Evidence-based recommendations for gene-specific ACMG/AMP variant classification from the ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel.

The ENIGMA research consortium develops and applies methods to determine clinical significance of variants in hereditary breast and ovarian cancer genes. An ENIGMA BRCA1/2 classification sub-group, formed in 2015 as a ClinGen external expert panel, evolved into a ClinGen internal Variant Curation Expert Panel (VCEP) to align with Food and Drug Administration recognized processes for ClinVar contributions. The VCEP reviewed American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) classification criteria for relevance to interpreting BRCA1 and BRCA2 variants. Statistical methods were used to calibrate evidence strength for different data types. Pilot specifications were tested on 40 variants and documentation revised for clarity and ease of use. The original criterion descriptions for 13 evidence codes were considered non-applicable or overlapping with other criteria. Scenario of use was extended or re-purposed for eight codes. Extensive analysis and/or data review informed specification descriptions and weights for all codes. Specifications were applied to pilot variants with pre-existing ClinVar classification as follows: 13 uncertain significance or conflicting, 14 pathogenic and/or likely pathogenic, and 13 benign and/or likely benign. Review resolved classification for 11/13 uncertain significance or conflicting variants and retained or improved confidence in classification for the remaining variants. Alignment of pre-existing ENIGMA research classification processes with ACMG/AMP classification guidelines highlighted several gaps in the research processes and the baseline ACMG/AMP criteria. Calibration of evidence strength was key to justify utility and strength of different data types for gene-specific application. The gene-specific criteria demonstrated value for improving ACMG/AMP-aligned classification of BRCA1 and BRCA2 variants.

The prognostic role of circulating tumour DNA detected prior to clinical diagnosis of colorectal cancer in the HUNT study.

BACKGROUND: Today, the prognostic tools available at the time of diagnosis in colorectal cancer (CRC) are limited. Better prognostic tools are a prerequisite for personalised treatment. This study aimed to investigate whether circulating tumour DNA (ctDNA) markers found in plasma before clinical diagnosis of CRC could contribute to the prediction of poor prognosis. METHODS: This observational cohort study included patients diagnosed with CRC stage I-III within 24 months following participation in the Trøndelag Health Study (n = 85). Known methylated ctDNA biomarkers of CRC were analysed by PCR in plasma. Outcomes were overall survival (OS), recurrence-free survival (RFS) and poor prognosis (PP). Candidate clinical and methylated ctDNA predictors of the outcomes were identified by Cox regression analyses. RESULTS: Methylated GRIA4 (HR 1.96 (1.06-3.63)), RARB (HR 9.48 (3.00-30.00)), SLC8A1 (HR 1.97 (1.03-3.77)), VIM (HR 2.95 (1.22-7.14)) and WNT5A (HR 5.83 (2.33-14.56)) were independent predictors of OS, methylated RARB (HR 9.67 (2.54-36.81)), SDC2 (HR 3.38 (1.07-10.66)), SLC8A1 (HR 2.93 (1.01-8.51)) and WNT5A (HR 6.95 (1.81-26.68)) were independent predictors of RFS and methylated RARB (HR 6.11 (1.69-22.18)), SDC2 (HR 2.79 (1.20-6.49)) and WNT5A (HR 5.57 (3.04-15.26)) were independent predictors of PP (p < 0.05). CONCLUSIONS: Prediagnostic ctDNA markers are promising contributors to predicting poor prognosis in CRC, potentially becoming one of the tools guiding more personalised treatment.

Congenital Hypodysfibrinogenemia due to γ326Cys→Tyr Mutation: Third Ever-Described Case Associated with Recurrent Venous Thrombosis and COVID Vaccine.

INTRODUCTION: Congenital fibrinogen disorders are a heterogenous group of fibrinogen defects. CASE PRESENTATION: Here, we describe hypodysfibrinogenemia in a 33-year-old female patient with provoked recurrent deep vein thrombosis (DVT) diagnosed based on decreased functional and antigenic fibrinogen levels with a decreased functional/antigenic fibrinogen ratio. Definitive diagnosis of congenital hypodysfibrinogenemia is done by genotyping using whole-exome sequencing, which identified the γ326Cys→Tyr mutation combined with single-nucleotide polymorphisms: rs2070011 and rs2070018 in FGA and rs1049636 in FGG. Fibrin structure assays showed reduced maximum polymerization rate. The mother of the proband shares the same γ326Cys→Tyr mutation and experienced a provoked DVT. CONCLUSION: Our case with DVT is the third ever-described occurrence of the mutation γ326Cys→Tyr that is associated with hypodysfibrinogenemia. The mechanism by which this mutation induces thrombosis remains unknown. Due to the high recurrence risk of thrombosis, the patient was treated with long-term reduced dose of rivaroxaban (10 mg daily) as secondary prophylaxis.

Colorectal cancer detected by liquid biopsy 2 years prior to clinical diagnosis in the HUNT study.

BACKGROUND: Colorectal cancer (CRC) is often diagnosed in advanced stages. Circulating tumour DNA (ctDNA) has been proposed as an early diagnostic biomarker. However, as a screening tool, ctDNA has mainly been studied in selected populations at the time of clinical diagnosis. The aim of this study was to detect CRC by known ctDNA markers up to 2 years prior to clinical diagnosis. METHODS: In this case-control study, methylated ctDNA markers were detected in plasma samples from 106 healthy controls and 106 individuals diagnosed with CRC within 24 months following participation in The Trøndelag Health Study. RESULTS: The most specific single markers were BMP3, FLI1, IKZF1, SFRP1, SFRP2, NPTX2, SLC8A1 and VIM (specificity >70%). When combining these into a panel, the CRC sensitivity was 43% (95% CI 42.7-43.4) and the CRC specificity was 86% (95% CI 85.7-86.2). The findings were reproduced in an independent validation set of samples. CONCLUSIONS: Detection of known methylated ctDNA markers of CRC is possible up to 2 years prior to the clinical diagnosis in an unselected population resembling the screening setting. This study supports the hypothesis that some patients could be diagnosed earlier, if ctDNA detection was part of the CRC screening programme.

Ovarian cancer pathology characteristics as predictors of variant pathogenicity in BRCA1 and BRCA2.

BACKGROUND: The distribution of ovarian tumour characteristics differs between germline BRCA1 and BRCA2 pathogenic variant carriers and non-carriers. In this study, we assessed the utility of ovarian tumour characteristics as predictors of BRCA1 and BRCA2 variant pathogenicity, for application using the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) variant classification system. METHODS: Data for 10,373 ovarian cancer cases, including carriers and non-carriers of BRCA1 or BRCA2 pathogenic variants, were collected from unpublished international cohorts and consortia and published studies. Likelihood ratios (LR) were calculated for the association of ovarian cancer histology and other characteristics, with BRCA1 and BRCA2 variant pathogenicity. Estimates were aligned to ACMG/AMP code strengths (supporting, moderate, strong). RESULTS: No histological subtype provided informative ACMG/AMP evidence in favour of BRCA1 and BRCA2 variant pathogenicity. Evidence against variant pathogenicity was estimated for the mucinous and clear cell histologies (supporting) and borderline cases (moderate). Refined associations are provided according to tumour grade, invasion and age at diagnosis. CONCLUSIONS: We provide detailed estimates for predicting BRCA1 and BRCA2 variant pathogenicity based on ovarian tumour characteristics. This evidence can be combined with other variant information under the ACMG/AMP classification system, to improve classification and carrier clinical management.

Feasibility and early clinical impact of precision medicine for late-stage cancer patients in a regional public academic hospital.

AIM: Our goal was to describe a precision medicine program in a regional academic hospital, characterize features of included patients and present early data on clinical impact. MATERIALS AND METHODS: We prospectively included 163 eligible patients with late-stage cancer of any diagnosis from June 2020 to May 2022 in the Proseq Cancer trial. Molecular profiling of new or fresh frozen tumor biopsies was done by WES and RNAseq with parallel sequencing of non-tumoral DNA as individual reference. Cases were presented at a National Molecular Tumor Board (NMTB) for discussion of targeted treatment. Subsequently, patients were followed for at least 7 months. RESULTS: 80% (N = 131) of patients had a successful analysis done, disclosing at least one pathogenic or likely pathogenic variant in 96%. A strongly or potentially druggable variant was found in 19% and 73% of patients, respectively. A germline variant was identified in 2.5%. Median time from trial inclusion to NMTB decision was one month. One third (N = 44) of patients who underwent molecularly profiling were matched with a targeted treatment, however, only 16% were either treated (N = 16) or are waiting for treatment (N = 5), deteriorating performance status being the primary cause of failure. A history of cancer among 1st degree relatives, and a diagnosis of lung or prostate cancer correlated with greater chance of targeted treatment being available. The response rate of targeted treatments was 40%, the clinical benefit rate 53%, and the median time on treatment was 3.8 months. 23% of patients presented at NMTB were recommended clinical trial participation, not dependent on biomarkers. CONCLUSIONS: Precision medicine in end-stage cancer patients is feasible in a regional academic hospital but should continue within the frame of clinical protocols as few patients benefit. Close collaboration with comprehensive cancer centers ensures expert evaluations and equality in access to early clinical trials and modern treatment.

Risks of breast and ovarian cancer for women harboring pathogenic missense variants in BRCA1 and BRCA2 compared with those harboring protein truncating variants.

PURPOSE: Germline genetic testing for BRCA1 and BRCA2 variants has been a part of clinical practice for >2 decades. However, no studies have compared the cancer risks associated with missense pathogenic variants (PVs) with those associated with protein truncating (PTC) variants. METHODS: We collected 582 informative pedigrees segregating 1 of 28 missense PVs in BRCA1 and 153 pedigrees segregating 1 of 12 missense PVs in BRCA2. We analyzed 324 pedigrees with PTC variants in BRCA1 and 214 pedigrees with PTC variants in BRCA2. Cancer risks were estimated using modified segregation analysis. RESULTS: Estimated breast cancer risks were markedly lower for women aged >50 years carrying BRCA1 missense PVs than for the women carrying BRCA1 PTC variants (hazard ratio [HR] = 3.9 [2.4-6.2] for PVs vs 12.8 [5.7-28.7] for PTC variants; P = .01), particularly for missense PVs in the BRCA1 C-terminal domain (HR = 2.8 [1.4-5.6]; P = .005). In case of BRCA2, for women aged >50 years, the HR was 3.9 (2.0-7.2) for those heterozygous for missense PVs compared with 7.0 (3.3-14.7) for those harboring PTC variants. BRCA1 p.[Cys64Arg] and BRCA2 p.[Trp2626Cys] were associated with particularly low risks of breast cancer compared with other PVs. CONCLUSION: These results have important implications for the counseling of at-risk women who harbor missense PVs in the BRCA1/2 genes.

Dysfibrinogenemia-Potential Impact of Genotype on Thrombosis or Bleeding.

The congenital dysfibrinogenemias, most often associated with bleeding disorders, encompass mutations in the amino-terminal end of fibrinogen α-chain consisting of Gly17-Pro18-Arg19-Val20, known as knob A, which is a critical site for fibrin polymerization. Here we review the studies reporting dysfibrinogenemia due to mutations affecting fibrinogen knob A and identified 29 papers. The number of reports on dysfibrinogenemias related to residues Gly17, Pro18, Arg19, and Val20 is 5, 4, 18, and 2, respectively. Dysfibrinogenemias related to residues Gly17, Pro18, and Val20 are exclusively associated with bleeding tendency. However, the clinical picture associated with dysfibrinogenemia related to residue Arg19 varies, with most patients suffering from bleeding tendencies, but also transitory ischemic attacks and retinal thrombosis may occur. The reason for this variation is unclear. To elaborate the genotype-phenotype associations further, we studied a Danish family with knob A-related dysfibrinogenemia caused by the Aα Arg19Gly (p.Arg19Gly) mutation using whole-exome sequencing and fibrin structure analysis. Our family is the first reported carrying the p.Arg19Gly mutation combined with one or more single nucleotide polymorphisms (SNP)s in FGA, FGB, and/or FGG and increased fibrin fiber thickness and fibrin mass-to-length ratio suffering from pulmonary emboli, suggesting that compound genotypes may contribute to the thrombogenic phenotype of these patients. Our review, accordingly, focuses on significance of SNPs, compound genotypes, and fibrin structure measures affecting the genotype-phenotype associations in fibrinogen knob A mutations.

The mutational profile of immune surveillance genes in diagnostic and refractory/relapsed DLBCLs.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid neoplasm among adults,and approximately 30-40% of patients will experience relapse while 5-10% will suffer from primary refractory disease caused by different mechanisms, including treatment-induced resistance. For refractory and relapsed DLBCL (rrDLBCL) patients, early detection and understanding of the mechanisms controlling treatment resistance are of great importance to guide therapy decisions. Here, we have focused on genetic variations in immune surveillance genes in diagnostic DLBCL (dDLBCL) and rrDLBCL patients to elaborate on the suitability of new promising immunotherapies. METHODS: Biopsies from 30 dDLBCL patients who did not progress or relapse during follow up and 17 rrDLBCL patients with refractory disease or who relapsed during follow up were analyzed by whole-exome sequencing, including matched individual germline samples to include only somatic genetic variants in downstream analysis of a curated list of 58 genes involved in major immune surveillance pathways. RESULTS: More than 70% of both dDLBCLs and rrDLBCLs harbored alterations in immune surveillance genes, but rrDLBCL tumor samples have a lower number of genes affected compared to dDLBCL tumor samples. Increased gene mutation frequencies in rrDLBCLs were observed in more than half of the affected immune surveillance genes than dDLBCLs. CONCLUSION: Genetic variants in the antigen-presenting genes affect a higher number of rrDLBCL patients supporting an important role for these genes in tumor progression and development of refractory disease and relapse.

Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing.

PURPOSE: Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic disorders (PGT-M). METHOD: PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS. RESULTS: Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1-6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results. CONCLUSION: These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS. TRIAL REGISTRATION NUMBER: N-20180001.

Use of genetic testing for hypolactasia trait in the North Denmark Region.

OBJECTIVE: Lactose intolerance (LI) may be considered in patients with unspecific gastrointestinal symptoms, but there is no clear consensus on when and how to diagnose the disorder. The LCT-13910 CC genotype is associated with acquired primary lactase deficiency (adult-type hypolactasia; ATH). We aimed to describe the number of tests and test results in the North Denmark Region considering patient age, geographical origin and repeated testing. METHODS: Retrospective evaluation of the polymerase chain reaction-based LCT-13910 genotype tests registered in the clinical laboratory information system (LABKA II) with data linkage to Danish nationwide registers. RESULTS: Between 18 May 2007 and 31 December 2018, a total of 23,560 individuals were tested. There was a sevenfold increase in the number of tests performed during the study period. About 9.8% of the tests performed in 2018 were repeated testing in the same individuals. Overall, 8.8% of tested individuals were younger than 5 years, 90.7% were of Danish origin and 5.5% originated from outside of Europe. The LCT-13910 CC genotype was identified in 13.3% of all tested individuals, in 16.0% of children younger than 5 years, in 6.8% of Danish individuals and in 90.9% originating from outside of Europe. CONCLUSIONS: In the North Denmark Region, a marked increase in the use of genetic testing for hypolactasia was observed and repeated testing was frequent. Furthermore, the use of the test and the test results were dependent on patient age and geographical origin. Results inform the debate on when and how to use genetic testing in the diagnosing of LI.

A systematic review on concurrent aneuploidy screening and preimplantation genetic testing for hereditary disorders: What is the prevalence of aneuploidy and is there a clinical effect from aneuploidy screening?

INTRODUCTION: In assisted reproductive technology, aneuploidy is considered a primary cause of failed embryo implantation. This has led to the implementation of preimplantation genetic testing for aneuploidy in some clinics. The prevalence of aneuploidy and the use of aneuploidy screening during preimplantation genetic testing for inherited disorders has not previously been reviewed. Here, we systematically review the literature to investigate the prevalence of aneuploidy in blastocysts derived from patients carrying or affected by an inherited disorder, and whether screening for aneuploidy improves clinical outcomes. MATERIAL AND METHODS: PubMed and Embase were searched for articles describing preimplantation genetic testing for monogenic disorders and/or structural rearrangements in combination with preimplantation genetic testing for aneuploidy. Original articles reporting aneuploidy rates at the blastocyst stage and/or clinical outcomes (positive human chorionic gonadotropin, gestational sacs/implantation rate, fetal heartbeat/clinical pregnancy, ongoing pregnancy, miscarriage, or live birth/delivery rate on a per transfer basis) were included. Case studies were excluded. RESULTS: Of the 26 identified studies, none were randomized controlled trials, three were historical cohort studies with a reference group not receiving aneuploidy screening, and the remaining were case series. In weighted analysis, 34.1% of 7749 blastocysts were aneuploid. Screening for aneuploidy reduced the proportion of embryos suitable for transfer, thereby increasing the risk of experiencing a cycle without transferable embryos. In pooled analysis the percentage of embryos suitable for transfer was reduced from 57.5% to 37.2% following screening for aneuploidy. Among historical cohort studies, one reported significantly improved pregnancy and birth rates but did not control for confounding, one did not report any statistically significant difference between groups, and one properly designed study concluded that preimplantation genetic testing for aneuploidy enhanced the chance of achieving a pregnancy while simultaneously reducing the chance of miscarriage following single embryo transfer. CONCLUSIONS: On average, aneuploidy is detected in 34% of embryos when performing a single blastocyst biopsy derived from patients carrying or affected by an inherited disorder. Accordingly, when screening for aneuploidy, the risk of experiencing a cycle with no transferable embryos increases. Current available data on the clinical effect of preimplantation genetic testing for aneuploidy performed concurrently with preimplantation genetic testing for inherited disorders are sparse, rendering the clinical effect from preimplantation genetic testing for aneuploidy difficult to access.

The BRCA1 c. 5096G>A p.Arg1699Gln (R1699Q) intermediate risk variant: breast and ovarian cancer risk estimation and recommendations for clinical management from the ENIGMA consortium.

BACKGROUND: We previously showed that the BRCA1 variant c.5096G>A p.Arg1699Gln (R1699Q) was associated with an intermediate risk of breast cancer (BC) and ovarian cancer (OC). This study aimed to assess these cancer risks for R1699Q carriers in a larger cohort, including follow-up of previously studied families, to further define cancer risks and to propose adjusted clinical management of female BRCA1*R1699Q carriers. METHODS: Data were collected from 129 BRCA1*R1699Q families ascertained internationally by ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium members. A modified segregation analysis was used to calculate BC and OC risks. Relative risks were calculated under both monogenic model and major gene plus polygenic model assumptions. RESULTS: In this cohort the cumulative risk of BC and OC by age 70 years was 20% and 6%, respectively. The relative risk for developing cancer was higher when using a model that included the effects of both the R1699Q variant and a residual polygenic component compared with monogenic model (for BC 3.67 vs 2.83, and for OC 6.41 vs 5.83). CONCLUSION: Our results confirm that BRCA1*R1699Q confers an intermediate risk for BC and OC. Breast surveillance for female carriers based on mammogram annually from age 40 is advised. Bilateral salpingo-oophorectomy should be considered based on family history.

Differential effect of surgical manipulation on gene expression in normal breast tissue and breast tumor tissue.

BACKGROUND: Gene expression profiles of normal and tumor tissue reflect both differences in biological processes taking place in vivo and differences in response to stress during surgery and sample handling. The effect of cold (room temperature) ischemia in the time interval between surgical removal of the specimen and freezing is described in a few studies. However, not much is known about the effect of warm (body temperature) ischemia during surgery. METHODS: Three women with primary operable breast cancer underwent in situ biopsies from normal breast and tumor tissue prior to radical mastectomy. Ex vivo biopsies from normal and tumor tissue were collected immediately after surgical excision. The putative effects on gene expression of malignancy (tumor versus normal), surgical manipulation (post- versus pre-surgical) and interaction between the two (differences in effect of surgical manipulation on tumor and normal samples) were investigated simultaneously by Generalized Estimating Equation (GEE) analysis in this self-matched study. RESULTS: Gene set enrichment analysis (GSEA) demonstrates a marked difference in effect of surgical manipulation on tumor compared to normal tissue. Interestingly, a large proportion of pathways affected by ischemia especially in tumor tissue are pathways considered to be specifically up regulated in tumor tissue compared to normal. CONCLUSION: The results of this study suggest that a large contribution to this differential expression originates from altered response to stress in tumor cells rather than merely representing in vivo differences. It is important to bear this in mind when using gene-expression analysis to deduce biological function, and when collecting material for gene expression profiling.

Promoter hypermethylation in plasma-derived cell-free DNA as a prognostic marker for pancreatic adenocarcinoma staging.

Correct staging of pancreatic cancer is paramount, as treatment is stage specific. However, minimally invasive tools to facilitate staging are lacking. DNA promoter hypermethylation is a hallmark of cancer. The aim of this study is to evaluate promoter hypermethylation in cell-free DNA as a prognostic marker for stage classification of pancreatic adenocarcinoma. Consecutive patients with pancreatic adenocarcinoma were prospectively included. Plasma samples were obtained before diagnostic work-up and treatment. Patients were staged according to the TNM classification. Methylation-specific PCR of 28 genes was performed. Prognostic prediction models for staging of pancreatic adenocarcinoma were developed by multivariable logistic regression analysis using stepwise backwards elimination. Ninety-five patients with pancreatic adenocarcinoma were included. The mean number of hypermethylated genes was identical for stage I, II and III disease (7.09 (95% CI; 5.51-8.66), 7.00 (95% CI; 5.93-8.07) and 6.77 (95% CI; 5.08-8.46)), respectively, and highly significantly different from stage IV disease (10.24 (95% CI; 8.88-11.60)). The prediction model (SEPT9v2, SST, ALX4, CDKN2B, HIC1, MLH1, NEUROG1, and BNC1) enabled the differentiation of stage IV from stage I-III disease (AUC of 0.87 (cut point 0.55; sensitivity 74%, specificity 87%)). Model (MLH1, SEPT9v2, BNC1, ALX4, CDKN2B, NEUROG1, WNT5A, and TFPI2) enabled the differentiation of stage I-II from stage III-IV disease (AUC of 0.82 (cut point 0.66; sensitivity 73%, specificity 80%)). Cell-free DNA promoter hypermethylation has the potential to be blood-based prognostic markers for pancreatic adenocarcinoma, as panels of hypermethylated genes enables the differentiation according to cancer stage. However, further validation is required.

Male breast cancer in BRCA1 and BRCA2 mutation carriers: pathology data from the Consortium of Investigators of Modifiers of BRCA1/2.

BACKGROUND: BRCA1 and, more commonly, BRCA2 mutations are associated with increased risk of male breast cancer (MBC). However, only a paucity of data exists on the pathology of breast cancers (BCs) in men with BRCA1/2 mutations. Using the largest available dataset, we determined whether MBCs arising in BRCA1/2 mutation carriers display specific pathologic features and whether these features differ from those of BRCA1/2 female BCs (FBCs). METHODS: We characterised the pathologic features of 419 BRCA1/2 MBCs and, using logistic regression analysis, contrasted those with data from 9675 BRCA1/2 FBCs and with population-based data from 6351 MBCs in the Surveillance, Epidemiology, and End Results (SEER) database. RESULTS: Among BRCA2 MBCs, grade significantly decreased with increasing age at diagnosis (P = 0.005). Compared with BRCA2 FBCs, BRCA2 MBCs were of significantly higher stage (P for trend = 2 × 10(-5)) and higher grade (P for trend = 0.005) and were more likely to be oestrogen receptor-positive [odds ratio (OR) 10.59; 95 % confidence interval (CI) 5.15-21.80] and progesterone receptor-positive (OR 5.04; 95 % CI 3.17-8.04). With the exception of grade, similar patterns of associations emerged when we compared BRCA1 MBCs and FBCs. BRCA2 MBCs also presented with higher grade than MBCs from the SEER database (P for trend = 4 × 10(-12)). CONCLUSIONS: On the basis of the largest series analysed to date, our results show that BRCA1/2 MBCs display distinct pathologic characteristics compared with BRCA1/2 FBCs, and we identified a specific BRCA2-associated MBC phenotype characterised by a variable suggesting greater biological aggressiveness (i.e., high histologic grade). These findings could lead to the development of gender-specific risk prediction models and guide clinical strategies appropriate for MBC management.

Comparison of mRNA splicing assay protocols across multiple laboratories: recommendations for best practice in standardized clinical testing.

BACKGROUND: Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. METHODS: We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. RESULTS: PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). CONCLUSIONS: We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

Early diagnosis of ovarian cancer based on methylation profiles in peripheral blood cell-free DNA: a systematic review.

Patients diagnosed with epithelial ovarian cancer (OC) have a 5-year survival rate of 49%. For early-stage disease, the 5-year survival rate is above 90%. However, advanced-stage disease accounts for most cases as patients with early stages often are asymptomatic or present with unspecific symptoms, highlighting the need for diagnostic tools for early diagnosis. Liquid biopsy is a minimal invasive blood-based approach that utilizes circulating tumor DNA (ctDNA) shed from tumor cells for real-time detection of tumor genetics and epigenetics. Increased DNA methylation of promoter regions is an early event during tumorigenesis, and the methylation can be detected in ctDNA, accentuating the promise of methylated ctDNA as a biomarker for OC diagnosis. Many studies have investigated multiple methylation biomarkers in ctDNA from plasma or serum for discriminating OC patients from patients with benign diseases of the ovaries and/or healthy females. This systematic review summarizes and evaluates the performance of the currently investigated DNA methylation biomarkers in blood-derived ctDNA for early diagnosis of OC. PubMed's MEDLINE and Elsevier's Embase were systematically searched, and essential results such as methylation frequency of OC cases and controls, performance measures, as well as preanalytical factors were extracted. Overall, 29 studies met the inclusion criteria for this systematic review. The most common method used for methylation analysis was methylation-specific PCR, with half of the studies using plasma and the other half using serum. RASSF1A, BRCA1, and OPCML were the most investigated gene-specific methylation biomarkers, with OPCML having the best performance measures. Generally, methylation panels performed better than single gene-specific methylation biomarkers, with one methylation panel of 103,456 distinct regions and 1,116,720 CpGs having better performance in both training and validation cohorts. However, the evidence is still limited, and the promising methylation panels, as well as gene-specific methylation biomarkers highlighted in this review, need validation in large, prospective cohorts with early-stage asymptomatic OC patients to assess the true diagnostic value in a clinical setting.