Development of the NOGGO GIS v1 Assay, a Comprehensive Hybrid-Capture-Based NGS Assay for Therapeutic Stratification of Homologous Repair Deficiency Driven Tumors and Clinical Validation

Abstract

The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25-85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The "NOGGO GIS v1 assay" performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.

Keywords: BRCA1; BRCA2; DNA repair; HRD; HRR; LOH; OS; PAOLA-1; PARP inhibition; PARPi; PFS; breast cancer; diagnostics; genomic instability; homologous repair deficiency; molecular pathology; ovarian cancer; somatic mutation.

Figure 1

Performance of assay. (A): Mean coverages reached using 50 ng and 100 ng of input DNA and sequencing 20 million read pairs in two independent replicates. As intended, mean coverages are twice as high in genes (exonic bases) compared to mean coverage in all targets (all bases). Using 100 ng input led to slightly higher mean coverages. (B): Percentage of unique reads for 50 and 100 ng of input DNA are shown in two independent replicates. Using 100 ng input DNA led to slightly higher complexity of the library measured in fraction of unique reads. (C): Percentage of Off-target reads are shown for two independent replicates. Off-target rates were stable and low (<20%) leading to efficient usage of the data. Using 100 ng of input led to slightly more off-target reads. (D): Coverage distribution of exonic positions in two independent replicates (dark and light blue, respectively). Blueboxes represent the 75% percentile with the median coverage depicted as black line. 50 ng (top) and 100 ng (bottom) DNA input are shown. Red dots represent the 95 percentile; hence, 95% of exonic positions had a coverage greater than the red dot. (E): Histogram of capture probes GC content in 5%tile bins are shown. (F): Normalized deduplicated coverage correlation of experimental replicates with 50 ng (left) and 100 ng (right) are shown. Colors from blue over purple to red reflect the degree of GC content (blue: low, red: high).

Figure 2

Cutoff determination: Cohen’s kappa coefficient was used to determine the maximum correspondence between Myriad MyChoice HRD status and NOGGO GIS (red line). In addition, sensitivity PPV, specificity, and NPV were calculated. These measures indicated that a NOGGO GIS cutoff between 83 and 85 led to the maximum correspondence between the two scores. PPV: positive predicted value; NPV: negative predicted value.

Figure 3

Results of the phase 2 blinded testing of 85 tumor samples from PAOLA-1 BRCA wild-type patients. (A) The numbers of true positives, false positives, false negatives and true negatives according to the Myriad MyChoice test are listed. (B) Calculated performance statistics using the numbers from the table A GIS: Genomic instability score; PPV: positive predicted value; NPV: negative predicted value.

Figure 4

PFS (A,B) and OS (C,D) data of 383 patients of the PAOLA-1 clinical trial analyzed with either Myriad MyChoice (cutoff ≥ 42) or the NOGGO GIS v1 assay (cutoff ≥ 83).

Figure 5

Subgroup analysis of patients successfully analyzed with the NOGGO GIS v1 assay for which no results with Myriad MyChoice could be obtained.