60 CHAPTER 3 24 would suggest a lack of (clinically meaningful) activity, while five or more patients with CB would suggest that further investigation of the drug in the tumor/variant cohort is warranted. The null hypothesis and alternative hypothesis to be tested are defined as CBR of 10% versus ≥ 30%. This monitoring rule has 85% power to reject the null hypothesis of a CBR of 10% when the true CBR is 30%, with a one-sided alpha error rate of 7.8%. Exact 95% confidence intervals (CI) were calculated using the Clopper-Pearson method. BASELINE TUMOR BIOPSIES AND BIOMARKER ANALYSIS At baseline, a new fresh frozen tumor biopsy was obtained from each patient. Biopsies were harvested and collected by the participating hospitals and sent to the Hartwig Medical Foundation ((HMF), Amsterdam, The Netherlands), together with a 10-ml blood sample to determine the background variation of the germline DNA of the patient. For WGS, a minimum tumor cell percentage of 30% is required. A 6-μm section was collected for haematoxylin and eosin (H&E) staining and estimation of tumor cellularity by an experienced pathologist. If the sample tumor cellularity was ≥ 30% and the DNA yield was ≥ 300 ng, WGS was performed. WGS data were analyzed using an optimized, high-quality bioinformatic pipeline31, and per patient a summarizing report of all relevant findings was created, including information on tumor purity, ploidy, somatic variants, copy number variations, mutational load, and more complex genomic features such as gene fusions, COSMIC mutational signatures32 and microsatellite (in) stability. A Classifier of HOmologous Recombination Deficiency (CHORD) for pan-cancer HRD detection, as recently developed by HMF, was computed for each sample, hereafter referred to as “HRD-score”33. Bi-allelic status of point mutations and the driver likelihood were assessed as described in previously published work31. All code and scripts used for analysis of the WGS data are available at GitHub (https://github.com/hartwigmedical/). Before biomarker analysis was performed, all WGS samples (baseline study samples and pre-enrollment WGS samples) were re-analyzed using the most recent HMF bioinformatics pipeline, including computation of the HRD-score for each sample. The investigators and an independent clinical molecular biologist reviewed the baseline biopsy WGS results and confirmed presence of the qualifying BRCA mutation, assessed bi-allelic status of BRCA LoF and explored other identified oncogenic driver alterations. In cases where no baseline WGS data were available (i.e. failed sequencing due to low tumor cellularity), the call for bi-allelic or mono-allelic BRCA LoF was made based on the pre-enrollment molecular data. If pre-enrollment WGS data was available, a HRD-score was computed from that sample. Recent reports show a high spatiotemporal preservation of genomic driver alterations36 which justifies this approach. ROLE OF FUNDING SOURCE This Investigator Initiated study receives funding from the Dutch Cancer Society (KWF), Barcode for Life and receives equal funding from a number of pharmaceutical companies, among which AstraZeneca. WGS was performed free of charge at HMF. Study medication was made available free of charge by the manufacturer.
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