NEW YORK – Cancer centers across the Netherlands are getting ready to implement widespread tumor DNA-based testing for BRCA1/2 testing in ovarian cancer patients — a strategy intended to identify those expected to benefit most from PARP inhibitors, while avoiding the time and cost of germline testing in BRCA1/2 mutation-free patients.
"We are in the middle of a project to implement universal tumor DNA BRCA1/2 testing of ovarian cancer, nationwide. All over the country this is going to be used," explained Nicoline Hoogerbrugge, a human genetic researcher at Radboud University Medical Center.
The initiative, spearheaded by the OPA Working Group in the Netherlands, stemmed from the realization that many ovarian cancer patients were coming in for testing twice: once to be tested for pathogenic germline variants in the BRCA1/2 genes and again for somatic tumor sequencing.
Together, the tests help to focus in on ovarian cancer patients with BRCA1/2 mutations who may benefit from PARP inhibitor treatment and to identify inherited BRCA1/2 risk variants that might also be present in patients' family members, informing future cancer screening recommendations for those relatives.
But trying to test each and every ovarian cancer patient with independent germline and somatic methods is expensive and inefficient, according to Hoogerbrugge.
She and her colleagues at Radboud University Medical Center and the Maastricht University Medical Center speculated that it would be more efficient and cost effective to look first for BRCA1 and BRCA2 mutations in the tumor and follow up positive cases with additional germline analysis.
With BRCA1/2 mutations typically turning up in roughly 18 to 24 percent of patients with ovarian cancer, the proportion of patients requiring hereditary testing could theoretically be curbed considerably if the tumor DNA approach was enhanced to reduce false-negative or false-positive BRCA mutation calls.
Consequently, the investigators first set out to develop a new "tumor-first" testing method that took DNA quality in formalin-fixed, paraffin-embedded (FFPE) samples and gene features into account. Their solution: a targeted, next-generation sequencing (NGS) method known as "single-molecule molecular inversion probe," or smMIP, which centers on hundreds of smMIPs specific to BRCA1 and BRCA2.
The team detailed that method in a paper in the journal Human Mutation in early 2017, validating smMIP on DNA from 127 retrospective FFPE ovarian carcinoma tumor samples obtained from 96 patients who had already gotten germline BRCA1/2 mutation testing.
"Our smMIP-based NGS approach provides analysis of both strands of the open reading frame of BRCA1 and BRCA2, enabling the discrimination between real variants and formalin-induced artefacts," Hoogerbrugge, senior author Marjolijn Ligtenberg, from Radboud University, and their co-authors explained.
Along with the tagged sequences — designed to pick up authentic BRCA1/2 mutations with high sensitivity while avoiding false-negative cases — the researchers relied on multiplex ligation-dependent probe amplification (MLST) to uncover informative deletions in BRCA1 exons, and methylation-specific MLST analyses to find risky BRCA1 promoter methylation shifts.
"We showed that smMIP testing for somatic mutations in tumor DNA from paraffin-embedded blocks is as good as germline testing in blood," Hoogerbrugge said. "When there is no somatic mutation, there is no germline mutation."
From there, the researchers looked at the possibility of using smMIP to assess BRCA1/2 mutation status in 315 ovarian cancer patients assessed at seven Dutch hospitals, focusing on everything from feasibility, uptake, and referral rates for genetic testing and counseling to effectiveness and diagnostic yield.
In that study, published in the Journal of the National Cancer Institute last May, the team got BRCA1/2 profiles from 305 of the 315 tumor samples, including 51 ovarian cancer patients found to have pathogenic BRCA1/2 variants. Follow-up testing indicated that more than half of those alterations were hereditary, while just over 43 percent of the BRCA1/2 mutations had a somatic source.
The work sparked a response from University of Rome researchers, who wrote a JNCI correspondence article calling for additional data, particularly when it came to issues such as test reliability and the relatively small proportion of patients and clinicians who provided feedback regarding the tumor-first workflow. In answer to that commentary, the Dutch team stressed the work done to validate and test the tumor-first approach at Radboud University Medical Center and other centers in the Netherlands in the years since the workflow was developed.
"We agree that to modify the clinical practice, it is essential that the test on FFPE tumor material as a prescreen be equally reliable as genetic predisposition testing on DNA derived from peripheral blood lymphocytes and be able to detect somatic alterations," the Radboud team wrote. "Provided that the universal tumor DNA testing workflow is implemented by a multidisciplinary team of experts active in the clinical care pathway, including an experienced laboratory specialist in cancer genetics, the workflow has shown to be a valid and reliable clinical workflow to prescreen for genetic predisposition testing and to stratify for PARP inhibitor therapy."
Hoogerbrugge cautioned that the tumor-first approach cannot automatically be applied to other cancer types, since it relies in part on the proportions of somatic and germline mutations present in an average tumor, which can vary between cancers.
"For ovarian cancer, this is very positive," she said. "For other cancers, that is different, and that's why we did not yet start this for other cancers."
While PARP inhibitors have been approved without a biomarker indication in some ovarian cancer settings, the drugs appear to be particularly beneficial for patients with BRCA1/2 mutations — a feature that makes cancer cells unable to repair DNA damage when the PARP pathway is also blocked.
Such advances have sparked treatment guidelines that call for BRCA1/2 germline mutation testing for ovarian cancer patients in the US, Europe, and beyond, as PARP inhibitors become a mainstay for treating BRCA-mutated ovarian cancers.
Meanwhile, Hoogerbrugge has heard from centers in the UK and continental Europe who are interested in, or have started adopting, their own tumor-first BRCA1/2 mutation detection workflows for ovarian cancer patients.
In the Netherlands, Hoogerbrugge has spent about a year developing relationships and getting buy-in from clinicians across the country, including medical oncologists, gynecologists, pathologists, clinical geneticists, and laboratory geneticists who would participate at different stages of the tumor-first treatment and testing pipeline.
Radboud secured a grant from the Dutch Cancer Society in late 2019 to support training for the multi-disciplinary clinical teams who will ultimately be tasked with implementing that workflow.
The approach is already being applied in a region of the Netherlands that includes roughly one-fifth of the country's population, Hoogerbrugge said. Centers in other parts of the country will follow suit, though the timing of the full national rollout may be delayed by the ongoing COVID-19 pandemic.
"We intended to start this summer," she said. "I don't have a clue if we are going to nail that date or not."