Kristina Pilekær Sørensen

Classifications within Molecular Subtypes Enables Identification of BRCA1/BRCA2 Mutation Carriers by RNA Tumor Profiling

Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority of BRCA1 tumors were basal-like while BRCA2 tumors were mainly luminal B. Using RNA profiles, we were able to distinguish BRCA1 tumors from sporadic tumors among basal-like tumors with 83% accuracy and BRCA2 from sporadic tumors among luminal B tumors with 89% accuracy. Furthermore, subtype-specific BRCA1/2 gene signatures were successfully validated in two independent data sets with high accuracies. Although additional validation studies are required, indication of BRCA1/2 involvement (“BRCAness”) by RNA profiling could potentially be valuable as a tool for distinguishing pathogenic mutations from benign variants, for identification of undetected mutation carriers, and for selecting patients sensitive to new therapeutics such as PARP inhibitors.

RNA profiling reveals familial aggregation of molecular subtypes in non-BRCA1/2 breast cancer families

BackgroundIn more than 70% of families with a strong history of breast and ovarian cancers, pathogenic mutation in BRCA1 or BRCA2 cannot be identified, even though hereditary factors are expected to be involved. It has been proposed that tumors with similar molecular phenotypes also share similar underlying pathophysiological mechanisms. In the current study, the aim was to investigate if global RNA profiling can be used to identify functional subgroups within breast tumors from families tested negative for BRCA1/2 germline mutations and how these subgroupings relate to different breast cancer patients within the same family.MethodsIn the current study we analyzed a collection of 70 frozen breast tumor biopsies from a total of 58 families by global RNA profiling and promoter methylation analysis.ResultsWe show that distinct functional subgroupings, similar to the intrinsic molecular breast cancer subtypes, exist among non-BRCA1/2 breast cancers. The distribution of subtypes was markedly different from the distribution found among BRCA1/2 mutation carriers. From 11 breast cancer families, breast tumor biopsies from more than one affected family member were included in the study. Notably, in 8 of these families we found that patients from the same family shared the same tumor subtype, showing a tendency of familial aggregation of tumor subtypes (p-value = 1.7e-3). Using our previously developed BRCA1/2-signatures, we identified 7 non-BRCA1/2 tumors with a BRCA1-like molecular phenotype and provide evidence for epigenetic inactivation of BRCA1 in three of the tumors. In addition, 7 BRCA2-like tumors were found.ConclusionsOur finding indicates involvement of hereditary factors in non-BRCA1/2 breast cancer families in which family members may carry genetic susceptibility not just to breast cancer but to a particular subtype of breast cancer. This is the first study to provide a biological link between breast cancers from family members of high-risk non-BRCA1/2 families in a systematic manner, suggesting that future genetic analysis may benefit from subgrouping families into molecularly homogeneous subtypes in order to search for new high penetrance susceptibility genes.

Acute hypoxia induces upregulation of microRNA-210 expression in glioblastoma spheroids.

AIM Tumor hypoxia and presence of tumor stem cells are related to therapeutic resistance and tumorigenicity in glioblastomas. The aim of the present study was therefore to identify microRNAs deregulated in acute hypoxia and to identify possible associated changes in stem cell markers. MATERIALS & METHODS Glioblastoma spheroid cultures were grown in either 2 or 21% oxygen. Subsequently, miRNA profiling was performed and expression of ten stem cell markers was examined. RESULTS MiRNA-210 was significantly upregulated in hypoxia in patient-derived spheroids. The stem cell markers displayed a complex regulatory pattern. CONCLUSION MiRNA-210 appears to be upregulated in hypoxia in immature glioblastoma cells. This miRNA may represent a therapeutic target although it is not clear from the results whether this miRNA may be related to specific cancer stem cell functions.

Microarray-Based RNA Profiling of Breast Cancer: Batch Effect Removal Improves Cross-Platform Consistency

Microarray is a powerful technique used extensively for gene expression analysis. Different technologies are available, but lack of standardization makes it challenging to compare and integrate data. Furthermore, batch-related biases within datasets are common but often not tackled. We have analyzed the same 234 breast cancers on two different microarray platforms. One dataset contained known batch-effects associated with the fabrication procedure used. The aim was to assess the significance of correcting for systematic batch-effects when integrating data from different platforms. We here demonstrate the importance of detecting batch-effects and how tools, such as ComBat, can be used to successfully overcome such systematic variations in order to unmask essential biological signals. Batch adjustment was found to be particularly valuable in the detection of more delicate differences in gene expression. Furthermore, our results show that prober adjustment is essential for integration of gene expression data obtained from multiple sources. We show that high-variance genes are highly reproducibly expressed across platforms making them particularly well suited as biomarkers and for building gene signatures, exemplified by prediction of estrogen-receptor status and molecular subtypes. In conclusion, the study emphasizes the importance of utilizing proper batch adjustment methods when integrating data across different batches and platforms.

De Novo Variants in GRIA4 Lead to Intellectual Disability with or without Seizures and Gait Abnormalities

Using trio whole-exome sequencing, we have identified de novo heterozygous pathogenic variants in GRIA4 in five unrelated individuals with intellectual disability and other symptoms. GRIA4 encodes an AMPA receptor subunit known as GluR4, which is found on excitatory glutamatergic synapses and is important for learning and memory. Four of the variants are located in the highly conserved SYTANLAAF motif in the transmembrane protein M3, and the fifth is in an extra-cellular domain. Molecular modeling of the altered protein showed that three of the variants in the SYTANLAAF motif orient toward the center of the pore region and most likely lead to disturbance of the gating mechanism. The fourth variant in the SYTANLAAF motif most likely results in reduced permeability. The variant in the extracellular domain potentially interferes with the binding between the monomers. On the basis of clinical information and genetic results, and the fact that other subunits of the AMPA receptor have already been associated with neurodevelopmental disorders, we suggest that pathogenic de novo variants in GRIA4 lead to intellectual disability with or without seizures, gait abnormalities, problems of social behavior, and other variable features.

Association of miR-548c-5p, miR-7-5p, miR-210-3p, miR-128-3p with recurrence in systemically untreated breast cancer

Current prognostic markers allocate the majority of lymph node (LN) negative and estrogen receptor (ER) positive breast cancer patients into the high-risk group. Accordingly, most patients receive systemic treatments although approximately 40% of these patients may have been cured by surgery and radiotherapy alone. Two studies identified seven prognostic microRNAs in systemically untreated, LN negative and ER positive breast cancer patients which may allow more precise patient classification. However, six of the seven microRNAs were analyzed in both studies but only found to be prognostic in one study. To validate their prognostic potential, we analyzed microRNA expression in an independent cohort (n = 110) using a pair-matched study design minimizing dependence of classical markers. The expression of hsa-miR-548c-5p was significantly associated with abridged disease-free survival (hazard ratio [HR]:1.96, p = 0.027). Contradicting published results, high hsa-miR-516-3p expression was associated with favorable outcome (HR:0.29, p = 0.0068). The association is probably time-dependent indicating later relapse. Additionally, re-analysis of previously published expression data of two matching cohorts (n = 100, n = 255) supports an association of hsa-miR-128-3p with shortened disease-free survival (HR:2.48, p = 0.0033) and an upregulation of miR-7-5p (p = 0.0038; p = 0.039) and miR-210-3p (p = 0.031) in primary tumors of patients who experienced metastases. Further analysis may verify the prognostic potential of these microRNAs.