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Challenges in next generation sequencing of homology recombination repair genomic variants in prostate cancer: A nationwide survey and calibration project in China

Background Homologous Recombination Repair (HRR) is the most reliable and important signaling pathway for repairing DNA damage. We initiated a calibration project to better understand the NGS landscape for HRR gene testing in China, provide indications for testing standardization, and guide clinical practice. Methods A questionnaire was used to collect laboratory information, panel design for HRR gene testing, tissue sample test parameters, plasma ctDNA sample test parameters, and procedures for variant interpretation. The testing quality of the participating laboratories was further evaluated by external quality assessment (EQA), which provided 5 FFPE slices and 5 mimic ctDNA samples as standard references for evaluation. Test results and reports were collected to assess laboratory performance. Results Our results showed that different laboratories had significant differences in sequencing platforms, library construction technologies, genes in the testing panel, detectable mutation types, probe coverage regions, sequencing parameters, variants interpretation guidelines, and positive test rates. For the EQA test, the overall pass rate was about 60%. The average accuracy for tissue samples and ctDNA samples was 79.55% and 74.13%, respectively. It is worth noting that variants in tandem repetition regions and splice sites, and those with low allele frequency were more prone to misdetection. The most common reasons for misdetection were as follows: the testing panel did not cover the genes or the whole exon and splice sites of the genes; the variants were misclassified as benign or likely benign, and the variants failed the QC criteria. Conclusions The discrepancies observed in our survey and EQA test affect the authenticity of HRR gene test results for prostate cancer, underlining the need to establish guidelines for HRR gene testing and variant interpretation in China, and to optimize HRR gene testing in clinical practice to improve management and patient care.


Introduction
The incidence of prostate cancer (PCa) is 15.6/100,000, and new cases exceed 110,000, leading to more than 50,000 deaths in China in 2020. 1 Although the majority of patients with locally advanced and metastatic disease initially respond to androgen deprivation therapy (ADT), most will progress into metastatic castration-resistant PCa (mCRPC). 2 However, some unique RNA-seq profiles of Pca had been detected, 3 and the instability of RNA limits its application in clinical testing. There had been some previous studies suggesting homology recombination repair (HRR) was the most important pathway to repair DNA double-strand break, which was one of the most genotoxic forms of DNA damage and tumori-genesis. 4 In the mCRPC stage, 25%e30% of patients carrying HRR gene mutations are sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy 5, . 6 Based on the positive results from PROfound trial Phase III, the PARP inhibitor Olaparib was approved by the United States Drug and Food Administration (FDA) in 2020 for mCRPC patients with deleterious or suspected deleterious HRR gene mutations who progressed on abiraterone or enzalutamide therapy. 7 The 2021 edition of the National Comprehensive Cancer Network (NCCN) and the European Association of Urology (EAU) Prostate Cancer Guidelines recommend that all patients with metastatic PCa should undergo tumor HRR gene testing 8, . 9 Next-generation sequencing (NGS) is increasingly used in cancer screening and early detection 10, . 11 Although HRR gene testing has been widely used in China, the concordance of testing results among different labs remains unclear. In fact, a survey of 158 laboratories that provided BRCA1/BRCA2 sequence analysis revealed significant differences in the platforms and variant detection used in 2018, 12 but there was no Chinese laboratory participated in this survey.
Though the failure rate of tumor tissue HRR gene testing is about one-third, 13 repeated biopsies are often impractical for mCRPC patients. Liquid biopsy is rapidly developing in tumor biomarker detection, with the advantages of easy sampling, minimally invasive, and reproducible. 14,15 HRR gene testing using plasma circulating tumor DNA (ctDNA) has high consistency with the test using tissue samples, and can be alternatively used in clinical practice when tissue is unavailable, especially in mCRPC patients. [16][17][18][19][20] The technical requirements for ctDNA testing are much stricter than tissue sample testing due to the low concentration and high fragmentation of tumor DNA in plasma. 21 Consequently, uncertainty exists regarding the reliability of ctDNA testing.
Because there are no guidelines or consensus statements available in China, we launched a nationwide HRR gene testing calibration project to better understand its landscape and quality. The study will provide a snapshot of the current status and the proficiency of HRR gene testing in China, and will serve as the basis to establish regulations or guidelines.

Survey
A total of 32 third-party laboratories that could provide PCa NGS-based HRR gene testing participated in our survey. The questionnaire is available on the following website: https://www.wjx. cn/jq/81953684.aspx. There are 43 questions in the questionnaire, including three sections: (1) basic laboratory information; (2) NGS testing information for PCa with HRR gene mutations (including HRR gene panel design information, tissue test parameters, plasma ctDNA test parameters); and (3) the verification and interpretation of HRR gene mutations.
The mutations in the reference standards were verified by Sanger sequencing. The details are reported in Supplementary  Tables 1 and 2. In summary, there are 22 mutation loci in the FFPE samples, involving 12 HRR genes. And the variant allele frequency (VAF) range is 3.60%-54.17%. The ctDNA mimic samples cover 40 mutation loci with the VAF range of 0.56%-49.58%. Only SNVs and indels were included. The expected results of the reference standards are summarized in Table 1.

Scoring criteria
The scoring criteria were based on the ability to detect the variants. A maximum of 5 points was assigned for each expected locus in the FFPE samples, and the total maximum score for all 22 mutation loci in the FFPE samples was 110 points (5*22). A maximum of 2.5 points was assigned for each expected variant in the mimic ctDNA samples, and the total maximum score for the mimic ctDNA samples was 100 points (2.5*40).
The participants were required to submit clinical reports and basic test information, quality control information, and variant interpretation information, which classified variants as pathogenic/ likely pathogenic variants or variants of uncertain significance (VUS). Since there is currently no specific guidance for the interpretation of HRR variants, this EQA project did not assess the interpretation capability. The information on variant classification was collected for data analysis.

Basic information of NGS laboratories
Among the 32 commercial NGS laboratories that participated in the survey, 43.75% of the laboratories were small-scale laboratories (<50 employees), 43.75% were medium-scale (50-500 employees), and 12.5% were large-scale laboratories (over 500 employees). 43.75% of the laboratories passed CAP (College of American Pathologists) certification, among which 18.75% had passed both CAP and CLIA (Clinical Laboratory Improvement Amendments) certi- fications, 12.5% passed ISO15189, and the remaining labs passed other certifications. All institutions had participated in and passed at least one external quality evaluation project for BRCA testing capability evaluation, and about 63% passed the EMQN external quality evaluation. In summary, although NGS testing in China is dominated by small and medium-sized facilities with relatively short operation time (<5 years), it puts forward high requirements for quality management.

Sequencing platform and library construction methodology
In terms of sequencing technology, Illumina was the most commonly used NGS platform (24 laboratories, 75%). 7 (22%) laboratories adopted the ThermoFisher platform, while 2 (6%) adopted the BGI (Beijing Genomics Institute) platform. The details were summarized in Table 2.
Overall, 96% of laboratories using Illumina and BGI NGS platforms adopted hybrid capture methodology to construct the sequencing libraries, and all laboratories using the ThermoFisher platforms adopted PCR amplicon methodology to construct libraries. Both library construction methodologies presented advantages and disadvantages, and some laboratories using the ThermoFisher platform did not provide plasma sample testing services.
Regarding the testing panels for tissue samples, 19 laboratories offered PCa-specific small panels, which were preferred by clinicians due to their higher cost-effectiveness. 13 laboratories used more expensive pan-tumor panels. Only 12 laboratories offered clinicians smaller panels for plasma testing. 24 laboratories used the same size panel for both tissue and plasma testing, although the design and quality control parameters of plasma and tissue samples may be different.

Panel design of HRR gene testing
In terms of gene coverage and probe design, 3 (9.3%) laboratories covered the 14 genes approved by the FDA for mCRPC in the testing panel, 26 (81.25%) laboratories covered more DDR genes (Fig. 1A).
Regarding the exon coverage of HRR genes, 22 (69%) laboratories covered all the exon regions of HRR genes, and 10 (31%) laboratories sequenced only the hot regions of more commonly mutated exons, based on the literature and clinical evidence (Fig. 1B). With regard to intron regions, 17 laboratories included all the canonical splice sites, of which 11 laboratories covered 20-50 bp downstream and upstream of the coding exons, 3 laboratories covered less than 20 bp of the intron region, and 3 laboratories covered only the canonical splice sites. 15 (47%) laboratories selected the covered intron regions, based on clinical evidence associated with individual genes (Fig. 1C). Since there are no specific hotspot loci or regions in the HRR genes, it is important to cover all the exons and splice sites to avoid potential false-negative results.
Several loss-of-function variant types have been identified, including single nucleotide variation (SNV), insertion-deletion (indel), copy number variation (CNV), and large genome rearrangement (LGR). In the requirements of mutation type detection, all laboratories could detect SNV and indel variants, and about 80% could detect CNV. The LGR detection remained a major challenge for NGS, and only 53% of laboratories could detect it (Fig. 1D).

HRR positive rate and interpretation guideline
From the survey results, 50% of laboratories did not perform statistical analysis on the HRR gene mutation rates in PCa and other tumor types due to the small sample size. However, some labo-  7 The difference in mutation frequency may be due to the inconsistency of gene panels and interpretation. Although there are no specific guidelines on HRR variants interpretation, the standards and guidelines of the American College of Medical Genetics and Genomics (ACMG guidelines) are wellaccepted in the practice [22][23][24][25] . "Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer" (AMP/ ASCO/CAP guideline) 26 is used as a guide for variant classification.
Only 25% of laboratories used only the ACMG guidelines, less than 5% used only the AMP/ASCO/CAP guideline, and 45% of laboratories used both. However, about 15% of laboratories also used other guidelines, and 5% of laboratories used their own standards. Due to the lack of public databases for HRR variant interpretation, the proportion of VUS is 10%-50%. Thus, it is important to establish public databases to facilitate the interpretation. However, only 60% of laboratories were willing to share their internal data. The test failure rate of tissue and plasma samples was approximately 5%-10%, which was far lower than the PROfound trial. 17 According to the survey, poor sample quality was the main reason, such as less tumor content, DNA degradation, genomic DNA contamination, and insufficient ctDNA extraction. Standardized sample handling is the key to improving the success rate of testing.   (Table 3). All 32 laboratories participated in tissue sample testing, and 62.5% of them passed the assessment ( Fig. 2A). 10 laboratories achieved a full score of 110, 15 laboratories achieved more than 100, and 20 laboratories got more than 90 points. The detection rate of each locus is shown in Fig. 2B and the mean is 79.55 ± 12.19%. Additionally, loci with a detection failure rate greater than 30% are listed in Table 4. Small indel variants are likely to be missed. The adjacent genome sequence of these loci were mainly tandem repeat sequences with poly-A or Poly-T regions (3), which may be challenging for some platforms. Sequencing depth and target region coverage rate had no significance on the testing score, while the library construction methodology was closely relevant. In 24 laboratories that adopted the hybrid capture method to construct libraries, the average score was 94.58, and the pass rate was 79%; while in 8 laboratories that adopted the PCR amplicon-based method, the average score was 66.88, and the pass rate was 12.5%.

EQA results for HRR testing using tissue samples
3.6. EQA results for HRR testing with plasma samples 23 laboratories participated in ctDNA sample testing. 10 laboratories achieved up to 90, 15 got >80 and the pass rate was about 65% (Fig. 3A). The detection rate of each variant loci is shown in Fig. 3B and the mean detection rate is 74.13 ± 17.78%. Information regarding the top missed variants is shown in Table 5. Missing loci were mainly distributed in variants of low allele frequency and indels in tandem repeat regions. These laboratories all adopted Illumina and BGI platforms, and no significance was found between testing score and sequencing depth, or target region coverage rate. The possible reasons for the missed variants were misclassification, low coverage, and below the QC criteria.

Discussion
This was the first nationwide survey and EQA involving HRR gene testing laboratories in China to evaluate laboratory practices and challenges in methodology, infrastructure, interpretation, and testing quality. In this survey, we observed inconsistencies in the HRR gene testing laboratories including sequencing platforms, library construction methods, gene panels, probe design, detectable mutation types, HRR positivity rate, variant interpretation, test failure rate, and quality control. In EQA, about 60% of laboratories passed the test. The most challenging variants for detection were small indels and variants located within the tandem repeat regions in tissue samples, as well as splice site and low allele frequency variants in ctDNA samples.
The most common NGS platform was Illumina (75%), followed by Thermo Fisher (22%) and BGI (6%). 96% of Illumina platforms used hybrid capture, while all Thermo Fisher platforms used PCR amplicon approaches. Compared with PCR amplicons, hybrid capture library creation generates higher capture efficiency, specificity, and repeatability, and is more suitable for detecting lower frequency variants, while PCR amplicon-based library preparation requires a much lower DNA loading amount, is easier to handle, has a shorter turn-around time, is more suitable for small panel testing, and is often used in hospital. 27 Indeed, the standard reference testing results showed a higher pass rate via hybrid capture for library construction.
In terms of gene panel design, over 90% of panels covered all the 14 HRR genes to guide Olaparib treatment for mCRPC, but not all laboratories covered all the exons and splice sites of these genes, which may lead to potential false negatives, and misdetection sites in their feedbacks. To avoid this, the panel design should include at least 14 HRR genes, covering all exon regions and splice sites.
With regard to the detectable mutation types, all panels could detect SNV and indel, about 50% of laboratories could detect LGR, and more than 80% could detect CNV. The main limitation of the reference standard was that the capability of LGR and CNV testing was not evaluated in this EQA project. Since LGR and CNV could cause loss of function, it is important to verify its panel testing capability. 28 For the positive rate, most laboratories did not provide statistical results due to the limited sample volumes and substantial variations. This may be explained by the inconsistencies across testing panels, platforms, and interpretation standards. A similar situation was also observed in a worldwide survey study, 47% of the non-US labs used ACMG guidelines and 38% of US labs followed their own measures. 29 The expert consensus in China on BRCA variant interpretation is that ACMG guidelines and AMP/ASCO/CAP guidelines should be combined and only pathogenic or likely pathogenic variants can guide treatment based on the clinical evidence. 30 Laboratories should be encouraged to establish personalized databases and standards based on these rules. In our survey, 24 labs submitted clinical reports and 19 labs reported VUS, while in a previous survey, 52% of US labs uncalculated VUS. 31 Since the uncertainty of VUS classification, it is challenging to estimate the risk of affected patients during clinical decision-making and genetic counseling 32, . 33 Variant interpretation will be improved through the coordination of clinicians, pathologists, and the establishment of a Chinese HRR gene database. Sharing data enables quality-control, peer-reviewed processes, providing opportunities to improve the strategic management of patient care. Unfortunately, our study revealed that over 80% of the data was not shared with public databases due to profit considerations.
Two-thirds of the laboratories carry out plasma ctDNA testing. Lacking matched tissue and plasma samples data is because of the low feasibility of tissue samples. In PROfound and TRITON2, the consistency between tissue samples and plasma samples was approximately 82%-91% 17, . 18 Additionally, the quantity and quality of ctDNA are critical 19, . 34 Therefore, the extraction protocol of cell-free DNA requires further optimization. 35 Clinical report standardization is also important for facilitating interpretation. In EQA testing, several issues were raised, including unnecessarily massive information, unacceptable errors, and missing critical information.
In summary, to improve HRR gene testing quality in China, it is necessary to establish standards for involved procedures. Our study is the first to report the nationwide EQA of HRR gene testing. The most common differences across laboratories included NGS platform, gene coverage, probe design, validation method, VUS analysis, interpretation, quality control, and data-sharing. Our results indicate the requirement for national guidelines to improve the clinical practice of HRR gene testing laboratories.