On-treatment Biomarkers Can Improve Prediction of Response to Neoadjuvant Chemotherapy in Breast Cancer

Richard J. Bownes; Arran K. Turnbull; Carlos Martinez-Perez; David A. Cameron; Andrew H. Sims; Olga Oikonomidou

Disclosures

Breast Cancer Res. 2019;21(73) 

In This Article

Results

Gene Expression Differences Between Responding and Non-responding Breast Cancer Tumours Treated With Chemotherapy are Subtle and Time Dependent

Unsupervised principal component analysis was first used to assess whether sequential patient-matched samples from patients receiving chemotherapy (Figure 1b) would cluster by time point or response status. There was no significant grouping of patients according to sampling time: pre, early, or later after chemotherapy in either the NEO or I-SPY 1 studies (Figure 1b). There were no significant differences between the two cohorts in terms of age, grade hormone receptor, and HER2 status, and the subset of patients with mid-chemo samples was not significantly different from the whole NEO cohort (Table 1). Patient-matched samples enable the pairwise analysis to look for consistent changes in the gene expression during treatment. Pairwise significance analysis of microarray analysis using a 10% false discovery rate (FDR) identified a relatively small proportion of overlapping upregulated (5%) and downregulated (4%) genes between the two studies. However, genes that were increased or decreased in response to treatment in one study were also clearly and consistently increased or decreased in the other study (Additional file 1: Figure S1A), further suggesting it would be difficult to discriminate responders from non-responders. Indeed, there was no clustering by response status before or during treatment (Additional file 1: Figure S1B). These results likely reflect the considerable inter-patient differences being substantially larger and more significant than the subtler commonalities in gene expression of a particular time point or response class of each tumour. More encouragingly, semi-supervised LFDA of each time point revealed significant separation on-treatment that was not apparent in pre-treatment samples; this indicated that there are meaningful differences between the classes, as early as 2 weeks on-treatment (Figure 2a). Complete responders and non-responsive patients were more clearly separated than partially responding patients. These results suggest that there is a potentially greater predictive value looking at on-treatment than pre-treatment biomarkers.

Figure 2.

Responders and non-responders are more distinct on than before treatment. a Supervised clustering using local Fisher discriminant analysis (LFDA) indicates that as early as 2 weeks on treatment, there is a visible separation of the response classes that were unseen in the pre-treatment samples in the NEO dataset. Red = non-responder, orange = partial responder, blue = complete responder. b Greater numbers of genes are under and overexpressed between responders and non-responders on treatment. The three lines represent different statistical thresholds (*p < 0.05, **p < 0.01, and ***p < 0.001 or FDR = 10%, FDR = 5%, and FDR = 1%, gene lists are in Additional file 4: Tables S2 and S3) in the NEO dataset. cSankey diagram illustrating the proportions of tumours that change or maintain PAM50 intrinsic subtype during chemotherapy treatment. Whilst basal subtypes remain mostly stable, the composition of the cohort changes with treatment time, which may help to identify responsive or non-responsive patients. PT = pre-treatment, T-ON = on-treatment

Responding and Non-responding Tumours are More Different Upon Exposure to Chemotherapy

In an attempt to quantify the molecular differences between the response groups at each time point, rank product analysis was performed at different standard p values (0.05, 0.01, and 0.001). This approach was hampered by different numbers of samples at each time point (with T2 having very few samples); however, the number of genes differentially expressed at all p values tended to be greater during rather than before treatment (Figure 2b). Similar results were also seen using 1%, 5%, and 10% FDR (Figure 2b). The biggest differences between the response classes were at TM (mid-chemo), which agrees with the LDFA results, which showed the least amount of overlap of the response classes at TM. Gene set enrichment analysis across the response classes at each time point also demonstrated more enriched pathways after 2 weeks of treatment (29), mid-chemo (30), and resection (29), compared to pre-treatment (18) (Additional file 2: Figure S2A). Next, we sought to examine common differentially expressed genes between responders and non-responders across the two datasets. Far more genes were commonly significantly differentially expressed (FDR = 10% between responders and non-responders on-treatment in the NEO and I-SPY 1 datasets compared with pre-treatment. In accordance with the LFDA results, more significantly differentially expressed genes (1814) were observed between on-treatment samples, with 6% (197), but only one was common between NEO and I-SPY pre-treatment (Additional file 2: Figure S2B and Additional file 4). Examination of the 468 most significantly differentially expressed genes (p < 0.001) between responders and non-responders in the NEO dataset at mid-chemo did not clearly distinguish between response groups or time points illustrated by the heatmap in Additional file 3: Figure S3, further demonstrating that identifying biomarkers of response to chemotherapy is very difficult.

We were also keen to evaluate whether the intrinsic subtype assigned to tumours would alter upon treatment. Looking at the NEO and I-SPY datasets, together we found that basal tumours were relatively stable with only 2/19 (11%) tumours changing. More tumours were classified as Luminal A or normal-like on-treatment, which likely reflects a reduction in the expression of proliferation genes during chemotherapy (Figure 2c).

AAGAB is a Promising Potential Novel On-treatment Biomarker of Response to Chemotherapy

The mid-chemo gene list from the NEO dataset (1102 genes, unadjusted p value = 0.01) was fed to a random forest model for further feature selection and classification and regression tree (CART) model, which reported AAGAB as the most predictive gene for response prediction in the NEO training dataset with 100% accuracy for pCR prediction on the mid-chemo samples (Figure 3a). Validation was conducted completely independently on publicly available sequentially sampled chemotherapy data from the I-SPY 1 Trial[10] and reported 76% accuracy using AAGAB at the same expression level on the scaled and centred expression data at the on-treatment time point prior to resection (T2). For comparison, the pre-treatment only sample gene lists were put through the same protocol in order to consider whether highly predictive models could be generated before chemotherapy. IGF1R was the most predictive pre-treatment marker with an accuracy of 74% and 63% in the NEO and I-SPY datasets, respectively (Table 2). AAGAB was the sixth most accurate predictor (65%, 57%); receiver operator curves show the relative specificity and sensitivity of this marker pre- and on-treatment (Figure 3b). Gene expression levels of AAGAB were lower in responders across all time points in the NEO cohort but were most significantly different at mid-chemo. In the I-SPY dataset, AAGAB was significantly lower before treatment and at excision (Figure 3c). We wondered whether AAGAB was lower in responders due to a reduction in proliferation, but Pearson correlation analysis with common proliferation-associated genes (TOP2A, BUB1, MKI67, MCM2, FOXM1, and PCNA) demonstrated no significant correlation to any of these genes (Figure 3d), suggesting that AAGAB is independent of proliferation. Survival analysis demonstrated that response status predicted by AAGAB level, at mid chemo in the NEO study and at 2 weeks in the I-SPY 1, was significantly associated with the outcome (NEO p = 0.048, I-SPY 1 p = 0.0036) (Figure 3e). Interestingly, the level of AAGAB before treatment was not associated with the outcome in either cohort (p = 0.71 and p = 0.2, Figure 3e). None of the other top 10 pre- or on-treatment markers was significantly associated with the outcome in both datasets (Table 2); only one gene (ARF5) was associated with the outcome in the NEO dataset (p = 0.004). Taken together, the single gene on-treatment biomarker AAGAB appears to outperform novel pre-treatment markers and established prognostic tests in predicting pCR and long-term outcome to chemotherapy.

Figure 3.

AAGAB is a promising on-treatment biomarker of chemotherapy response and outcome. a CART analysis identified AAGAB as a possible biomarker from the Edinburgh NEO dataset and was 100% accurate at predicting pCR in the training data and 76% accurate in the I-SPY 1 validation set. b The ROC curves highlight the difference in on-treatment and pre-treatment accuracy and selectivity. c Strip charts showing the level of AAGAB in responding and non-responding patients across time points. dAAGAB showed no significant (Pearson) correlation with established markers of proliferation in the NEO dataset, indicating it does not seem to be a downstream proxy of their regulation. e Kaplan-Meier plots demonstrate that on-treatment, but not pre-treatment, levels of AAGAB were significantly associated with the outcome in both cohorts. p values are log-rank test

Comparison of pre- and On-treatment Predictions of Response and Outcome

We were also keen to assess whether estimations of established prognostic signatures might be different upon treatment and if on-treatment might be more accurate. All and almost all responding patients were predicted to have poor outcomes with the estimated Mammaprint,[19] PAM50,[20] or rorS[21] signatures in pre-treatment samples of the NEO cohort, whereas around half of the responding patients were predicted as good outcome using on-treatment data (Figure 4a). Overall accuracy improved by 2–8% using on- rather than pre-treatment data; however, improvement in the predictive power of these tests was not uniform between response classes. Good outcome predictions for responders to neoadjuvant chemotherapy saw an aggregate increase in predictive power from 11 to 44.4%, whilst poor outcome predictions for non-responders saw a moderate decrease in accuracy, 75 to 63%. None of the gene expression signatures either pre- or on-treatment or established prognostic markers (NPI, Grade, Her2 status) was significantly associated with the outcome in contrast to the remarkable performance of on-treatment measurement of AAGAB (Figure 4b).

Figure 4.

On-treatment signatures more accurately predict pathological response and outcome than pre-treatment. a A greater proportion of patients with pathological response are predicted as responders with estimations of molecular signatures on-treatment than pre-treatment. Concordance between patients predicted as high and low risk across time is poor, but the positive predictive value of these tests increase with treatment. For PAM50 subtypes, normal-like and Luminal A are considered good prognosis and basal/Luminal B/HER2-enriched are considered poor outcome. Red = predicted poor outcome, blue = predicted good outcome. b Forrest plots to compare molecular signatures and AAGAB before and on-treatment combining both datasets, except where indicated* due to individual sample data unavailable for I-SPY 1 patients

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