Joakim Lundeberg

Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring.

Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.

Fabrication and functionalization of PCB gold electrodes suitable for DNA-based electrochemical sensing.

The request of high specificity and selectivity sensors suitable for mass production is a constant demand in medical research. For applications in point-of-care diagnostics and therapy, there is a high demand for low cost and rapid sensing platforms. This paper describes the fabrication and functionalization of gold electrodes arrays for the detection of deoxyribonucleic acid (DNA) in printed circuit board (PCB) technology. The process can be implemented to produce efficiently a large number of biosensors. We report an electrolytic plating procedure to fabricate low-density gold microarrays on PCB suitable for electrochemical DNA detection in research fields such as cancer diagnostics or pharmacogenetics, where biosensors are usually targeted to detect a small number of genes. PCB technology allows producing high precision, fast and low cost microelectrodes. The surface of the microarray is functionalized with self-assembled monolayers of mercaptoundodecanoic acid or thiolated DNA. The PCB microarray is tested by cyclic voltammetry in presence of 5 mM of the redox probe K3Fe(CN6) in 0.1 M KCl. The voltammograms prove the correct immobilization of both the alkanethiol systems. The sensor is tested for detecting relevant markers for breast cancer. Results for 5 nM of the target TACSTD1 against the complementary TACSTD1 and non-complementary GRP, MYC, SCGB2A1, SCGB2A2, TOP2A probes show a remarkable detection limit of 0.05 nM and a high specificity.

Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury

Summary CNS injury often severs axons. Scar tissue that forms locally at the lesion site is thought to block axonal regeneration, resulting in permanent functional deficits. We report that inhibiting the generation of progeny by a subclass of pericytes led to decreased fibrosis and extracellular matrix deposition after spinal cord injury in mice. Regeneration of raphespinal and corticospinal tract axons was enhanced and sensorimotor function recovery improved following spinal cord injury in animals with attenuated pericyte-derived scarring. Using optogenetic stimulation, we demonstrate that regenerated corticospinal tract axons integrated into the local spinal cord circuitry below the lesion site. The number of regenerated axons correlated with improved sensorimotor function recovery. In conclusion, attenuation of pericyte-derived fibrosis represents a promising therapeutic approach to facilitate recovery following CNS injury.

Design and assessment of binary DNA for nanopore sequencing

DNA sequencing using an array of nanometer-sized pores (nanopores) offer an exciting option for third-generation sequencing, which will allow faster and cheaper sequencing with minimal sample pre-processing. When a voltage is applied through a nanopore in a conducting fluid, a slight electric current is observed, the strength of which depends on the structure of the nanopore. When a DNA molecule passed through a nanopore, with an applied voltage, the current detected through the nanopore will differ for each base due to their differential effect on the structure of nanopore. However, current nanopore platforms cannot fully differentiate between single nucleotides due to their fast passage through the nanopore. To overcome this, Meller et al. proposed a strategy where the sequence detection is fluorescence based and involves passage of predesigned short oligonucleotides, so-called binary DNAs, encoding nucleotides [1]. In the current model, there are two binary DNA sequences and their binary combination encodes the DNA bases. Two molecular beacons complementary to the binary DNAs, each carrying a different fluorophore are also required for the fluorescence detection of DNA sequences. The genomic DNA is sheared and every 24 bases of sequence is converted to a DNA molecule consisting of corresponding binary DNAs encoding the matching bases. The binary-encoded DNA is then hybridised with molecular beacons. Once the binary-encoded DNA passes through the nanopore, the beacons are separated from the original strand and a short burst of light is emitted which is then used to determine the base information to sequence the DNA. We have designed sequences that serve as binary DNAs and corresponding molecular beacons. They have balanced GC content, minimal secondary structure, no cross-hybridisation and no occurrence in the human genome. A software pipeline either generates random DNA sequences or parses sequences from Archaea genomes. It then filters sequences by their GC content, repeat content, complexity, self hybridisation and occurrence in the human genome. Sequences that meet the desired criteria are then paired and tested further (e.g. cross-hybridisation). One set of a successful pair of binary DNA was assessed using the Biacore instrument, which allows interaction analysis in a label-free fashion. Our results confirmed that there is no cross hybridisation between the binary DNA sequences and they have similar hybridisation kinetics. We are currently testing the performance of molecular beacons and different hybridisation conditions (e.g. salt concentration, temperature). Our aim is to sequence an actual binary converted DNA using an array of nanopores.

Abstract 5028: Whole exome sequencing and genetic association of gemcitabine/carboplatin induced thrombocytopenia in non-small cell lung cancer patients

Introduction: Chemotherapy induced myelosuppression is a clinically relevant problem for cancer treatments, affecting both quality of life and response to treatment. Severe hematological toxicities such as thrombocytopenia can lead to dose reductions, postponement or cessation of treatment and there is a need to identify patients at risk before the start of treatment to allow for personalized medicine. The aim of this study was to use whole exome sequencing to identify genetic markers for gemcitabine and carboplatin induced thrombocytopenia that can be used to guide future treatments. Methods: 212 non-small cell lung cancer patients treated with gemcitabine and carboplatin were included. Blood samples were whole exome sequenced using Nextera Rapid Capture Exome kit and Illumina HiSeq 2500. Patients’ thrombocyte counts were measured at baseline and during the first treatment cycle. The thrombocyte nadir value and the relative decrease of the nadir value from baseline were used as thrombocytopenia toxicity parameters. Coding SNVs as well as whole gene-regions and complete pathways were investigated using the following statistical methods: A) Association of common SNVs/INDELs with thrombocytopenia using linear regression in PLINK. B) Association of the combined effect of common and rare variants within a gene-region with thrombocytopenia using the region based association test SKATO, in the R-package SKAT. C) All genes including SNVs/INDELs in A) or a gene-regions in B) with p Results: The study associated A) 103 SNVs/INDELs and B) 21 genes to thrombocytopenia (p Single SNVs, INDELs and genes can be of importance for induced toxicity. However, special interest should be given to the variants and genes in the pathways above. Two of the pathways link back to thrombocytopenia via platelet production and hemostasis and include, JMJD1C and DOCK8, previously associated with thrombocyte formation and mean platelet volume. The pathway concerning folate metabolism links back to gemcitabine’s and carboplatin’s mode of action via the involvement of folate in thymidylate and DNA synthesis. Further, folate is necessary for the rapid synthesis of blood cells in the bone marrow. Conclusion: The identified genetic markers and pathways are associated with chemotherapy induced thrombocytopenia and provide a strong foundation for further investigation. Citation Format: Niclas Bjorn, Anna Svedberg, Benjamin Sigurgeirsson, Sailendra Pradhananga, Eva Branden, Hirsh Koyi, Rolf Lewensohn, Luigi De Petris, Cristina Rodriguez-Antona, Maria Apellaniz-Ruiz, Joakim Lundeberg, Henrik Green. Whole exome sequencing and genetic association of gemcitabine/carboplatin induced thrombocytopenia in non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5028. doi:10.1158/1538-7445.AM2017-5028

Abstract 5026: Association to drug-induced leukopenia using whole-exome sequencing of non-small cell lung cancer patients on gemcitabine/carboplatin regimen

A classical non-small cell lung cancer (NSCLC) chemotherapy treatment is gemcitabine in combination with carboplatin. The treatment is known to cause severe hematological toxicity such as leukopenia, which can lead to chemotherapy cessation or even death. It would therefore a priori be of advantage to identify patients at risk of severe leukopenia to allow for a personalized treatment approach. In this study we aim to identify genetic markers for chemotherapy induced leukopenia in non-small cell lung cancer. In total, 212 non-small cell lung cancer patients treated with gemcitabine and carboplatin regimen were included in the study. Whole blood extracted DNA was prepared with Nextera Rapid Capture kit and whole exome sequenced using Illumina HiSeq 2500. Leukopenia was assessed from leukocyte particle count at baseline and the first cycle nadir values. The statistical approach was to study association of single common variants (MAF > 0.01) to leukopenia using linear regression in PLINK and association of genes (common and rare variants) to leukopenia using SKATO in the R-package SKAT. An enrichment analysis, with input from the association results (p The single variant analysis of common variants identified 133 variants (p The top pathway, HIF-1-alpha transcription factor network, overlaps with four genes (HDAC7, NDRG1, HK2 and CP). It can be of interest to leukopenia as regulation of HIF-1 alpha is essential for maintenance of hematopoietic stem cells in the bone marrow hypoxic niche. It has also been shown that HIF-1 alpha knocked mice are more sensitive to myelosuppressive treatment compared to unknocked mice, supporting the involvement of the pathway in chemotherapy induced myelosuppression. We have identified variants located in genes and pathways likely to be involved in leukocyte sensitivity after exposure of gemcitabine and carboplatin. A prediction model has been created to estimate the risk that patients will suffer from severe leukopenia. The biological connection of the pathway HIF-1-alpha transcription factor network to leukopenia supports the relevance of these results. Further studies on this are of interest to improve identification of patients at high risk of severe leukopenia after gemcitabine and carboplatin treatment. Citation Format: Anna Svedberg, Benjamin Sigurgeirsson, Niclas Bjorn, Sailendra Pradhananga, Eva Branden, Hirsh Koyi, Rolf Lewensohn, Luigi De Petris, Cristina Rodriguez-Antona, Maria Apellaniz-Ruiz, Joakim Lundeberg, Henrik Green. Association to drug-induced leukopenia using whole-exome sequencing of non-small cell lung cancer patients on gemcitabine/carboplatin regimen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5026. doi:10.1158/1538-7445.AM2017-5026