670 & ID 628903 and the Cancer Institute NSW Grant ID 12/RIG/1-17 & 15/RIG/1-16.

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