Thymosin beta 15A (TMSB15A) is a predictor of chemotherapy response in triple-negative breast cancer

Abstract

Background: Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed.

Methods: Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts (n=86 and n=55). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n=212, and GeparQuattro, n=383).

Results: A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts (P<0.0001; P<0.0042). In the GeparTrio FFPE training cohort, a significant linear correlation between TMSB15A expression and pCR was apparent in triple-negative breast cancer (TNBC) (n=61, P=0.040). A cutoff point was then defined that divided TNBC into a low and a high expression group (pCR rate 16.0% vs 47.2%). Both linear correlation of TMSB15A mRNA levels (P=0.017) and the pre-defined cutoff point were validated in 134 TNBC from GeparQuattro (pCR rate 36.8% vs 17.0%, P=0.020). No significant predictive capacity was observed in luminal carcinomas from GeparTrio and GeparQuattro.

Conclusion: In TNBC, TMSB15A gene expression analysis might help to select patients with a high chance for pCR after NACT.

Figure 1

Consort statement: TMSB15A was a predictive factor in fresh frozen samples from a population-based cohort treated with EC (n=86), as well as in a subset of GeparTrio treated with TAC (fresh frozen tissue, n=55). Gene expression analysis was transferred to a qRT–PCR platform using 212 FFPE samples from GeparTrio. Biological tumour type was established by qRT–PCR of ESR1 and HER2 using pre-defined cutoff points, and logistic regression analysis for determination of the predictive value of TMSB15A gene expression was performed in luminal (n=151) and TNBC (n=61) separately. In the GeparTrio training cohort, a subtype-specific cutoff point for TMSB15A gene expression was determined additionally. The findings from GeparTrio were validated in 383 FFPE samples from GeparQuattro, comprising 249 luminal and 134 TNBC.

Figure 2

Distributions of TMSB15A gene expression in dependence of pCR in the EC cohort and the TAC cohort bold lines: medians; whiskers, 10–90th percentile; FC= fold change, P, Mann–Whitney test. (B, C) Reciever operator characteristic (ROC) curves for TMSB15A gene expression in the EC and TAC cohort. Abbreviation: Auc=area under the curve.

Figure 3

(A) Distribution of TMSB15A expression in luminal carcinomas and TNBC from GeparTrio. Dots indicate individual tumours. Red lines, medians. P, Mann–Whitney test. (B) Upper panel: determination of a cutoff point of TMSB15A gene expression for TNBC in GeparTrio. All possible cutoff points were considered and the corresponding odds ratios were calculated and plotted. Each data point in the line gives the corresponding OR and the 95% CI (dotted lines) on the y-axis. Vertical line: most significant split. Lower panel: waterfall plot for TNBC in GeparTrio charts each tumour as a vertical bar. Green bars represent cases with correct pCR classification, red bars represent cases with wrong pCR classification. Sensitivity and specificity of the cutoff point are indicated. (C) Pathological complete response (pCR) rates in dependence of TMSB15A status in TNBC from GeparTrio. For dichotomisation into a TMSB15A low and high expression group, the subtype-specific cutoff point was used (11.7 ΔCT). P-values were calculated by logistic regression. *Indicates significant values. The colour reproduction of this figure available at the British Journal of Cancer online.

Figure 4

(A) Distribution of TMSB15A expression in luminal cancers and TNBC from GeparQuattro. Dots indicate individual tumours. Red lines, medians. P, Mann–Whitney test. (B) Waterfall plot for TNBC in GeparQuattro charts each tumour as a vertical bar. Green bars represent cases with correct pCR classification, red bars represent cases with wrong pCR classification. Sensitivity and specificity of the cutoff point are indicated. (C) Pathological complete response (pCR) rates in dependence of TMSB15A status in TNBC from GeparQuattro. For dichotomisation into a TMSB15A low and high expression group, the cutoff point of 11.7 ΔCT, which had been pre-defined in GeparTrio, was used. P-values were calculated by logistic regression. *Indicates significant values. The colour reproduction of this figure available at the British Journal of Cancer online.

Figure 5

(A) TMSB15A mRNA levels (Affymetrix HG U133A microarray; probe set 205347_s_at) in MCF-7 and Cal-51 breast cancer cell lines. For clones of wild type (wt), cells were measured (dots), four clones of paclitaxel-resistant (res) Cal-51 were measured twice (dots). Expression levels in wt and res Cal-51 were compared by Mann–Whitney test. Bold lines, mean; FC=fold change. (B) TMSB15A mRNA expression in parental Cal-51 cells transfected with siRNA against TMSB15A (si TMSB15A) relative to cells transfected with control siRNA (si control). P, columnar t-test. (C) Dose–response curves for wild-type Cal-51 treated with increasing concentrations of paclitaxel (left panel) and doxorubicin (right panel) 24 h after siRNA transfection. Comparison of si control (bold grey line) with si TMSB15A treated cells (bold black line) were made by paired t-test. Dashed line, IC50.