A commentary on the discrepancy between blood and tumorBRCA testing: an open question Elisa De Paolis1, Claudia Marchetti2;3, Paola Concolino1, Giovanni Scambia2;3, Andrea Urbani1,3, Anna Fagotti2;3, Angelo Minucci1*1Molecular and Genomic Diagnostics Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy;2Division of Oncological Gynecology, Department of Women’s and Children’s Health, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy;3Catholic University of the Sacred Heart, Rome, Italy.Running title: Understanding discordant BRCA test cases.*Corresponding author:Angelo MinucciMolecular and Genomic Diagnostics Unit, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.angelo.minucci@policlinicogemelli.itMolecular evaluation of BRCA1/2 (BRCA ) genes represents a well-known example of precision oncology. The availability of Poly ADP Ribose Polymerase inhibitors (PARPi) as target therapy option for several BRCA mutated cancers types (e.g. ovarian, breast, prostate, and pancreatic)1 changed the course ofBRCA testing over the last years. In this context, an emerging path of molecular evaluation is represented by the BRCA testing performed directly on tumor tissue (tBRCA ): this increased the chance to identify more patients with higher likelihood of benefiting from PARPi treatment. This approach leads to the simultaneous identification of both constitutional and somatically acquired variants, with a lower turnaround time: the identification of BRCApathogenic variants (PVs) could lead to a secondary “reflex” germlineBRCA (gBRCA) testing in order to assess personal and familiar risks. In contrast, performing gBRCA as first molecular test causes the loss of a relevant proportion of patients with tissue acquired BRCA PVs, needing of a following tumor test2;3.However, challenges exist in tBRCA that may lead to inefficient germline variant call. A recently published paper by Kordes et al. reported a pancreatic adenocarcinoma patient with a germlinenovel BRCA2 c.516+4A>G variant classified as deleterious by the authors based on in silico and functional data4. Also the tumor tissue was sequenced in order to achieve the enrolment criteria for a clinical trial of Olaparib in combination with pembrolizumab (KEYLYNK-007). Unexpectedly, the germline variant was not stated in the final report. The authors took into account all the relevant basis of the experienced discrepancy, without identifying a confident reason.In our opinion it is crucial to investigate about the reliability of tBRCA in the identification of both somatic and germline variants. Inspired by the recently published commentary of Gourley5 and taking into account that several troubling cases of discrepancy between blood and tBRCA testing have been reported in literature, we collected the recent relevant studies covering the comparison between gBRCA and tBRCA to give a critical opinion about some shared key points of the somatic testing that could affect the final genotyping and reporting (Table 1).Major reasons of discrepancies are related to: (1) differences in input DNA quality, (2) type of BRCA gene alteration, (3) inherent limitations of the Next Generation Sequencing (NGS), (4) bioinformatics pipeline features (e.g. the ability to predict the occurrence of Copy Number Alterations (CNAs) and the evaluation of the intron/exon boundaries), and finally (5) the issues related to the BRCAvariants interpretation and classification.To date, tBRCA testing is mainly performed on two sample types: Fresh Frozen Tissue (FFT) and Formalin-Fixed Paraffin-Embedded (FFPE). Here, we focused on tBRCA performed on FFPE being the most common tissue type used for clinical diagnostic purpose. Pre-analytical procedures regarding fixation step, tissue section size, and neoplastic cell content assessment, are well-known crucial aspects of the tBRCA testing reliability. In fact, sub-optimal DNA quality represents a relevant reason of inaccuracy of tBRCA and also it is the cause of around 5% of FFPE tBRCA NGS testing fails, with the consequent need of additional new samples9. In Bekos et al. only the retesting of a newly extracted tumor DNA solved two cases of discrepancies with gBRCA : the BRCA1c.1881_1884del variant was not recognized due to poor NGS quality data related to the input materials, as well as for theBRCA2 c.8537_8538del variant2. Also in Careet al. the test failure rate was related to fixation methods or storage of FFPE material8. Ad hocrecommendations for the “ideal” starting tissue material are available9;14.Furthermore, different approaches should be used in the analytical step for the BRCA genes amplification and sequencing, with several types of sequencing chemistries (e.g. amplicon-based, capture-based), platforms (e.g. Illumina, IonTorrent) and data analysis pipelines (e.g. full-coding regions or hot spot analysis, different size of splice site region analysed, CNAs detection). Each one of these could be characterized by specific pitfalls that affect the downstream bioinformatics variants filtering and calling. For example, in amplicon-based approaches, a reason leading to the missing of a variant detection may be related to the experimental design of the primers distribution along the genomic region of interest. Variants located at the 3’ or 5’ ends of overlapping amplicons could be covered by only one read and could be consequently identified with a “strand bias” flag and filtered out at the bioinformatics quality check3.Also the use of different bioinformatics pipeline for the NGS data analysis derived from the germline and the somatic tests of the same patient could be the cause of apparent inconsistent results. For example, in a large cohort of patients affected by several types of malignancies and analysed for the evaluation of the utility of germline test following tumor test, Lincoln et al. identified several cases of discrepancies between the two tests (n=4)15. Among these, the germline BRCA2 c.8967_8973del variant was not detected in tumor sequencing due to the characteristic of somatic panel (hot spot type), not comparable to the germline one. Moreover, in case of discrepancy involving splice site variants could be useful to check the concordance of the splice site region size included in the germline and somatic bioinformatics pipelines3. Regarding data analysis, it should be acknowledged that some tumor testing platforms filter out germline variants in the final reports in order to improve the accuracy of somatic variant calling.A well-known cause of gBRCA /tBRCA non-concordance resulted from the challenge in the bioinformatics calling of CNAs in tissue samples2;3;15. NGS sensitivity in CNAs detection mostly depends on DNA quality, tumor heterogeneity, library preparation, type of algorithms, and size of rearrangement. As a consequence, the somatic bioinformatics pipeline must require computational algorithms developed ad hoc and specific characteristics of sequencing raw data (e.g. maximum amount, coverage uniformity and sufficient reads depth)1. Even if the majority of methods are optimized for somatic CNAs identification6;8, attention should be given in the comparison of blood and tissue tests results13. As an example, Bekos et al. failed to identify in the tumor sample a verified pathogenic germline deletion of BRCA1 exon 20. Only a careful re-evaluation of the bioinformatics variant calls finally revealed the deletion and leaded to the correction of the report2.Relevant role in the evaluation of non-concordant results is played by the post-analytical step involving the BRCA variants interpretation. Complex issues underlying the classification ofBRCA variants exist. The American Collage of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) have established the best practice for germline variant interpretation providing the well-known classification using a five-tier system16. Conversely, the interpretation of somatic variants should be focused on their impact on clinical care. Specifically, evidence-based categorization of somatic variants released by the AMP, the American Society of Clinical Oncology (ASCO), and the College of American Pathologists (CAP) includes a four-tier system: (1) variants of strong clinical significance (level A and B of evidence); (2) variants of potential clinical significance (level C and D of evidence); (3) variants of unknown clinical significance; (4) benign of likely benign variants17. To date, with the publication of an increasing number of large-scale tumor sequencing projects, a plenty of information is being collected into several public databases useful for the querying about the significance of aBRCA variant. Cancer-specific variant databases are available as: BRCAexchange, OncoKB, Catalog of Somatic Mutations in Cancer, My Cancer Genome, cBioPortal, Memorial Sloan Kettering Cancer Center, International Cancer Genome Consortium, and VARSOME. Likewise, constitutional variant databases available are mainly: ClinVar, Human Gene Mutation Database, ENIGMA, Leiden Open Variation Database, and VARSOME. Differences in the germline- and somatic-based annotation may exist between the abovementioned tools. Consequently, the risk of non-concordant annotations of a BRCA variant could occur. This is crucial in the comparison between the same molecular test performed by different labs and it is exacerbated in the case of tBRCA and gBRCA concordance evaluation: variants that met germline guidelines16 to be considered pathogenic may not meet the criteria17 to be considered oncogenic in the somatic test. This situation could more likely affect the missense Variants of Unknown Significance (VUSs)15. As reported by Bekos et al. , after the inclusion of BRCA VUSs in the secondary data analyses, the concordance rate of tumor testing compared to germline one decreased, mainly due to VUSs classification2. Moreover, in a large study investigating the differences in variant interpretation between germline and somatic variants accounted in several cancer-related genes, Moodyet al. highlighted a relevant percentage of discrepancies in variants classification. Among these, the authors reported fourBRCA2 variants with discordant somatic/germline annotations15.In a retrospective cohort of 57 subjects tested for both germline and somatic BRCA status, Kim et al. highlighted one case of a germline variant not identified in the tissue evaluation10. This discrepancy derived from a true reversion of the germline BRCA1 variant accounted via restoration of the wild-type allele in the tissue cells. Finally, tBRCAshould follow specific criteria that maximize molecular information, improving the clinical relevance of the test and giving a more comprehensive interpretation of each variant. With these purposes, peculiar role is played by the “naturally occurring” BRCAsplicing isoforms. As we recently described for the BRCA1c.788G >T variant, complex considerations should be done for rare variants that not only are different germline and somatic annotations, but also are characterized by variability in final effect and annotation in the context of all gene relevant transcripts19.In conclusion, we underline as the systematic and careful checking of tumor tissue suitability could prevent and solve non-concordance cases. Moreover, the robust identification of BRCA variants in FFPE sample correlates with the confidence of the bioinformatics pipeline adopted for the variant filtering and calling, especially for the CNAs detection. In addition, translation of variant calls into clinical decisions relies on proper annotations and discrepancies in classifications of specific variants between tumor and germline contexts could represent a relevant pitfall.We argue that only harmonized guidelines encompassing the abovementioned methodological and post-analytical steps could solve the BRCAgermline and somatic testing bias. In our laboratory, BRCAgenetic testing is routinely performed on blood, FFT and FFPE samples1. In many cases, we routinely analyze matched blood and tissue samples belonging from the same patient, in order to perform an efficient BRCA test comprehensive of both germline and somatic evaluation. This approach pointed out also the relevance of multi-disciplinary and skilled resources for a solid molecular characterization of the tumor. Together with the need of standardization, we suggest as performing BRCA molecular test at both germline and somatic levels in the same laboratory could improve the reliability of the entire molecular path taken by the patient and his clinicians.Disclosure of interests: none declared.Contribution to Authorship: E.D.P. and A.M. conceived of the presented paper and wrote the manuscript with support from P.C. and C.M.. G.S., A.U. and A.F. supervised the project. All authors discussed, edited and contributed to the final manuscript.Details of Ethics Approval: not applicable.Funding: no funding was received for this commentary.