BRCA1/2 Testing Landscape in Ovarian Cancer: A Nationwide, Real-World Data Study

Simple Summary Nowadays, tumor tests to analyze DNA in tumor cells from epithelial tubal/ovarian cancers (EOCs) are performed in many centers to detect tumor pathogenic variants (TPVs) in the BRCA1/2 genes. Information on the presence of these TPVs guides treatment options and further genetic testing in patients and relatives. However, there is no standardization of testing procedures, and information about how testing is performed is limited. Therefore, we described how BRCA1/2 tumor testing is performed in 999 EOC patients in the Netherlands in 2019 using real-world clinical data. Tumor tests were performed for 502 patients (50.2%) and TPVs were detected in 14.7% of the tests. This study shows that there is variability in the execution of BRCA1/2 tumor tests, but there were no indications for quality differences. Adequate reporting and quality monitors are essential to ensure that all centers perform reliable tumor tests to ultimately identify all patients with BRCA1/2 TPVs. Abstract Analyzing BRCA1/2 tumor pathogenic variants (TPVs) in epithelial tubal/ovarian cancers (EOCs) has become an essential part of the diagnostic workflow in many centers to guide treatment options and genetic cascade testing. However, there is no standardization of testing procedures, including techniques, gene assays, or sequencers used, and data on the execution of tumor tests remains scarce. Therefore, we evaluated characteristics of BRCA1/2 tumor testing in advanced-stage EOC with real-world national data. Pathology reports of patients diagnosed with EOC in 2019 in the Netherlands were obtained from the Dutch Pathology Registry (PALGA), and data regarding histological subtype and BRCA1/2 tumor tests were extracted. A total of 999 patients with advanced-stage EOC were included. Tumor tests were performed for 502 patients (50.2%) and BRCA1/2 TPVs were detected in 14.7%. Of all tests, 48.6% used hybrid capture techniques and 26.5% used PCR-based techniques. More than half of the tests (55.0%) analyzed other genes in addition to BRCA1/2. Overall, this study highlights the heterogeneity in the execution of BRCA1/2 tumor tests. Despite a lack of evidence of quality differences, we emphasize that adequate reporting and internal and external quality monitors are essential for the high-quality implementation and execution of reliable BRCA1/2 tumor testing, which is crucial for identifying all patients with BRCA1/2 TPVs.


Introduction
In recent years, testing on BRCA1/2 pathogenic variants (PVs) in patients diagnosed with epithelial tubal/ovarian cancer (EOC) has become increasingly important.While germline testing for BRCA1/2 PVs has been available for EOC patients in most medical centers for over a decade, the introduction of the poly (ADP-ribose) polymerase inhibitor (PARPi) therapy has created the need to also identify patients with somatic PVs, as tumors with BRCA1/2 PVs (somatic and germline) exhibit superior sensitivity towards this therapy [1][2][3][4][5].Clinically meaningful overall survival benefits have been reported in EOC patients with a germline or somatic BRCA1/2 PV receiving the PARPi olaparib for two years; 67.0% of the patients receiving olaparib were alive after seven years, versus 46.5% of the patients in the placebo arm [1].
The American Society of Clinical Oncology (ASCO) and the European Society of Medical Oncology (ESMO) recommend performing both somatic and germline testing in EOC patients [6,7].This identifies patients with genetic predisposition to the disease, which could have clinical implications for the family members of the patient, but also identifies those patients who are likely to benefit from PARPi therapy.To efficiently perform both tests, many centers analyze DNA from tumor samples by first using next-generation sequencing (NGS) and subsequently analyzing germline pathogenic variants (GPVs) only in those with a PV in the tumor (also referred to as tumor PV (TPV)).Patients with a positive family history and/or no or inconclusive tumor test results are also eligible for germline testing.This sequential workflow reduces the number of referrals for genetic counseling and germline testing, as well as the associated patient burden, and is considered cost-effective [8][9][10].
In the Netherlands, this tumor-first workflow is fully implemented in specialized centers.However, there is no standardization of testing procedures, including the techniques, gene assays, or sequence machines used for the analyses.In addition to a lack of national guidelines on testing procedures, data on the performance of the tumor tests, as well as test outcomes, throughout the Netherlands remain scarce.For these reasons, we evaluated the execution of BRCA1/2 TPV testing in the Netherlands with real-world data from 2019 and provided insight into the number of BRCA1/2 TPVs detected, and the techniques used in Dutch testing centers.

Materials and Methods
Patients diagnosed with EOC in 2019 in the Netherlands were identified with the help of the nationwide network and registry of histo-and cytopathology in the Netherlands (PALGA) [11].The PALGA database contains excerpts of all pathology reports from pathology departments in the Netherlands and has had full national coverage since 1991.The pathology laboratories in the Netherlands are ISO-15189-certified and the quality of the accredited laboratories is evaluated through various ways, including internal and external audits as well as interlaboratory quality comparisons [12].
Anonymous pathology reports regarding the execution of BRCA tumor tests were retrieved from PALGA for all patients diagnosed with EOC in 2019 in the Netherlands.These pathology reports were subsequently linked to data from the Netherlands Cancer Registry [13] to obtain the FIGO stage for each patient.Patients were excluded if they were diagnosed with FIGO stage I or II EOC, as these patients had no indication for adjuvant PARPi therapy [14].
For all included patients, data regarding the histological subtype of the tumor and the BRCA1/2 tumor NGS analyses were obtained from the pathology reports.These data include, amongst other variables, the following: tumor NGS analysis performed (yes/no); tumor NGS results; technique used; and genes analyzed.Additionally, it was checked whether the tumor test was complemented with a BRCA1 multiplex ligationdependent probe amplification (MLPA) analysis (yes/no) and, if yes, the MLPA test result was collected (BRCA1 PV: yes/no).Information on the detection of variants of unknown clinical significance (VUS) was also collected when reported in the pathology reports.Dutch pathology laboratories follow national guidelines for establishing the classification and relevance of detected variants, which include close collaboration with the genetics departments of medical centers [15].In the case of a detected BRCA1/2 TPV in the EOC of an ambiguous or unspecified histological subtype (e.g., carcinoma not otherwise specified (NOS)), an expert pathologist reviewed the pathology reports and further classified the tumor, if possible, based on the information from the corresponding pathology reports and according to the World Health Organization's classification of the female genital tumors of 2020 [16].
The following endpoints were analyzed in this cohort of advanced-stage EOC patients: (1) the number of diagnosed EOC by histological subtype; (2) the prevalence of BRCA1/2 TPVs and VUS by histological subtype; (3) the (reporting of) techniques and platforms used in BRCA1/2 tumor NGS analyses, including the specific genes analyzed; and (4) the lead times of the BRCA1/2 tumor analyses.
Data were reported as frequencies and percentages and lead times as mean, standard deviation and minimum and maximum values.Information on the type of technique used for target enrichment was collected and classified as hybrid capture techniques or polymerase chain reaction (PCR)-based amplicon techniques.The number of TPVs detected by BRCA1 MLPA analysis was reported separately from those detected by NGS.The number of BRCA1/2 TPVs was compared between the hybrid capture and PCR-based techniques using the Chi-square exact test.The distribution of the histological subtype was also compared between the hybrid capture and PCR-based techniques using Fisher's exact test.The lead time of the BRCA1/2 tumor analyses was defined as the number of days between the date of the receival of tumor material in pathology centers and the reported date of the tumor test results.The lead time could only be calculated when both dates were reported.

Results
The PALGA search identified 1308 women who were diagnosed with EOC in 2019 in The Netherlands (Figure 1).After the exclusion of patients with early-stage disease (FIGO I/II) (n = 309; 23.6%), a total of 999 EOC patients were included.Most EOC patients were diagnosed with high-grade serous carcinoma (n = 682; 68.3%), followed by carcinoma NOS (n = 65; 6.5%) and low-grade serous carcinoma (n = 46; 4.6%) (Table 1).The histological subtype was not reported in the pathology report for 5.8% of the patients.
significance (VUS) was also collected when reported in the pathology reports.Dutch pathology laboratories follow national guidelines for establishing the classification and relevance of detected variants, which include close collaboration with the genetics departments of medical centers [15].
In the case of a detected BRCA1/2 TPV in the EOC of an ambiguous or unspecified histological subtype (e.g., carcinoma not otherwise specified (NOS)), an expert pathologist reviewed the pathology reports and further classified the tumor, if possible, based on the information from the corresponding pathology reports and according to the World Health Organization's classification of the female genital tumors of 2020 [16].
The following endpoints were analyzed in this cohort of advanced-stage EOC patients: (1) the number of diagnosed EOC by histological subtype; (2) the prevalence of BRCA1/2 TPVs and VUS by histological subtype; (3) the (reporting of) techniques and platforms used in BRCA1/2 tumor NGS analyses, including the specific genes analyzed; and (4) the lead times of the BRCA1/2 tumor analyses.
Data were reported as frequencies and percentages and lead times as mean, standard deviation and minimum and maximum values.Information on the type of technique used for target enrichment was collected and classified as hybrid capture techniques or polymerase chain reaction (PCR)-based amplicon techniques.The number of TPVs detected by BRCA1 MLPA analysis was reported separately from those detected by NGS.The number of BRCA1/2 TPVs was compared between the hybrid capture and PCR-based techniques using the Chi-square exact test.The distribution of the histological subtype was also compared between the hybrid capture and PCR-based techniques using Fisher's exact test.The lead time of the BRCA1/2 tumor analyses was defined as the number of days between the date of the receival of tumor material in pathology centers and the reported date of the tumor test results.The lead time could only be calculated when both dates were reported.

Results
The PALGA search identified 1308 women who were diagnosed with EOC in 2019 in The Netherlands (Figure 1).After the exclusion of patients with early-stage disease (FIGO I/II) (n = 309; 23.6%), a total of 999 EOC patients were included.Most EOC patients were diagnosed with high-grade serous carcinoma (n = 682; 68.3%), followed by carcinoma NOS (n = 65; 6.5%) and low-grade serous carcinoma (n = 46; 4.6%) (Table 1).The histological subtype was not reported in the pathology report for 5.8% of the patients.BRCA1/2 tumor NGS analyses were performed for 502 patients (50.3%), and a total of 62 TPVs were detected (12.4% of all NGS analyses); 31 TPVs in BRCA1 and 31 TPVs in BRCA2 (Table 2).A complementary BRCA1 MLPA analysis was performed for 344 patients (34.4%) and it detected an additional 12 BRCA1 TPVs (3.5% of all MLPA analyses).Combining the TPVs detected through NGS and MLPA analyses (n = 74), most BRCA1/2 TPVs were detected in high-grade serous carcinoma (n = 67; 90.5%) (Supplementary Table S1).The remaining seven TPVs were detected in low-grade serous carcinoma (n = 1; 1.4%), endometrioid carcinoma (n = 1; 1.4%), clear cell carcinoma (n = 1; 1.4%), carcinosarcoma (n = 3; 4.1%) and carcinoma NOS (n = 1; 1.4%).In addition, the detection of six VUS was reported in the pathology reports.Five VUS (83.3% of all VUS) were reported in high-grade serous carcinoma and one VUS (16.7%) in endometrioid carcinoma.Caution must be taken when interpreting the number of VUS reported in this study, since reporting a detected VUS in the pathology report is not universally adopted by all the testing centers.Figure 2 visualizes the distribution of the different techniques applied for the target enrichment and the genes analyzed in the EOC tumor tests.Of the 502 BRCA1/2 tumor analyses performed in our cohort, a total of 244 analyses (48.6%) were performed using the hybrid capture technique and 133 analyses (26.5%) using the PCR-based technique (Figure 2).Information on the target enrichment technique applied was missing for a substantial proportion of the performed BRCA1/2 tumor analyses (n = 125, 24.9%).The proportion of BRCA1/2 TPVs detected did not significantly differ between the hybrid capture and PCR-based techniques (12.3% and 21.1%, respectively; p-value = 0.078), neither did the distribution of histological subtypes between the hybrid capture and PCR-based  S2).Of all NGS analyses, 42.6% analyzed exclusively the BRCA1/2 genes, and 55.0% used a more comprehensive panel, also including genes other than BRCA1/2 (referred to as BRCA1/2+ in Figure 2).For 2.4% of all NGS analyses, the specific genes analyzed were not reported.
ing genes other than BRCA1/2 (referred to as BRCA1/2+ in Figure 2).For 2.4% of all NGS analyses, the specific genes analyzed were not reported.
More than half of the BRCA1/2 tumor analyses were performed on an Illumina platform (Illumina, Inc., San Diego, CA, USA) (n = 262; 52.2%), 15.1% on an Ion Torrent platform (Thermo Fisher Scientific Inc., Waltham, MA, USA), and in more than 30% of the BRCA1/2 tumor analyses, the platform used was not specified (n = 156; 31.1%)(Supplementary Table S4).Lead times were analyzed for the cases where the dates of the receival of the tumor material and test results were reported in the pathology report (n = 376).The mean lead time was 38.3 days (SD = 64.2days), ranging from 0 days to 525 days (Supplementary Table S5). 1 (100.0) 1 The denominator used for calculating percentages for assays is the type of technique applied; the denominator used for calculating percentages for genes is the assay used.Abbreviations: smMIP, single-molecule molecular inversion probe; PCR, polymerase chain reaction.

Discussion
The current study provides insight into the execution and outcomes of BRCA1/2 tumor analyses in patients diagnosed with advanced-stage EOC in 2019 in the Netherlands.Of the 999 advanced stage EOC patients included in this study, BRCA1/2 tumor NGS analyses were performed for 502 patients (50.3%).Most importantly, this study shows that substantial variety exists in the execution of tumor analyses in EOC regarding the techniques and assays used, and the (types of) genes analyzed.
To the best of our knowledge, this study is the first to provide insight into the nationwide landscape of BRCA1/2 tumor testing in EOC.Besides providing insight into the applied techniques, assays and analyzed genes, this study also highlights the lack of uniform reporting in pathology reports, despite high-quality centralized care and the utilization of a national pathology registration database.A great proportion of the pathology reports lacked information on the techniques and assays used for the analyses, the analyzed genes, and dates of, for example, the test results.For this reason, lead times could only be analyzed for a subset of 376 tests.Importantly, lead time is included in the criteria of the national quality control standards that are currently being implemented [17].Inadequate reporting limited quality assessment in the current study and, more importantly, could have clinical implications for patients, as it limits the exchange of diagnostic information between clinicians and the tailoring of a patient's treatment.Considering the increased sensitivity of patients with BRCA1/2 TPVs towards PARPi therapy, and the possible heredity of the disease, the complete reporting of these analyses is extremely important.Fortunately, studies show that pathology reporting in oncology is changing from a narrative approach to standardized synoptic reporting, leading to a significantly increased completeness of the pathology reports [18][19][20].
A complementary BRCA1 MLPA analysis was performed for 344 patients.In these patients, the MLPA analysis detected an additional 3.5% of BRCA1 TPVs.MLPA analyses are generally applied to detect large rearrangements, such as the deletions or duplications of complete exons or multiple exons.While NGS is considered a reliable tool to detect point mutations, which comprise most BRCA1/2 PVs, the NGS is less sensitive to detecting large rearrangements.The large arrangements particularly occur in the BRCA1 gene and are known to be more prevalent in certain populations, including the Dutch population [21][22][23][24].
Conducting NGS with a complementary MLPA analysis is frequently regarded as offering a comprehensive evaluation of potential genomic changes in the BRCA1/2 genes, whereas when solely NGS is employed, potential TPVs may be missed.Estimates of the prevalence of large genomic rearrangements in BRCA1 in EOC specifically remain limited, which makes it challenging to estimate the number of TPVs missed when not performing an MLPA analysis alongside the NGS.A Slovakian study performed MLPA analyses in 39 tumor samples of high-grade serous ovarian cancer and detected one pathogenic BRCA1 deletion (2.6%) [25].Pathogenic large rearrangements were also analyzed in 20,000 ovarian tumors with NGS and were detected in 0.7% of the cases, which reflected a total of 6.3% of all BRCA1/2 TPVs detected in the cohort [26].This relatively low percentage could be explained by the lower sensitivity of NGS in detecting large deletions and duplications and may, therefore, underestimate the prevalence of these large rearrangements.Furthermore, the presence of founder mutations, as established in BRCA1 in the Netherlands [24], increases the number of TPVs to be detected by MLPA analysis and should be considered when making direct comparisons.
The proportion of BRCA1/2 TPVs detected did not significantly differ between the hybrid capture technique, which constituted solely smMIP-based assays [27], and the PCR-based techniques and does not, therefore, indicate significant performance differences between the techniques regarding BRCA1/2 TPV yield.It must be noted that for a thorough comparison of BRCA1/2 yield in hybrid capture versus the PCR-based technique, a more diverse inclusion of tests using the hybrid capture technique is preferred.Few studies have compared the overall performance of hybrid capture and PCR-based approaches in detecting PVs.A better overall performance was reported for the hybrid capture technique in detecting BRCA1/2 PVs from formalin-fixed paraffin-embedded (FFPE) EOC tumor samples compared to the PCR-based technique [28], and similar findings were reported in a study assessing the detection of actionable mutations in lymphoma [29].In general, these studies linked the PCR-based technique to a lower sensitivity due to amplicon dropout and insufficient coverage.On the other hand, PCR-based techniques are also reported to be suitable for the accurate detection of BRCA1/2 PVs [30].Moreover, it requires lower quality and quantity of DNA and is significantly less time consuming, which are important parameters for a laboratory to consider when choosing between methods [31][32][33].The results of the current study do not show quality differences between the techniques, thereby justifying the use of both techniques in BRCA1/2 TPV detection.The selection of methods, genes and sequence machines is often carried out by individual laboratories and is generally based on several aspects, including reliability, lead times and costs.The latter could not be evaluated in the current study since this information was not available.In the Netherlands, pathology laboratories are free to choose techniques given the technique is validated, and the national quality control standard for molecular diagnostics ensures that these techniques meet high-quality criteria [17].This subsequently guarantees high-quality diagnostics and care for all patients.
A total of 74 BRCA1/2 TPVs were detected in this cohort of Dutch EOC patients (14.7% of all tests), of which 90.5% were detected in high-grade serous carcinoma.Our overall proportion of BRCA1/2 TPVs in EOC, unselected for histotype, is similar to the 13% proportion we reported previously in a Dutch multi-center study that included a consecutive series of EOC patients [34].The proportion is slightly lower compared to the 16.7% proportion in EOC reported by another Dutch study, which included a complementary MLPA analysis for all cases, and also lower compared to the 19% proportion reported in the United States [8,35].
This study shows that EOC tumor tests for BRCA1/2 TPV detection were already performed for 50% of all patients before this was officially recommended by national and international guidelines [6,7,36].Currently, tumor testing is implemented nationwide; testing is centralized mostly in academic hospitals, and comprehensive gene panels are more frequently applied.It should be noted that the BRCA1/2 tumor test rate of 50.3% reported in this study does not imply that only half of all patients received BRCA1/2 testing.
The timeframe analyzed here was before national guidelines recommended tumor testing in EOC; therefore, it is likely that medical centers followed former guidelines and referred patients for genetic counseling and germline testing instead [37].
This study has several strengths and limitations.Strengths include the analysis of realworld clinical data with full nationwide coverage of all EOC pathology reports.Linking the data from narrative pathology reports to clinical characteristics such as the FIGO stage allowed us to tailor this evaluation to the population of interest, namely FIGO stage III/IV patients.Nevertheless, data requests from national registries, such as PALGA, are subject to prespecified timeframes and the possibility exists that tumor tests were requested beyond this timeframe for the patients in our population.This may have led to an underestimation of the proportion of EOC patients who received a tumor test.Moreover, data collection for the current study entirely depended on the data reported in the pathology reports.This limited the possibility to evaluate the additional technical aspects of the execution of the tumor tests and restricted the evaluation to the endpoints reported in the current study.Finally, this study analyzed the execution of tumor tests before the full completion of the nationwide implementation of the tumor testing; therefore, it is likely that not all the centers that are currently performing tumor testing were included.Repeating our analyses with the data obtained after the full completion of the national implementation and comparing that data to the results reported in this study can provide valuable insights into the changes in the execution of tumor tests in EOC over time.

Conclusions
This study highlights the heterogeneity in the execution of EOC tumor testing in the Netherlands in 2019 despite the centralization of testing in specialized centers.The findings of this study are not indicative of any quality differences between the techniques used.Furthermore, nationally implemented quality control standards ensure the high-quality implementation of reliable BRCA1/2 tumor testing.This is crucial for identifying all patients with BRCA1/2 TPVs to provide high-quality care, as well as for guiding genetic cascade testing to ultimately prevent cancer in unaffected relatives with BRCA1/2 GPVs.

Figure 1 .
Figure 1.Flowchart of stage III/IV EOC patient selection.Figure 1. Flowchart of stage III/IV EOC patient selection.

Figure 1 .
Figure 1.Flowchart of stage III/IV EOC patient selection.Figure 1. Flowchart of stage III/IV EOC patient selection.

Figure 2 .
Figure 2. Distribution of the techniques applied and genes analyzed in EOC tumor tests.

Figure 2 .
Figure 2. Distribution of the techniques applied and genes analyzed in EOC tumor tests.

Table 1 .
Number of EOC diagnoses by histological subtype in the Netherlands in 2019.

Table 2 .
Number of BRCA1/2 tumor tests performed and the prevalence of BRCA1/2 TPVs and VUS.

Table 3 .
Assays used and genes tested in epithelial ovarian tumor analyses using hybrid capture (n = 244) or PCR-based techniques (n = 133).