The AURORA pilot study for molecular screening of patients with advanced breast cancer-a study of the breast international group

Abstract

Several studies have demonstrated the feasibility of molecular screening of tumour samples for matching patients with cancer to targeted therapies. However, most of them have been carried out at institutional or national level. Herein, we report on the pilot phase of AURORA (NCT02102165), a European multinational collaborative molecular screening initiative for advanced breast cancer patients. Forty-one patients were prospectively enroled at four participating centres across Europe. Metastatic tumours were biopsied and profiled using an Ion Torrent sequencing platform at a central facility. Sequencing results were obtained for 63% of the patients in real-time with variable turnaround time stemming from delays between patient consent and biopsy. At least one clinically actionable mutation was identified in 73% of patients. We used the Illumina sequencing technology for orthogonal validation and achieved an average of 66% concordance of substitution calls per patient. Additionally, copy number aberrations inferred from the Ion Torrent sequencing were compared to single nucleotide polymorphism arrays and found to be 59% concordant on average. Although this study demonstrates that powerful next generation genomic techniques are logistically ready for international molecular screening programs in routine clinical settings, technical challenges remain to be addressed in order to ensure the accuracy and clinical utility of the genomic data.

Fig. 1

Logistics and feasibility of the study. a logistic workflow of the study, b inclusion criteria and number of patients with successful results, c anatomical distribution of biopsied lesions with breakdown by recruiting centre, d global distribution of patients by recruiting centre, e turnaround time for each patient with breakdown by recruiting centre, f median global turnaround time by recruiting centre, and g median delivery turnaround time by recruiting centre. In a, the Illumina sequencing and Affymetrix OncoScan SNP arrays were done as batch processes. 19 patients were sequenced by Illumina targeted NGS and 18 patients were genotyped using the Affymetrix SNP arrays. However, these are not fully overlapping subsets and only 14 (54%) patients had the full set of all three data types. In a and b, the Ion Torrent sequencing results were obtained in real-time and in (e), the darker shades indicate delivery turnaround time (TAT) whilst the lighter shades indicate global turnaround time. The colour codes for the recruiting centres in c to g are indicated in (d)

Fig. 2

Mutation detection from the OncoDEEP clinical cancer panel and CNA from SNP arrays. a genes for which at least one mutation was indexed across the 26 patients, b and c comparison of cancer cell fraction and genomic mass respectively obtained from two widely used algorithms for estimating CNA from SNP arrays. In a, asterisk indicates potentially hypermutated patients. In b and c, each dot represents a sample. In d–g, each dot represents a gene from the list of clinically actionable or biologically relevant targets and is the mean of 100 bootstrap replicates such that for each replicate, the external cohorts are matched for ER and HER2 status. The size of each dot is proportional to the standard error of the mean frequency estimate

Fig. 3

Orthogonal cross-testing of substitution calls using Illumina NGS. a distribution of % concordance for single nucleotide substitutions indexed from the Ion Torrent and Illumina NGS platforms using different mutation callers, b % error rate of the different mutation callers over three substitution clusters compared to the Ion Torrent sequencing, c correlation of VAF for substitutions indexed from the Ion Torrent and Illumina NGS using a majority rule of any two of three mutation callers, d comparison of sequencing coverage between the Ion Torrent and Illumina NGS for substitutions in cluster 1, e empirical cumulative distribution of theoretical residual coverage from the Illumina sequencing for substitutions in cluster 1 and cluster 2 false negatives, f comparison of sequencing coverage from the Ion Torrent NGS for substitutions in cluster 2 false positives, and g comparison of sequencing coverage from the Illumina NGS for substitutions in cluster 2 false negatives. In a, the different mutation callers and any combination thereof are colour coded and indicated at the bottom. The leftmost panel gives the number of substitutions called. In b–g, the cluster numbers are relative to (c). Substitutions in clusters 1 and 3 are exclusive to one of the sequencing platforms and have 0% VAF in the alternate data. Substitutions in cluster 2 are those found by either or both NGS platforms and have non-zero VAF in both sequencing data. False negatives in cluster 2 are substitutions indexed by Ion Torrent NGS only whilst false positives are substitutions indexed by Illumina NGS only using a given mutation caller or any combination thereof. The size of each dots is proportional to the difference in coverage between the two sequencing platforms. In e, the residual coverage is obtained by subtracting the theoretical coverage required to achieve 99% power for indexing a substitution given one mutated copy out of n total copies from the observed value of sequencing depth. Only two substitutions in cluster 1 failed the criteria of positive residual for detection and are non-callable loci

Fig. 4

Genome-wide copy number profiles inferred from SNP arrays and targeted NGS data. Genome-wide Log₂ ratio profiles of patient IJB0021 obtained using a SNP array b Ion Torrent and c Illumina targeted NGS data. The solid vertical lines represent chromosome boundaries whilst the dashed horizontal lines indicate canonical copy numbers inferred from the cancer cell fraction and ploidy of the sample, both estimated from the SNP array. The solid horizontal lines represent the segmented Log₂ ratios. Correlation of segmented Log₂ ratios between d the SNP array and the Ion Torrent platform, e the SNP arrays and the Illumina platform, and f the two NGS platforms for the same case patient. In a–c, the loci are sorted according to their coordinate on the human genome reference hg19/GRCh37. For ease of representation, they are plotted by indices

Fig. 5

Comparison of copy number profiles from SNP arrays and targeted sequencing data. a distribution of Spearman’s correlation coefficient comparing the segmented Log₂ ratios from the SNP arrays and the NGS data across patients with all three data types. The segmented Log₂ ratios were further categorised into copy number aberration calls i.e., −1, 0 and +1 and compared patient wise between the SNP arrays and the NGS data. The resulting distributions of accuracy values are depicted in b. For a small set of 15 clinically actionable or biologically relevant genes, the accuracy values comparing SNP arrays and NGS data were computed using all available samples and displayed individually for each gene in c. d, e correlation of accuracy values measured genome-wide for each patient as in b vs. the number of aberrations as determined by the corresponding NGS platform. f, g correlation of accuracy for each of the 15 clinically actionable or biologically relevant genes vs. the frequency of aberrations for the same genes measured using the SNP array. In c, each value of accuracy was generated by 100 bootstrap replicates. The values displayed represent the mean of the replicates and the error bars represent the standard error of this estimate