BRCA1 germline mutation and glioblastoma development: report of cases

BMC Cancer 15.1 (2015): 1-7.

Boukerroucha M, Josse C, Segers K, El-Guendi S, Frères P, Jerusalem G, Bours V.

Background Germline mutations in breast cancer susceptibility gene 1 (BRCA1) increase the risk of breast and ovarian cancers. However, no association between BRCA1 germline mutation and glioblastoma malignancy has ever been highlighted. Here we report two cases of BRCA1 mutated patients who developed a glioblastoma multiform (GBM). Cases presentation: Two patients diagnosed with triple negative breast cancer (TNBC) were screened for BRCA1 germline mutation. They both carried a pathogenic mutation introducing a premature STOP codon in the exon 11 of the BRCA1 gene. Few years later, both patients developed a glioblastoma and a second breast cancer. In an attempt to clarify the role played by a mutated BRCA1 allele in the GBM development, we investigated the BRCA1 mRNA and protein expression in breast and glioblastoma tumours for both patients. The promoter methylation status of this gene was also tested by methylation specific PCR as BRCA1 expression is also known to be lost by this mechanism in some sporadic breast cancers. Conclusion: Our data show that BRCA1 expression is maintained in glioblastoma at the protein and the mRNA levels, suggesting that loss of heterozygosity (LOH) did not occur in these cases. The protein expression is tenfold higher in the glioblastoma of patient 1 than in her first breast carcinoma, and twice higher in patient 2. In agreement with the high protein expression level in the GBM, BRCA1 promoter methylation was not observed in these tumours. In these two cases, despite of a BRCA1 pathogenic germline mutation, the tumour-suppressor protein expression is maintained in GBM, suggesting that the BRCA1 mutation is not instrumental for the GBM development.

Quantifying BRCA1 and BRCA2 mRNA Isoform Expression Levels in Single Cells

Int J Mol Sci.

2019 Feb 06

Lattimore VL, Pearson JF, Morley-Bunker AE, Investigators kConFab, Spurdle, Robinson AB, Currie BA, Walker MJ, Logan C.
PMID: 30736279 | DOI: 10.3390/ijms20030693

BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting.

Clinical significance of BRCA1 and BRCA2 mRNA and protein expression in patients with sporadic gastric cancer

Oncology Letters

2019 Mar 08

Kim H, Hwang I, Min H, Bang Y and Kim W
| DOI: 10.3892/ol.2019.10132

Evaluation of BRCA1-related molecular features and microRNAs as prognostic factors for triple negative breast cancers.

BMC Cancer.

2015 Oct 21

Boukerroucha M, Josse C, ElGuendi S, Boujemla B, Frères P, Marée R, Wenric S, Segers K, Collignon J, Jerusalem G, Bours V.
PMID: 26490435 | DOI: 10.1186/s12885-015-1740-9.

Abstract

BACKGROUND:
The BRCA1 gene plays a key role in triple negative breast cancers (TNBCs), in which its expression can be lost by multiple mechanisms: germinal mutation followed by deletion of the second allele; negative regulation by promoter methylation; or miRNA-mediated silencing. This study aimed to establish a correlation among the BRCA1-related molecular parameters, tumor characteristics and clinical follow-up of patients to find new prognostic factors.

METHODS:
BRCA1 protein and mRNA expression was quantified in situ in the TNBCs of 69 patients. BRCA1 promoter methylation status was checked, as well as cytokeratin 5/6 expression. Maintenance of expressed BRCA1 protein interaction with BARD1 was quantified, as a marker of BRCA1 functionality, and the tumor expression profiles of 27 microRNAs were determined.

RESULTS:
miR-548c-5p was emphasized as a new independent prognostic factor in TNBC. A combination of the tumoral expression of miR-548c and three other known prognostic parameters (tumor size, lymph node invasion and CK 5/6 expression status) allowed for relapse prediction by logistic regression with an area under the curve (AUC) = 0.96. BRCA1 mRNA and protein in situ expression, as well as the amount of BRCA1 ligated to BARD1 in the tumor, lacked any associations with patient outcomes, likely due to high intratumoral heterogeneity, and thus could not be used for clinical purposes.

CONCLUSIONS:
In situ BRCA1-related expression parameters could be used for clinical purposes at the time of diagnosis. In contrast, miR-548c-5p showed a promising potential as a prognostic factor in TNBC.

Functional ex vivo assay to select Homologous Recombination deficient breast tumors for PARP inhibitor treatment

Clin Cancer Res. 2014 Jun 24

Naipal KA, Verkaik NS, Ameziane N, van Deurzen CH, Ter Brugge P, Meijers M, Sieuwerts AM, Martens J, O'Connor MJ, Vrieling H, Hoeijmakers JH, Jonkers J, Kanaar R, de Winter J, Vreeswijk M, Jager A, van Gent DC.
PMID: 24963051

Purpose: Poly(ADP-Ribose) Polymerase (PARP) inhibitors are promising targeted treatment options for hereditary breast tumors with a Homologous Recombination (HR) deficiency caused by BRCA1 or BRCA2 mutations. However, the functional consequence of BRCA gene mutations is not always known and tumors can be HR deficient for other reasons than BRCA gene mutations. Therefore, we aimed to develop a functional test to determine HR activity in tumor samples to facilitate selection of patients eligible for PARP inhibitor treatment. Experimental design: We obtained 54 fresh primary breast tumor samples from patients undergoing surgery. We determined their HR capacity by studying the formation of ionizing radiation induced foci (IRIF) of the HR protein RAD51 after ex vivo irradiation of these organotypic breast tumor samples. Tumors showing impaired RAD51 IRIF formation were subjected to genetic and epigenetic analysis. Results: Five out of 45 primary breast tumors with sufficient numbers of proliferating tumor cells were RAD51 IRIF formation deficient (11%, 95%CI: 5%-24%). This HR defect was significantly associated with Triple Negative Breast Cancer (OR:57, 95%CI: 3.9-825, p=0.003). Two out of five HR deficient tumors were not caused by mutations in the BRCA genes, but by BRCA1 promoter hypermethylation. Conclusion: The functional RAD51 IRIF assay faithfully identifies HR deficient tumors and has clear advantages over gene sequencing. It is a relatively easy assay that can be performed on biopsy material, making it a powerful tool to select patients with an HR-deficient cancer for PARP inhibitor treatment in the clinic.

IRX5 promotes DNA damage repair and activation of hair follicle stem cells

Stem cell reports

2023 Apr 07

Chen, JK;Wiedemann, J;Nguyen, L;Lin, Z;Tahir, M;Hui, CC;Plikus, MV;Andersen, B;
PMID: 37084727 | DOI: 10.1016/j.stemcr.2023.03.013

The molecular mechanisms allowing hair follicles to periodically activate their stem cells (HFSCs) are incompletely characterized. Here, we identify the transcription factor IRX5 as a promoter of HFSC activation. Irx5-/- mice have delayed anagen onset, with increased DNA damage and diminished HFSC proliferation. Open chromatin regions form near cell cycle progression and DNA damage repair genes in Irx5-/- HFSCs. DNA damage repair factor BRCA1 is an IRX5 downstream target. Inhibition of FGF kinase signaling partially rescues the anagen delay in Irx5-/- mice, suggesting that the Irx5-/- HFSC quiescent phenotype is partly due to failure to suppress Fgf18 expression. Interfollicular epidermal stem cells also show decreased proliferation and increased DNA damage in Irx5-/-mice. Consistent with a role for IRX5 as a promoter of DNA damage repair, we find that IRX genes are upregulated in many cancer types and that there is a correlation between IRX5 and BRCA1 expression in breast cancer.

Functional ex vivo assay reveals homologous recombination deficiency in breast cancer beyond BRCA gene defects

Clin Cancer Res.

2018 Aug 23

Meijer TG, Verkaik NS, Sieuwerts AM, van Riet J, Naipal KAT, van Deurzen CHM, den Bakker M, Sleddens HFBM, Dubbink HJ, den Toom TD, Dinjens WNM, Lips EH, Nederlof PM, Smid M, van de Werken HJG, Kanaar R, Martens JWM, Jager A, van Gent DC.
PMID: 30139880 | DOI: 10.1158/1078-0432.CCR-18-0063

Abstract

PURPOSE:

Tumors of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double strand break (DSB) repair and high sensitivity to Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. In the present study we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancers (BrC) tissue: the RECAP test.

METHODS:

Fresh samples of 170 primary BrC were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring BRCA deficiencies, mutational signatures, tumor infiltrating lymphocytes (TILs) and microsatellite instability (MSI).

RESULTS:

RECAP was completed successfully in 148 out of 170 samples (87%). 24 tumors showed HRD (16%), while 6 tumors were HR intermediate (HRi) (4%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas 7 HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts (p=0.023) compared to HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in BrC (1%) (p=0.017).

CONCLUSION:

RECAP is a robust functional HR assay detecting both BRCA1/2 deficient and BRCA1/2 proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.

Increased gene expression variability in BRCA1-associated and basal-like breast tumours

Breast cancer research and treatment

2021 Jul 21

Wiggins, GAR;Black, MA;Dunbier, A;Morley-Bunker, AE;kConFab Investigators, ;Pearson, JF;Walker, LC;
PMID: 34287743 | DOI: 10.1007/s10549-021-06328-y

Inherited variants in the cancer susceptibility genes, BRCA1 and BRCA2 account for up to 5% of breast cancers. Multiple gene expression studies have analysed gene expression patterns that maybe associated with BRCA12 pathogenic variant status; however, results from these studies lack consensus. These studies have focused on the differences in population means to identified genes associated with BRCA1/2-carriers with little consideration for gene expression variability, which is also under genetic control and is a feature of cellular function.We measured differential gene expression variability in three of the largest familial breast cancer datasets and a 2116 breast cancer meta-cohort. Additionally, we used RNA in situ hybridisation to confirm expression variability of EN1 in an independent cohort of more than 500 breast tumours.BRCA1-associated breast tumours exhibited a 22.8% (95% CI 22.3-23.2) increase in transcriptome-wide gene expression variability compared to BRCAx tumours. Additionally, 40 genes were associated with BRCA1-related breast cancers that had ChIP-seq data suggestive of enriched EZH2 binding. Of these, two genes (EN1 and IGF2BP3) were significantly variable in both BRCA1-associated and basal-like breast tumours. RNA in situ analysis of EN1 supported a significant (p = 6.3 × 10-04) increase in expression variability in BRCA1-associated breast tumours.Our novel results describe a state of increased gene expression variability in BRCA1-related and basal-like breast tumours. Furthermore, genes with increased variability may be driven by changes in DNA occupancy of epigenetic effectors. The variation in gene expression is replicable and led to the identification of novel associations between genes and disease phenotypes.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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Probes for BRCA1

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Abstract

CONCLUSIONS:
In situ BRCA1-related expression parameters could be used for clinical purposes at the time of diagnosis. In contrast, miR-548c-5p showed a promising potential as a prognostic factor in TNBC.

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China

Probes for BRCA1

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Abstract

CONCLUSIONS:In situ BRCA1-related expression parameters could be used for clinical purposes at the time of diagnosis. In contrast, miR-548c-5p showed a promising potential as a prognostic factor in TNBC.

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China

CONCLUSIONS:
In situ BRCA1-related expression parameters could be used for clinical purposes at the time of diagnosis. In contrast, miR-548c-5p showed a promising potential as a prognostic factor in TNBC.