Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter

Background

Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na⁺ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined.

Materials and Methods

The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers.

Results

G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005).

Discussion

Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.     

In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.     

Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.A far greater understanding of proteins interacting in complexes in cells and tissues is needed to explain the functional states of cells. Accordingly, there is a pressing need for improved methods to study protein interaction complexes to explain disease mechanisms; however, suitable methods have been lacking, particularly for clinical material. As an example, proteins in the epidermal growth factor receptor (EGFR)¹ family have traditionally been used as clinical markers. However, in many cases this has proven of limited prognostic value, and activity markers such as receptor interactions are attracting increasing interest (1, 2). Methods such as FRET-based detection (3) or the VeraTag assay (4) can be used to investigate protein complex formations in patient tissues. However, such techniques are not suitable to measure several concurrent protein complexes as required to characterize the balance between different segments of a signaling pathway or between different pathways. FRET-based methods are difficult to use with clinical material and have very limited multiplexing capabilities. The VeraTag assay can be multiplexed, but it fails to provide spatial information of the complexes and thus cannot distinguish between cancer cells and surrounding stroma. Similarly, bulk measures of protein complexes via e.g. co-immunoprecipitation and mass spectrometry (5) disregard cell-to-cell variations and the subcellular distributions of protein complexes. Moreover, such methods are poorly suited for analyzing precious clinical material as too much sample material is needed for the analysis.To enable parallel analyses directly in tumor tissue of multiple protein complexes involved in signaling pathways, we have developed a multiplex version of the in situ proximity ligation assay (PLA)¹ (6). In situ PLA has previously been used for localized detection of proteins, protein complexes, and post-translational modifications in cells and tissues (6). Because of its intrinsic requirement for dual target recognition by pairs of antibodies and the use of rolling circle amplification (RCA) to substantially amplify signals, the assay allows detection of endogenous protein complexes or post-translational modifications in fixed cells and tissue sections (7, 8) or Western blot membranes (9). The basis of in situ PLA is the detection of a target molecule through the use of a pair of PLA probes, i.e. target-specific affinity reagents such as antibodies to which DNA oligonucleotides have been attached (Fig. 1). We describe herein how tag sequences in the oligonucleotides of each PLA probe, uniquely identifying these probes, can be propagated into the single-stranded RCA products that result when two PLA probes have bound complex-forming proteins. The amplified tags in the RCA products can then be visualized using detection oligonucleotides, labeled with different fluorophores, to uniquely recognize the tag sequences. This multiplex readout makes it possible to compare levels of protein complexes between individual cells by identifying the PLA probes that gave rise to the signals.Open in a separate windowFig. 1.Parallel detection of protein complexes using multiplex in situ PLA. Groups of PLA probes are used to detect all binary complexes between a protein X and any of the proteins A–C. Using oligonucleotides attached to specific antibodies as templates, two linear connector oligonucleotides and one probe-specific tag oligonucleotide are enzymatically joined into a DNA circle that subsequently templates RCA. The RCA products, whose repeated sequences identify the protein in complex with protein X, can be visualized by hybridization of three tag-specific detection oligonucleotides labeled with distinct fluorophores.To test our probe design, we targeted the well characterized EGFR family. This family consists of four transmembrane tyrosine kinase receptors (EGFR, HER2, HER3, and HER4), involved in the regulation of fundamental cellular functions such as cell growth, survival, death, differentiation, and proliferation (10). Increased expression, or aberrant regulation, of the receptors has been implicated in a broad range of human malignancies, including breast cancer, where overexpression of HER2 is associated with a poor prognosis (11). Members of the EGFR family can interact in different constellations, with HER2 as the preferred interaction partner (12), activating several signaling pathways. These interactions between different members of the EGFR family and with associated proteins have been studied extensively in many different types of cells and tissues with a range of methods (2–4, 13), including in situ PLA (14–17).Using multiplex in situ PLA, we successfully visualized multiple protein complexes in cultured cells and in fresh frozen tissue sections, illustrating the potential to study the balance between alternative protein complexes in clinical specimens to identify cellular phenotypes.     

Quantification of Normal Cell Fraction and Copy Number Neutral LOH in Clinical Lung Cancer Samples Using SNP Array Data

Background

Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.

Principal Finding

Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.

Conclusion

Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.     

Targeted resequencing of candidate genes using selector probes

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R² = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.     

Allele-specific copy number analysis of tumor samples with aneuploidy and tumor heterogeneity

We describe a bioinformatic tool, Tumor Aberration Prediction Suite (TAPS), for the identification of allele-specific copy numbers in tumor samples using data from Affymetrix SNP arrays. It includes detailed visualization of genomic segment characteristics and iterative pattern recognition for copy number identification, and does not require patient-matched normal samples. TAPS can be used to identify chromosomal aberrations with high sensitivity even when the proportion of tumor cells is as low as 30%. Analysis of cancer samples indicates that TAPS is well suited to investigate samples with aneuploidy and tumor heterogeneity, which is commonly found in many types of solid tumors.     

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.     

Allele-specific copy number analysis of tumor samples with aneuploidy and tumor heterogeneity

We describe a bioinformatic tool, Tumor Aberration Prediction Suite (TAPS), for the identification of allele-specific copy numbers in tumor samples using data from Affymetrix SNP arrays. It includes detailed visualization of genomic segment characteristics and iterative pattern recognition for copy number identification, and does not require patient-matched normal samples. TAPS can be used to identify chromosomal aberrations with high sensitivity even when the proportion of tumor cells is as low as 30%. Analysis of cancer samples indicates that TAPS is well suited to investigate samples with aneuploidy and tumor heterogeneity, which is commonly found in many types of solid tumors.