A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A4G and BRCA1 c.5194À12G4A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977À7C4G induced in frame inclusion of 6 nt from the 3¢ end of intron 17. The novel variants BRCA2 c.520C4T and BRCA2 c.7992T4A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions. Keywords: variants of unknown significance; splicing defects; splicing reporter minigene; breast and ovarian cancer; BRCA1 and BRCA2 INTRODUCTIONThe interpretation of variants of unknown significance (VUS) found in the molecular screenings of the breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, is essential for genetic counseling of patients and their families and for the implementation of new therapies targeted to carriers of BRCA mutations, such as those based on poly-(ADP-ribose) polymerase inhibitors. 1 The number of these variants already exceeds that of the reported pathogenic mutations, and is expected to increase rapidly with the use of new high throughput sequencing technologies that are based on massive parallel sequencing. 2 It is now widely accepted that RNA analyses should be used to improve the assessment of pathogenicity of sequence variation, because a large fraction of sequence variants, both intronic and exonic, may induce splicing defects. However, in many cases, patient RNA is either not available or it has been obtained in ways that do not ensure its stability. Moreover, it is sometimes difficult to detect the mRNA affected by a truncating mutation because of the activation of the nonsense-mediated mRNA decay pathway. 3 Functional assays of the effect of VUS on RNA splicing, using patient genomic DNA and a splicing reporter hybrid minigene,

Acquired estrogen receptor gene (ESR1) mutations have been recently reported as a marker of resistance to aromatase inhibitors in hormone receptor positive metastatic breast cancer. We retrospectively considered seven patients treated for metastatic breast cancer with available samples from the primary tumor before any treatment, cryopreserved metastasis removed during progression and concomitant plasmas. All these seven patients were in disease progression after previous exposure to aromatase inhibitors for at least 6 months, and were assessed for ESR1 mutations detection in tumor and circulating DNA. For these patients, Sanger sequencing identified four metastases with clear ESR1 mutation and one possible, whereas digital PCR identified six mutated metastases. Then, under blind conditions and using digital PCR, corresponding circulating ESR1 mutations were successfully detected in four of these six metastatic breast cancer patients. Moreover, in two patients with serial blood samples following treatments exposure, the monitoring of circulating ESR1 mutations clearly predicted disease evolution. In the context of high interest for ESR1 mutations, our results highlight that these acquired recurrent mutations may be tracked in circulating tumor DNA and may be of clinical relevance for metastatic breast cancer patient monitoring.Nearly 70% of breast cancers are hormone receptor positive (HR1) and potentially sensitive to hormonal therapy. Aromatase inhibitors (AI) or tamoxifen is the recommended first line hormonal therapy in postmenopausal HR1 metastatic breast cancer (MBC).1,2 Nevertheless, disease progression is usually observed within a few months after treatment initiation.3,4 Acquired resistance to hormonal therapy may be based on activating mutations in the estrogen receptor gene (ESR1). These mutations have been detected in HR1 MBC patients previously exposed to hormonal therapy, including treatment with an AI in all cases. The frequencies of ESR1 mutations was 25% (nine of 36), 38% (five of 13) and 54% (six of 11) among these cohorts. [5][6][7] Approximately 12 ESR1 point mutations have been described, with a hot spot confined to codons 537 and 538 in exon 8. 8 These mutations result in a ligand-independent estrogen receptor (ER) activity. In vitro and preclinical data suggest that ESR1 mutations lead to complete AI resistance and to partial resistance to ER agonists and antagonists.9,10 The detection of ESR1-activating mutations may be relevant for guiding clinicians between endocrine and nonendocrine therapy.11 Thus far, ESR1 genotyping must be performed on metastases: this method is hardly compatible with repetitive sampling analyses for disease monitoring. Circulating tumor DNA (ctDNA) analysis is considered a promising tool for providing relevant prognostic and/or predictive information instead of tumor sampling. 12,13

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