Olaparib Addition to Maintenance Bevacizumab Therapy in Ovarian Carcinoma With BRCA-Like Genomic Aberrations

Key Points Question Is olaparib maintenance treatment associated with longer survival in patients with ovarian cancer tumors with and without a previously established BRCA-like genomic profile? Findings This cohort study was a secondary biomarker-by-treatment interaction analysis of 469 patients from a randomized clinical trial comparing the bevacizumab plus olaparib and bevacizumab plus placebo groups. Patients with BRCA-like tumors had a longer progression-free survival after olaparib vs placebo, but there was no significant difference in patients with a non–BRCA-like tumor after olaparib vs placebo. Meaning The findings of this study suggest that the BRCA-like classifier can be used as a biomarker for olaparib maintenance therapy.

This supplementary material has been provided by the authors to give readers additional information about their work.

Low coverage sequencing methods
The 85 phase 2 samples were sequenced at the Netherlands Cancer Institute (NKI) as described before After validation of concordance between the NKI and Cologne Center for Genomics (CCG) sequencing facilities (eMaterials), the phase 3 samples were sequenced at CCG as follows.A total of 384 DNA samples (100 ng each) isolated from formalin fixed paraffin embedded (FFPE) tumors were provided by ARCAGY Research (8 Rue Lamennais, 75008 Paris, France).All DNA samples were treated with NEBNext FFPE DNA Repair Mix (M6630L; New England Biolabs GmbH, Frankfurt am Main, Germany) according to the manufacturer´s protocol.Mechanical shearing of DNA was performed using a Bioruptor NGS sonication system (UCD-600; Diagenode, 4102 Seraing (Ougrée), Belgium).DNA fragmentation was evaluated using a Tape Station 4200 (G2991BA, Agilent Technologies, Santa Clara, CA, USA) and (high-sensitivity) D1000 ScreenTapes Agilent).The TruSeq Nano DNA High Throughput Library Prep Kit (96 samples) was used (20015965, Illumina, San Diego, CA, USA) for library preparation.The library pool was quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System and then sequenced on an Illumina NovaSeq 6000 sequencing instrument with a PE100 sequencing protocol (one SP flow cell).

Proof of concept: decentralized distribution
Previously, we used a HiSeq2500 sequencer with a 65-bp read length at the Netherlands Cancer Institute (NKI) (1).As a proof of concept of decentralized distribution and to investigate the robustness of the assay, we conducted sequencing of Phase 3 samples at the Cologne Center for Genomics (CCG).For higher throughput, we used an Illumina NovaSeq6000 sequencer.We sequenced 47 paired samples from the AGOTR1 study using the NovaSeq system (see Materials and Methods) and compared the classification results with those previously obtained using the HiSeq system (1).Readouts for successful validation comprised the similarity of the (average) profiles between both facilities/techniques and the concordance of classification.
We plotted the average log ratio and segmented log ratio copy number profiles of the samples sequenced at NKI and CCG.Visual inspection confirmed that the average profiles overlapped (Figure 1).When we plotted the sorted and sorted segmented log ratios, there was a strong correlation between the two platforms, with the (segmented) log ratios mostly on the identity line of the plot (Figure 2).Notably, this measure resembles quantile (normalized) plots of the distribution and not the actual distribution or correlation between the profiles, that is, the range and centering of the (segmented) log ratios obtained by the two sequencing facilities.We plotted the distributions of the log ratios and segmented log ratios (eFigure 3).The distributions are very similar.
Given that the obtained copy number aberration profiles appeared to be very similar, specifically, the segmented ratios used for classification, we classified the 47 samples and cross-tabulated the results in eTable 1. 46/47 samples (98%) showed the same BRCA1-like classification, see eTable 1.We plotted the copy number aberration profile (eFigure 4) of the discordant sample and observed both in the visual assessment and confirmed by the density plot (eFigure 5) that the CCG-sequenced profile had reduced amplitude.Although the overall validation was successful, there was residual experimental variation despite the profile passing quality control.
eFigure 5. Density plot of sample discordance between NKI and CCG.
Reduced amplitude for the CCG sample is observed, e.g. at the peak around -0.5 and in the values > 0.25.

PFS1 (investigator assessment based on RECIST version 1.1)
Average Unsegmented and Segmented (B) Copy Number Profiles of 47 Samples Sequenced at the NKI and CCG Sites eFigure 2. Correlation Between the Distribution of 47 Samples Sequenced at the NKI and CCG eFigure 3. Distribution of (Segmented) Log Ratios of 47 Samples Sequenced at the NKI and CCG eTable 1. BRCA1-Like Classification of 47 Samples Sequenced at NKI and CCG eFigure 4. Copy Number Profiles of Discordant Samples Sequenced at the NKI and CCG eFigure 5. Density Plot of Sample Discordance Between NKI and CCG eFigure 6. Flow Diagram of Samples in the Study eTable 2. Programming Rules for Censoring PFS1 eTable 3. Cross

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2024 Schouten PC et al.JAMA Network Open.eFigure 1. Copy number aberration profiles of 47 samples sequenced at the NKI and CCG.Average unsegmented (A) and segmented (B) copy number profiles of 47 samples sequenced at the NKI (blue) and CCG (orange) sites.

eFigure 2 .
Correlation between the distribution of the 47 samples sequenced at the NKI and CCG.Sorted average log ratios (A) and segmented log ratios (B) of both centers.The diagonal line represents x= y.

eTable 1 .
BRCA1-like classification of 47 samples sequenced at NKI and Table of BRCA-like Status and Myriad MyChoice CDx eTable 4. Univariable Cox Regression Analysis of Samples With Discordant BRCA-Like and Myriad MyChoice CDx Results

without documented progression based on investigator RECIST assessment eTable 3. Cross table of BRCA-like status and Myriad MyChoice CDx
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