Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant.

BACKGROUND: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. METHODS: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. RESULTS: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. CONCLUSIONS: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

Non-BRCA1/BRCA2 high-risk familial breast cancers are not associated with a high prevalence of BRCAness.

BACKGROUND: Familial breast cancer is in most cases unexplained due to the lack of identifiable pathogenic variants in the BRCA1 and BRCA2 genes. The somatic mutational landscape and in particular the extent of BRCA-like tumour features (BRCAness) in these familial breast cancers where germline BRCA1 or BRCA2 mutations have not been identified is to a large extent unknown. METHODS: We performed whole-genome sequencing on matched tumour and normal samples from high-risk non-BRCA1/BRCA2 breast cancer families to understand the germline and somatic mutational landscape and mutational signatures. We measured BRCAness using HRDetect. As a comparator, we also analysed samples from BRCA1 and BRCA2 germline mutation carriers. RESULTS: We noted for non-BRCA1/BRCA2 tumours, only a small proportion displayed high HRDetect scores and were characterized by concomitant promoter hypermethylation or in one case a RAD51D splice variant previously reported as having unknown significance to potentially explain their BRCAness. Another small proportion showed no features of BRCAness but had mutationally active tumours. The remaining tumours lacked features of BRCAness and were mutationally quiescent. CONCLUSIONS: A limited fraction of high-risk familial non-BRCA1/BRCA2 breast cancer patients is expected to benefit from treatment strategies against homologue repair deficient cancer cells.

Heterogeneity and tumor evolution reflected in liquid biopsy in metastatic breast cancer patients: a review.

Breast cancer is a spatially and temporally dynamic disease in which differently evolving genetic clones are responsible for progression and clinical outcome. We review tumor heterogeneity and clonal evolution from studies comparing primary tumors and metastasis and discuss plasma circulating tumor DNA as a powerful real-time approach for monitoring the clonal landscape of breast cancer during treatment and recurrence. We found only a few early studies exploring clonal evolution and heterogeneity through analysis of multiregional tissue biopsies of different progression steps in comparison with circulating tumor DNA (ctDNA) from blood plasma. The model of linear progression seemed to be more often reported than the model of parallel progression. The results show complex routes to metastasis, however, and plasma most often reflected metastasis more than primary tumor. The described patterns of evolution and the polyclonal nature of breast cancer have clinical consequences and should be considered during patient diagnosis and treatment selection. Current studies focusing on the relevance of clonal evolution in the clinical setting illustrate the role of liquid biopsy as a noninvasive biomarker for monitoring clonal progression and response to treatment. In the clinical setting, circulating tumor DNA may be an ideal support for tumor biopsies to characterize the genetic landscape of the metastatic disease and to improve longitudinal monitoring of disease dynamics and treatment effectiveness through detection of residual tumor after resection, relapse, or metastasis within a particular patient.

Whole blood transcriptional profiling reveals highly deregulated atherosclerosis genes in Philadelphia-chromosome negative myeloproliferative neoplasms.

BACKGROUND: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a huge comorbidity burden, including an increased risk of cardiovascular diseases. Recently, chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in MPN but also potentially having an impact upon the development of accelerated (premature) atherosclerosis. OBJECTIVES: Since chronic inflammation, atherosclerosis, and atherothrombosis are prevalent in MPNs and we have previously shown oxidative stress genes to be markedly upregulated in MPNs, we hypothesized that genes linked to development of atherosclerosis might be highly deregulated as well. METHODS: Using whole blood gene expression profiling in patients with essential thrombocythemia (ET; n = 19), polycythemia vera (PV; n = 41), or primary myelofibrosis (PMF; n = 9), we herein for the first time report aberrant expression of several atherosclerosis genes. RESULTS: Of 84 atherosclerosis genes, 45, 56, and 46 genes were deregulated in patients with ET, PV, or PMF, respectively. Furthermore, BCL2L1, MMP1, PDGFA, PTGS1, and THBS4 were progressively significantly upregulated and BCL2 progressively significantly downregulated from ET over PV to PMF (all FDR <0.05). CONCLUSIONS: We have for the first time shown massive deregulation of atherosclerosis genes in MPNs, likely reflecting the inflammatory state in MPNs in association with in vivo activation of leukocytes, platelets, and endothelial cells being deeply involved in the atherosclerotic process.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

Differential regulation of the DNA methylome in adults born during the Great Chinese Famine in 1959-1961.

BACKGROUND: Extensive epidemiological studies have established the association between exposure to early-life adversity and health status and diseases in adults. Epigenetic regulation is considered as a key mediator for this phenomenon but analysis on humans is sparse. The Great Chinese Famine lasting from 1958 to 1961 is a natural string of disasters offering a precious opportunity for elucidating the underlying epigenetic mechanism of the long-term effect of early adversity. METHODS: Using a high-throughput array platform for DNA methylome profiling, we conducted a case-control epigenome-wide association study on early-life exposure to Chinese famine in 79 adults born during 1959-1961 and compared to 105 unexposed subjects born 1963-1964. RESULTS: The single CpG site analysis of whole epigenome revealed a predominant pattern of decreased DNA methylation levels associated with fetal exposure to famine. Four CpG sites were detected with p < 1e-06 (linked to EHMT1, CNR1, UBXN7 and ESM1 genes), 16 CpGs detected with 1e-06 < p < 1e-05 and 157 CpGs with 1e-05 < p < 1e-04, with a predominant pattern of hypomethylation. Functional annotation to genes and their enriched biological pathways mainly involved neurodevelopment, neuropsychological disorders and metabolism. Multiple sites analysis detected two top-rank differentially methylated regions harboring RNF39 on chromosome 6 and PTPRN2 on chromosome 7, both showing epigenetic association with stress-related conditions. CONCLUSION: Early-life exposure to famine could mediate DNA methylation regulations that persist into adulthood with broad impacts in the activities of genes and biological pathways. Results from this study provide new clues to the epigenetic embedding of early-life adversity and its impacts on adult health.

Tumour-infiltrating CD4-, CD8- and FOXP3-positive immune cells as predictive markers of mortality in BRCA1- and BRCA2-associated breast cancer.

BACKGROUND: The prognostic value of tumour-infiltrating lymphocytes (TILs) in breast cancer is well-established. However, the investigation of specific T-cell subsets exclusively in BRCA-associated breast cancer is sparse. METHODS: Tumour tissues from 414 BRCA-mutated breast cancer patients were analysed by immunohistochemistry and digital image analysis for expression of CD4, CD8 and FOXP3 immune markers. Distribution of CD4-, CD8- and FOXP3-positive cells and clinicopathological characteristics were assessed according to groups of low or high expression. The prognostic value was evaluated as continuous variables in univariate and multivariate analyses of overall survival and disease-free survival. RESULTS: Both CD4 and CD8 expression are associated with histological diagnosis, tumour grade and oestrogen and progesterone receptor expression status. CD4 expression is associated with BRCA gene status. A high percentage of tumour-infiltrating CD4-, CD8- or FOXP3-positive cells is significantly associated with lower mortality in BRCA1- and BRCA2-associated breast cancer and CD8-positive cells are associated with disease-free survival. No heterogeneity according to BRCA gene status was found for the prognostic value of the immune markers. CONCLUSIONS: The results support a prognostic role of specific T-cell subsets in BRCA-associated breast cancer and the promising potential of targeting the immune system in the treatment of these patients.

Epithelial ovarian cancer and the use of circulating tumor DNA: A systematic review.

OBJECTIVE: One way to improve the survival rate of epithelial Ovarian Cancer (EOC) is by identifying effective biomarkers useful at different stages and time points of the disease. A potential biomarker is circulating tumor DNA (ctDNA) in plasma or serum. In this systematic review, we provide an overview of applications of ctDNA in EOC to discuss the direction of future research in this field. METHODS: We performed a systematic search in Pubmed, Embase, and Scopus to identify relevant clinical studies eligible for inclusion. Furthermore, the references in the identified studies and relevant reviews were assessed to identify additional studies. The PRISMA guideline was employed to perform the systematic review, and data from the studies were extracted using piloted data extraction forms. RESULTS: A total of 36 observational studies were included. The concordance between tumor and ctDNA was assessed in 19 studies, early diagnosis in 1, diagnosis in 23, monitoring of treatment response in 7, detection of reversion mutations in 3, prognosis in 9, but no studies assessed early detection of recurrence. Data from the studies were reported descriptively. The studies had a large variation in the methods used for ctDNA analysis and limited sample sizes of 10-126 patients. Overall, the studies show that ctDNA is a potential biomarker for EOC useful in several settings during assessment and treatment of these patients. CONCLUSIONS: Although the identified studies are limited in number and their methods for ctDNA analysis vary, it is clear that ctDNA as a biomarker for EOC is promising for several applications in diagnostics, monitoring of treatment response, and prognostics. However, more studies are needed to establish the ideal methods and settings for the clinical use of ctDNA in EOC.

Ruxolitinib and interferon-α2 combination therapy for patients with polycythemia vera or myelofibrosis: a phase II study.

We report the final 2-year end-of-study results from the first clinical trial investigating combination treatment with ruxolitinib and low-dose pegylated interferon-α2 (PEG-IFNα2). The study included 32 patients with polycythemia vera and 18 with primary or secondary myelofibrosis; 46 patients were previously intolerant of or refractory to PEGIFNα2. The primary outcome was efficacy, based on hematologic parameters, quality of life measurements, and JAK2 V617F allele burden. We used the 2013 European LeukemiaNet and International Working Group- Myeloproliferative Neoplasms Research and Treatment response criteria, including response in symptoms, splenomegaly, peripheral blood counts, and bone marrow. Of 32 patients with polycythemia vera, ten (31%) achieved a remission which was a complete remission in three (9%) cases. Of 18 patients with myelofibrosis, eight (44%) achieved a remission; five (28%) were complete remissions. The cumulative incidence of peripheral blood count remission was 0.85 and 0.75 for patients with polycythemia vera and myelofibrosis, respectively. The Myeloproliferative Neoplasm Symptom Assessment Form total symptom score decreased from 22 [95% confidence interval (95% CI):, 16-29] at baseline to 15 (95% CI: 10-22) after 2 years. The median JAK2 V617F allele burden decreased from 47% (95% CI: 33-61%) to 12% (95% CI: 6-22%), and 41% of patients achieved a molecular response. The drop-out rate was 6% among patients with polycythemia vera and 32% among those with myelofibrosis. Of 36 patients previously intolerant of PEG-IFNα2, 31 (86%) completed the study, and 24 (67%) of these received PEG-IFNα2 throughout the study. In conclusion, combination treatment improved cell counts, reduced bone marrow cellularity and fibrosis, decreased JAK2 V617F burden, and reduced symptom burden with acceptable toxicity in several patients with polycythemia vera or myelofibrosis. #EudraCT2013-003295-12.

Effect of thrombopoietin receptor agonists on markers of coagulation and P-selectin in patients with immune thrombocytopenia.

Thrombopoietin-receptor-agonists (TPO-RA) are effective treatments of immune thrombocytopenia (ITP). Previous long-term TPO-RA clinical trials have shown that thrombotic events occurred in 6% of TPO-RA-treated ITP patients. To explore the increased risk of thrombosis, the effects of TPO-RA on markers of coagulation and P-selectin were studied. The study comprised two ITP cohorts and controls. Cohort 1 included 26 patients with sequential samples acquired before and during treatment with TPO-RA. Cohort 2 included a single sample in 18 patients on TPO-RA for more than one year. Thrombin generation (endogenous thrombin potential (ETP)) prothrombin fragments 1 + 2 (F1+2), D-dimer, and plasminogen-activator-inhibitor-1 (PAI-1) were measured as well as soluble P-selectin (sP-selectin). Sequential expression of encoding genes for P-selectin (SELP) and PAI-1 (SERPINE1) was determined in four patients in cohort 1. Significantly higher levels of F1+2, D-dimer, and PAI-1 were found in ITP patients before TPO-RA treatment and in patients on long-term TPO-RA treatment than in controls. Pre-treatment levels of sP-selectin did not differ from controls. Analysis of longitudinal trends showed an increase in platelet count, sP-selectin, and PAI-1 after initiation of TPO-RA, followed by gradual decline. Platelet count and sP-selectin remained at higher levels throughout the study, whereas PAI-1 did not. Levels of other studied parameters did not show significant changes after initiation of treatment. Expression of SELP was up-regulated after initiation of TPO-RA, while the expression of SERPINE1 showed no significant changes. In conclusion, elevated pre-treatment levels of F1+2, D-dimer and PAI-1 are compatible with ITP being an intrinsically pro-thrombotic condition. After TPO-RA treatment, there were no significant changes in markers of coagulation activation or fibrinolysis, except for an initial increase in PAI-1 and a significant increase in sP-selectin both of which may contribute to increased thrombotic risk associated with TPO-RA treatment in ITP.