Somatic mRNA Analysis of BRCA1 Splice Variants Provides a Direct Theranostic Impact on PARP Inhibitors

Chevalier, Louise-Marie; Billaud, Amandine; Fronteau, Sabrina; Dauvé, Jonathan; Patsouris, Anne; Verriele, Véronique; Morel, Alain

doi:10.1007/s40291-020-00452-z

Cited by 3 publications

(5 citation statements)

References 21 publications

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“…The sensitivity of the patient 4, who relapsed after only 3 months on PARP inhibitor, was consistent with these hypotheses (Fig. 3 ) and might also reflect the low level of expression of the splice variant compared to the full-length transcript despite the high variant frequency in the sample (analysis of the patient’s RNA by RT-qPCR and previously published) [ 20 ]. The second intermediate variant, p.Leu1080=, is located in the middle of exon 11 of BRCA1 .…”

Section: Discussionsupporting

confidence: 83%

“…We then also studied other 10 variants of BRCA1 previously classified as VUS or not previously reported in the databases (ClinVar, BRCA exchange, UMD) (Table 1 ). References mentioning these variants and their potential functional impact are listed in Table 1 where available [ 20 , 25 , 36 – 52 ]. These 23 VUS and the 10 other variants we used for validating our system affected different domains of the proteins and were distributed along the entire length of these genes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Somatic variants, which may be detected in only one or two individuals, also exist and many such variants remain unreported. Functional testing in vitro is currently based on transcriptional activation [ 14 , 15 ], HR activity [ 16 , 17 ], and splicing [ 18 – 20 ]. However, such tests are not necessarily compatible with clinical management in terms of the time taken to obtain results to guide treatment.…”

Section: Introductionmentioning

confidence: 99%

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Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

et al. 2021

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Background Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using BRCA1 and BRCA2 variants, which have consequences on PARP inhibitor sensitivity, and POLE variants, potential biomarkers of immunotherapy response, we developed a rapid functional assay based on CRISPR-Cas9 genome editing to determine the functional consequences of these variants having potentially direct implications on patients’ access to targeted therapies. Methods We first evaluated the functional impact of 26 BRCA1 and 7 BRCA2 variants by editing and comparing NGS results between the variant of interest and a silent control variant. Ten of these variants had already been classified as benign or pathogenic and were used as controls. Finally, we extended this method to the characterization of POLE VUS. Results For the 23 variants that were unclassified or for which conflicting interpretations had been reported, 15 were classified as functionally normal and 6 as functionally abnormal. Another two variants were found to have intermediate consequences, both with potential impacts on splicing. We then compared these scores to the patients’ responses to PARP inhibitors when possible. Finally, to prove the application of our method to the classification of variants from other tumor suppressor genes, we exemplified with three POLE VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four POLE variants previously classified in databases were also evaluated. However, we found evidence of a discordance with the classification, variant p.Leu424Val being found here functionally normal. Conclusions Our new rapid functional assay can be used to characterize the functional implication of BRCA1 and BRCA2 variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients’ responses to PARP inhibitors, where accessible, was consistent with our functional score evaluation and confirmed the accuracy of our protocol. This method could potentially be extended to the classification of VUS from all essential tumor suppressor genes and can be performed within a timeframe compatible with clinical applications, thereby having a direct theranostic impact.

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Section: Discussionsupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

et al. 2021

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“…Nine mutations of BRCA1 were detected from the sequencing process in the same patients’ samples ( Table 1 ). Two, namely c.5277 + 65C > T and c.5277 + 67T > C were new, not previously identified mutations, whereas seven (M3-M9) had been previously identified and characterized (NCBI; , accessed on 13 September 2021) [ 45 , 46 ]. All of the detected mutations were found in the heterozygous state.…”

Section: Resultsmentioning

confidence: 99%

“…M4 (A > T mutation) ( , accessed on 13 September 2021) is a splice acceptor variant. In Chevalier et al, the reported A > G mutation (c.5194-2A > G) caused a change in the splice acceptor region which affects alternative splicing and is thus identified as pathogenic [ 46 ]. TNBC patients bearing the M4 ( p = 0.008), M7 ( p = 0.019) and M8 ( p = 0.019) mutations were also correlated with decreased OS.…”

Section: Discussionmentioning

confidence: 99%

PARP-1 Expression and BRCA1 Mutations in Breast Cancer Patients’ CTCs

Sklias

Vardas

Pantazaka

et al. 2022

Cancers

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BRCA1 and PARP are involved in DNA damage repair pathways. BRCA1 mutations have been linked to higher likelihood of triple negative breast cancer (TNBC). The aim of the study was to determine PARP-1 expression and BRCA1 mutations in circulating tumor cells (CTCs) of BC patients. Fifty patients were enrolled: 23 luminal and 27 TNBC. PARP expression in CTCs was identified by immunofluorescence. Genotyping was performed by PCR-Sanger sequencing in the same samples. PARP-1 expression was higher in luminal (61%) and early BC (54%), compared to TNBC (41%) and metastatic (33%) patients. In addition, PARP-1 distribution was mostly cytoplasmic in luminal patients (p = 0.024), whereas it was mostly nuclear in TNBC patients. In cytokeratin (CK)-positive patients, those with the CK+PARP+ phenotype had longer overall survival (OS, log-rank p = 0.046). Overall, nine mutations were detected; M1 and M2 were completely new and M4, M7 and M8 were characterized as pathogenic. M7 and M8 were predominantly found in metastatic TNBC patients (p = 0.014 and p = 0.002). Thus, PARP-1 expression and increased mutagenic burden in TNBC patients’ CTCs, could be used as an indicator to stratify patients regarding therapeutic approaches.

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L’instabilité génomique, paramètre limitant l’efficacité des thérapies ciblées en oncologie

et al. 2020

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Somatic mRNA Analysis of BRCA1 Splice Variants Provides a Direct Theranostic Impact on PARP Inhibitors

Cited by 3 publications

References 21 publications

Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

PARP-1 Expression and BRCA1 Mutations in Breast Cancer Patients’ CTCs

L’instabilité génomique, paramètre limitant l’efficacité des thérapies ciblées en oncologie

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