Leonardo Meza-zepeda - Academia.edu (original) (raw)

Papers by Leonardo Meza-zepeda

Muscle cells have different phenotypes adapted to different usage and can be grossly divided into... more Muscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A gen...

Blood

Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relap... more Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Both transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: We performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients. An average sequencing coverage of 700X was achieved for both ...

Blood

Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only ... more Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only 10% of these patients reach a 3-year progression-free survival compared to 65% at diagnosis. Our ability to distinguish patients at risk for relapse remains based on clinical staging. We hypothesized that identifying genetic alterations in serial tumour biopsies at diagnosis and relapse would improve our ability to identify high-risk patients, make therapeutic selections and reveal molecular markers for chemo-immunotherapy resistant tumours. However, relatively few relapsed/refractory biopsies have been sequenced. A unique, clinically annotated, Nordic DLBCL cohort was used to identify significantly mutated genes, assess potential driver genes, comprehensively examine clonal evolution, and gauge the importance of clinical relapsed sampling. Methods To address the lack of information on the molecular foundations of relapsed/refractory DLBCL, we performed whole exome sequencing (WES) on 42...

Molecular Aspects of Medicine

Cells

Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLP... more Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLPS) with amplified FRS2, but we previously only demonstrated transient cytostatic effects when treating FRS2-amplified DDLPS cells with NVP-BGJ398. Methods: Effects of the more potent FGFR inhibitor LY2874455 were investigated in three DDLPS cell lines by measuring effects on cell growth and apoptosis in vitro and also testing efficacy in vivo. Genome, transcriptome and protein analyses were performed to characterize the signaling components in the FGFR pathway. Results: LY2874455 induced a stronger, longer-lasting growth inhibitory effect and moderate level of apoptosis for two cell lines. The third cell line, did not respond to FGFR inhibition, suggesting that FRS2 amplification alone is not sufficient to predict response. Importantly, efficacy of LY2874455 was confirmed in vivo, using an independent FRS2-amplified DDLPS xenograft model. Expression of FRS2 was similar in the responding ...

Nature Communications

Inhibitory signaling during natural killer (NK) cell education translates into increased responsi... more Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca 2+ stores in primary NK cells reduces target-specific Ca 2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI (3,5)P 2 synthesis, or genetic silencing of the PI(3,5)P 2-regulated lysosomal Ca 2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.

Inhibitory signaling during natural killer (NK) cell education translates into increased responsi... more Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzyme-B dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signal...

International journal of molecular sciences, Jan 23, 2018

The BRAF mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas an... more The BRAF mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma (A673) and atypical synovial sarcoma (SW982), were treated with vemurafenib and the effects on cell growth, apoptosis, cell cycle progression and cell signaling were determined. Vemurafenib induced a strong cytostatic effect in SA-4 cells, mainly due to cell cycle arrest, whereas only moderate levels of apoptosis were observed. However, a high dose was required compared to BRAF mutated melanoma cells, and removal of vemurafenib demonstrated that the continuous presence of drug was required for sustained growth inhibition. A limited growth inhibition was observed in the other three cell lines. Protein analyses demonstrated reduced phosphorylation of ERK during treatment with vemurafenib in all the four sarco...

European urology, 2018

Most primary prostate cancers are multifocal with individual tumors harboring different aggressiv... more Most primary prostate cancers are multifocal with individual tumors harboring different aggressiveness; however, the genomic heterogeneity among these tumors is poorly understood. To better understand the biological basis for clinical variability among different lesions, we sought to comprehensively characterize the heterogeneity of somatic gene mutations in multifocal prostate cancer. High-coverage whole-exome sequencing of 153 frozen tissue samples, taken from two to three distinct tumor foci and one non-cancerous area from each of 41 patients, covering a total of 89 tumor foci. State-of-the-art bioinformatics tools for mutation calling and copy number determination from whole-exome sequencing data. We found a very high degree of interfocal heterogeneity among tumors, that is, 76% of pairwise-compared tumor foci from the same prostatectomy specimen had no point mutations in common and DNA copy number changes were rarely shared across cancer foci. The few point mutations shared acr...

Scientific reports, Jan 28, 2018

Sample pooling enabled by dedicated indexes is a common strategy for cost-effective and robust hi... more Sample pooling enabled by dedicated indexes is a common strategy for cost-effective and robust high-throughput sequencing. Index misassignment leading to mutual contamination between pooled samples has however been described as a general problem of the latest Illumina sequencing instruments utilizing exclusion amplification. Using real-life data from multiple tumour sequencing projects, we demonstrate that index misassignment can induce artefactual variant calls closely resembling true, high-quality somatic variants. These artefactual calls potentially impact cancer applications utilizing low allelic frequencies, such as in clonal analysis of tumours. We discuss the available countermeasures with an emphasis on improved library indexing methods, and provide software that can assist in the identification of variants that may be consequences of index misassignment.

Molecular Cancer

Background: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Compre... more Background: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. Methods: We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. Results: The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA-and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFβ signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately ¼ of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene-and protein-expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. Conclusions: This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.

Genome medicine, May 24, 2017

Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a co... more Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations-the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers. A total of 333 primary MSI+ colorectal tumors from multiple cohorts were analyzed by multilevel genomics and computational modeling-including mutation profiling, clonality modeling, and neoantigen prediction in a subset of the tumors, as well as gene expression profiling for consensus molecular subtypes (CMS) and immune cell infiltration. Novel, frequent frameshift mutations in four cancer-critical genes were identified by deep exome sequencing, including in CRTC1, BCL9, JAK1, and PTCH1. JAK1 loss-of-function mutations were validated with an overall frequency of 20% in Norwegian and British patients, and mutated tumors had up-regulation of transc...

Haematologica, Jul 6, 2017

Circulating tumor DNA (ctDNA) is a promising biomarker to monitor tumor load and genome alteratio... more Circulating tumor DNA (ctDNA) is a promising biomarker to monitor tumor load and genome alterations. We have explored the presence of ctDNA in multiple myeloma patients and its relation to disease activity during long-term follow-up. We used digital droplet PCR to monitor recurrent mutations, mainly in mitogen activated protein kinase pathway genes NRAS, KRAS and BRAF. Mutations were identified by next generation sequencing or PCR of bone marrow plasma cells, and their presence analyzed in 251 archived serum samples obtained from 20 patients during up to 7 years. In 17 of 18 patients, mutations identified in bone marrow during active disease were also found in a time-matched serum sample. The concentration of mutated alleles in serum correlated with the fraction in bone marrow plasma cells (r=0.507, n=34, p<0.002). There was a striking covariation between circulating mutation levels and M protein in 10 out of 11 patients with sequential samples. When relapse evaluation by ctDNA a...

BMC Cancer, 2017

Background: Many patients experience local recurrence or metastases after receiving potentially c... more Background: Many patients experience local recurrence or metastases after receiving potentially curative treatment, and early detection of these events is important for disease control. Recent technological advances make it possible to use blood plasma containing circulating cell-free tumour DNA (ctDNA) as a liquid biopsy. In this case report we show how serial liquid biopsies can be used to monitor the disease course and detect disease recurrence in a sarcoma patient. Case presentation: A 55-year-old male presented with a rapidly growing, painful palpable mass in the left groin region, and a biopsy revealed a high-grade malignant spindle cell sarcoma. No metastases were detected on radiologic imaging scans. Using targeted resequencing with a custom 900 cancer gene panel, eight somatic mutations among them KRAS and NF1, were identified in the primary tumour. Targeted resequencing of plasma cell-free DNA (ctDNA) collected before and after surgery and at disease progression confirmed the presence of six of eight mutations at all three time points. The ctDNA level, estimated from the somatic allele frequencies of these six mutations, was high in plasma taken at the time of surgery, at levels similar to the primary tumour. Detection of low levels of ctDNA three days after surgery indicated persistent microscopic disease. Repeated radiologic imaging six weeks postoperatively showed widespread metastatic disease in the lungs, skeleton and the pelvic region. At this time point there was a dramatic increase in the ctDNA level, reflecting the disease progression of the patient. The patient had an unusually aggressive cancer, and succumbed to the disease 13 weeks after surgery. Conclusions: This case report demonstrated that targeted resequencing of ctDNA from longitudinal collected plasma can be used to monitor disease progression in a soft tissue sarcoma patient, including manifestation of metastatic disease. The ctDNA represented the genomic profile of the tumour, supporting clinical use of liquid biopsies to identify tumour-specific mutations as well as recurrent disease.

PloS one

Osteosarcoma (OS) is the most common primary malignant tumor of bone, showing complex chromosomal... more Osteosarcoma (OS) is the most common primary malignant tumor of bone, showing complex chromosomal rearrangements but with few known consistent changes. Deeper biological understanding is crucial to find new therapies to improve patient survival. We have sequenced the whole exome of two primary tumors (before and after chemotherapy), one metastatic tumor and a matched normal sample from two OS patients, to identify mutations involved in cancer biology. The metastatic samples were also RNA sequenced. By RNA sequencing we identified dysregulated expression levels of drug resistance- and apoptosis-related genes. Two fusion transcripts were identified in one patient (OS111); the first resulted in p53 inactivation by fusing the first exon of TP53 to the fifth exon of FAM45A. The second fusion joined the two first exons of FGFR1 to the second exon of ZNF343. Furthermore, FGFR1 was amplified and highly expressed, representing a potential treatment target in this patient. Whole exome sequenc...

Oncotarget, 2014

Sarcomas are rare cancers with limited treatment options. Patients are generally treated by chemo... more Sarcomas are rare cancers with limited treatment options. Patients are generally treated by chemotherapy and/or radiotherapy in combination with surgery, and would benefit from new personalized approaches. In this study we demonstrate the potential of combining personal genomic characterization of patient tumors to identify targetable mutations with in vitro testing of specific drugs in patient-derived cell lines. We have analyzed three metastases from a patient with highgrade metastatic dedifferentiated liposarcoma (DDLPS) by exome and transcriptome sequencing as well as DNA copy number analysis. Genomic aberrations of several potentially targetable genes, including amplification of KITLG and FRS2, in addition to amplification of CDK4 and MDM2, characteristic of this disease, were identified. We evaluated the efficacy of drugs targeting these aberrations or the corresponding signaling pathways in a cell line derived from the patient. Interestingly, the pan-FGFR inhibitor NVP-BGJ398, which targets FGFR upstream of FRS2, strongly inhibited cell proliferation in vitro and induced an accumulation of cells into the G0 phase of the cell cycle. This study indicates that FGFR inhibitors have therapeutic potential in the treatment of DDLPS with amplified FRS2.

Genes, Chromosomes and Cancer, 2016

Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyp... more Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology. © 2016 Wiley Periodicals, Inc.

Frontiers in Genetics, 2016

Background: TP53 mutations are among the most common mutations found in lung cancers, identified ... more Background: TP53 mutations are among the most common mutations found in lung cancers, identified as an independent prognostic factor in many types of cancers. The purpose of this study was to investigate the frequency and prognostic impact of TP53 mutations in never-smokers and in different histological subtypes of lung cancer. Methods: We analyzed tumor tissue from 394 non-small cell carcinomas including adenocarcinomas (n = 229), squamous cell carcinomas (n = 112), large cell carcinomas (n = 30), and others (n = 23) for mutations in TP53 by the use of Sanger sequencing (n = 394) and next generation sequencing (n = 100). Results: TP53 mutations were identified in 47.2% of the samples, with the highest frequency (65%) of mutations among squamous cell carcinomas. Among neversmokers, 36% carried a TP53 mutation, identified as a significant independent negative prognostic factor in this subgroup. For large cell carcinomas, a significantly prolonged progression free survival was found for those carrying a TP53 mutation. In addition, the frequency of frameshift mutations was doubled in squamous cell carcinomas (20.3%) compared to adenocarcinomas (9.1%). Conclusion: TP53 mutation patterns differ between the histological subgroups of lung cancers, and are also influenced by smoking history. This indicates that the histological subtypes in lung cancer are genetically different, and that smoking-induced TP53 mutations may have a different biological impact than TP53 mutations occurring in never-smokers.

Oncotarget, Jan 15, 2015

Liposarcoma (LPS) is the most common type of soft tissue sarcoma accounting for 20% of all adult ... more Liposarcoma (LPS) is the most common type of soft tissue sarcoma accounting for 20% of all adult sarcomas. Due to absence of clinically effective treatment options in inoperable situations and resistance to chemotherapeutics, a critical need exists to identify novel therapeutic targets. We analyzed LPS genomic landscape using SNP arrays, whole exome sequencing and targeted exome sequencing to uncover the genomic information for development of specific anti-cancer targets. SNP array analysis indicated known amplified genes (MDM2, CDK4, HMGA2) and important novel genes (UAP1, MIR557, LAMA4, CPM, IGF2, ERBB3, IGF1R). Carboxypeptidase M (CPM), recurrently amplified gene in well-differentiated/de-differentiated LPS was noted as a putative oncogene involved in the EGFR pathway. Notable deletions were found at chromosome 1p (RUNX3, ARID1A), chromosome 11q (ATM, CHEK1) and chromosome 13q14.2 (MIR15A, MIR16-1). Significantly and recurrently mutated genes (false discovery rate < 0.05) incl...

Journal of medical case reports, Jan 9, 2015

Renal cell carcinoma with the distinct type of t(6;11)(p21;q12) translocation (transcription fact... more Renal cell carcinoma with the distinct type of t(6;11)(p21;q12) translocation (transcription factor EB) is a rare neoplasm. In the present case study, we show for the first time an autophagy signature in a patient with transcription factor EB renal cell carcinoma. We attempted to characterize the mutational and expressional features of a t(6;11)(p21;q12) renal cell carcinoma, in an effort to address the potential for molecular guidance of personalized medical decision for a case in this renal cell carcinoma category. We report the case of a 42-year-old white man who had a late relapse of his renal cell carcinoma. The first diagnosis of clear cell renal carcinoma was derived from a histological examination; analyzing the metastasis and going back to the primary tumor it turned out to be a transcription factor EB-renal cell carcinoma. The treatment plan included local radiation and systemic therapy. As part of the multimodal approach, tumor samples for genetic assessment were obtained...

Muscle cells have different phenotypes adapted to different usage and can be grossly divided into... more Muscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A gen...

Blood

Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relap... more Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Both transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: We performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients. An average sequencing coverage of 700X was achieved for both ...

Blood

Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only ... more Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only 10% of these patients reach a 3-year progression-free survival compared to 65% at diagnosis. Our ability to distinguish patients at risk for relapse remains based on clinical staging. We hypothesized that identifying genetic alterations in serial tumour biopsies at diagnosis and relapse would improve our ability to identify high-risk patients, make therapeutic selections and reveal molecular markers for chemo-immunotherapy resistant tumours. However, relatively few relapsed/refractory biopsies have been sequenced. A unique, clinically annotated, Nordic DLBCL cohort was used to identify significantly mutated genes, assess potential driver genes, comprehensively examine clonal evolution, and gauge the importance of clinical relapsed sampling. Methods To address the lack of information on the molecular foundations of relapsed/refractory DLBCL, we performed whole exome sequencing (WES) on 42...

Molecular Aspects of Medicine

Cells

Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLP... more Background: FGFR inhibition has been proposed as treatment for dedifferentiated liposarcoma (DDLPS) with amplified FRS2, but we previously only demonstrated transient cytostatic effects when treating FRS2-amplified DDLPS cells with NVP-BGJ398. Methods: Effects of the more potent FGFR inhibitor LY2874455 were investigated in three DDLPS cell lines by measuring effects on cell growth and apoptosis in vitro and also testing efficacy in vivo. Genome, transcriptome and protein analyses were performed to characterize the signaling components in the FGFR pathway. Results: LY2874455 induced a stronger, longer-lasting growth inhibitory effect and moderate level of apoptosis for two cell lines. The third cell line, did not respond to FGFR inhibition, suggesting that FRS2 amplification alone is not sufficient to predict response. Importantly, efficacy of LY2874455 was confirmed in vivo, using an independent FRS2-amplified DDLPS xenograft model. Expression of FRS2 was similar in the responding ...

Nature Communications

Inhibitory signaling during natural killer (NK) cell education translates into increased responsi... more Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca 2+ stores in primary NK cells reduces target-specific Ca 2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI (3,5)P 2 synthesis, or genetic silencing of the PI(3,5)P 2-regulated lysosomal Ca 2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.

Inhibitory signaling during natural killer (NK) cell education translates into increased responsi... more Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzyme-B dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signal...

International journal of molecular sciences, Jan 23, 2018

The BRAF mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas an... more The BRAF mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma (A673) and atypical synovial sarcoma (SW982), were treated with vemurafenib and the effects on cell growth, apoptosis, cell cycle progression and cell signaling were determined. Vemurafenib induced a strong cytostatic effect in SA-4 cells, mainly due to cell cycle arrest, whereas only moderate levels of apoptosis were observed. However, a high dose was required compared to BRAF mutated melanoma cells, and removal of vemurafenib demonstrated that the continuous presence of drug was required for sustained growth inhibition. A limited growth inhibition was observed in the other three cell lines. Protein analyses demonstrated reduced phosphorylation of ERK during treatment with vemurafenib in all the four sarco...

European urology, 2018

Most primary prostate cancers are multifocal with individual tumors harboring different aggressiv... more Most primary prostate cancers are multifocal with individual tumors harboring different aggressiveness; however, the genomic heterogeneity among these tumors is poorly understood. To better understand the biological basis for clinical variability among different lesions, we sought to comprehensively characterize the heterogeneity of somatic gene mutations in multifocal prostate cancer. High-coverage whole-exome sequencing of 153 frozen tissue samples, taken from two to three distinct tumor foci and one non-cancerous area from each of 41 patients, covering a total of 89 tumor foci. State-of-the-art bioinformatics tools for mutation calling and copy number determination from whole-exome sequencing data. We found a very high degree of interfocal heterogeneity among tumors, that is, 76% of pairwise-compared tumor foci from the same prostatectomy specimen had no point mutations in common and DNA copy number changes were rarely shared across cancer foci. The few point mutations shared acr...

Scientific reports, Jan 28, 2018

Sample pooling enabled by dedicated indexes is a common strategy for cost-effective and robust hi... more Sample pooling enabled by dedicated indexes is a common strategy for cost-effective and robust high-throughput sequencing. Index misassignment leading to mutual contamination between pooled samples has however been described as a general problem of the latest Illumina sequencing instruments utilizing exclusion amplification. Using real-life data from multiple tumour sequencing projects, we demonstrate that index misassignment can induce artefactual variant calls closely resembling true, high-quality somatic variants. These artefactual calls potentially impact cancer applications utilizing low allelic frequencies, such as in clonal analysis of tumours. We discuss the available countermeasures with an emphasis on improved library indexing methods, and provide software that can assist in the identification of variants that may be consequences of index misassignment.

Molecular Cancer

Background: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Compre... more Background: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. Methods: We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. Results: The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA-and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFβ signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately ¼ of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene-and protein-expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. Conclusions: This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.

Genome medicine, May 24, 2017

Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a co... more Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations-the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers. A total of 333 primary MSI+ colorectal tumors from multiple cohorts were analyzed by multilevel genomics and computational modeling-including mutation profiling, clonality modeling, and neoantigen prediction in a subset of the tumors, as well as gene expression profiling for consensus molecular subtypes (CMS) and immune cell infiltration. Novel, frequent frameshift mutations in four cancer-critical genes were identified by deep exome sequencing, including in CRTC1, BCL9, JAK1, and PTCH1. JAK1 loss-of-function mutations were validated with an overall frequency of 20% in Norwegian and British patients, and mutated tumors had up-regulation of transc...

Haematologica, Jul 6, 2017

Circulating tumor DNA (ctDNA) is a promising biomarker to monitor tumor load and genome alteratio... more Circulating tumor DNA (ctDNA) is a promising biomarker to monitor tumor load and genome alterations. We have explored the presence of ctDNA in multiple myeloma patients and its relation to disease activity during long-term follow-up. We used digital droplet PCR to monitor recurrent mutations, mainly in mitogen activated protein kinase pathway genes NRAS, KRAS and BRAF. Mutations were identified by next generation sequencing or PCR of bone marrow plasma cells, and their presence analyzed in 251 archived serum samples obtained from 20 patients during up to 7 years. In 17 of 18 patients, mutations identified in bone marrow during active disease were also found in a time-matched serum sample. The concentration of mutated alleles in serum correlated with the fraction in bone marrow plasma cells (r=0.507, n=34, p<0.002). There was a striking covariation between circulating mutation levels and M protein in 10 out of 11 patients with sequential samples. When relapse evaluation by ctDNA a...

BMC Cancer, 2017

Background: Many patients experience local recurrence or metastases after receiving potentially c... more Background: Many patients experience local recurrence or metastases after receiving potentially curative treatment, and early detection of these events is important for disease control. Recent technological advances make it possible to use blood plasma containing circulating cell-free tumour DNA (ctDNA) as a liquid biopsy. In this case report we show how serial liquid biopsies can be used to monitor the disease course and detect disease recurrence in a sarcoma patient. Case presentation: A 55-year-old male presented with a rapidly growing, painful palpable mass in the left groin region, and a biopsy revealed a high-grade malignant spindle cell sarcoma. No metastases were detected on radiologic imaging scans. Using targeted resequencing with a custom 900 cancer gene panel, eight somatic mutations among them KRAS and NF1, were identified in the primary tumour. Targeted resequencing of plasma cell-free DNA (ctDNA) collected before and after surgery and at disease progression confirmed the presence of six of eight mutations at all three time points. The ctDNA level, estimated from the somatic allele frequencies of these six mutations, was high in plasma taken at the time of surgery, at levels similar to the primary tumour. Detection of low levels of ctDNA three days after surgery indicated persistent microscopic disease. Repeated radiologic imaging six weeks postoperatively showed widespread metastatic disease in the lungs, skeleton and the pelvic region. At this time point there was a dramatic increase in the ctDNA level, reflecting the disease progression of the patient. The patient had an unusually aggressive cancer, and succumbed to the disease 13 weeks after surgery. Conclusions: This case report demonstrated that targeted resequencing of ctDNA from longitudinal collected plasma can be used to monitor disease progression in a soft tissue sarcoma patient, including manifestation of metastatic disease. The ctDNA represented the genomic profile of the tumour, supporting clinical use of liquid biopsies to identify tumour-specific mutations as well as recurrent disease.

PloS one

Osteosarcoma (OS) is the most common primary malignant tumor of bone, showing complex chromosomal... more Osteosarcoma (OS) is the most common primary malignant tumor of bone, showing complex chromosomal rearrangements but with few known consistent changes. Deeper biological understanding is crucial to find new therapies to improve patient survival. We have sequenced the whole exome of two primary tumors (before and after chemotherapy), one metastatic tumor and a matched normal sample from two OS patients, to identify mutations involved in cancer biology. The metastatic samples were also RNA sequenced. By RNA sequencing we identified dysregulated expression levels of drug resistance- and apoptosis-related genes. Two fusion transcripts were identified in one patient (OS111); the first resulted in p53 inactivation by fusing the first exon of TP53 to the fifth exon of FAM45A. The second fusion joined the two first exons of FGFR1 to the second exon of ZNF343. Furthermore, FGFR1 was amplified and highly expressed, representing a potential treatment target in this patient. Whole exome sequenc...

Oncotarget, 2014

Sarcomas are rare cancers with limited treatment options. Patients are generally treated by chemo... more Sarcomas are rare cancers with limited treatment options. Patients are generally treated by chemotherapy and/or radiotherapy in combination with surgery, and would benefit from new personalized approaches. In this study we demonstrate the potential of combining personal genomic characterization of patient tumors to identify targetable mutations with in vitro testing of specific drugs in patient-derived cell lines. We have analyzed three metastases from a patient with highgrade metastatic dedifferentiated liposarcoma (DDLPS) by exome and transcriptome sequencing as well as DNA copy number analysis. Genomic aberrations of several potentially targetable genes, including amplification of KITLG and FRS2, in addition to amplification of CDK4 and MDM2, characteristic of this disease, were identified. We evaluated the efficacy of drugs targeting these aberrations or the corresponding signaling pathways in a cell line derived from the patient. Interestingly, the pan-FGFR inhibitor NVP-BGJ398, which targets FGFR upstream of FRS2, strongly inhibited cell proliferation in vitro and induced an accumulation of cells into the G0 phase of the cell cycle. This study indicates that FGFR inhibitors have therapeutic potential in the treatment of DDLPS with amplified FRS2.

Genes, Chromosomes and Cancer, 2016

Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyp... more Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology. © 2016 Wiley Periodicals, Inc.

Frontiers in Genetics, 2016

Background: TP53 mutations are among the most common mutations found in lung cancers, identified ... more Background: TP53 mutations are among the most common mutations found in lung cancers, identified as an independent prognostic factor in many types of cancers. The purpose of this study was to investigate the frequency and prognostic impact of TP53 mutations in never-smokers and in different histological subtypes of lung cancer. Methods: We analyzed tumor tissue from 394 non-small cell carcinomas including adenocarcinomas (n = 229), squamous cell carcinomas (n = 112), large cell carcinomas (n = 30), and others (n = 23) for mutations in TP53 by the use of Sanger sequencing (n = 394) and next generation sequencing (n = 100). Results: TP53 mutations were identified in 47.2% of the samples, with the highest frequency (65%) of mutations among squamous cell carcinomas. Among neversmokers, 36% carried a TP53 mutation, identified as a significant independent negative prognostic factor in this subgroup. For large cell carcinomas, a significantly prolonged progression free survival was found for those carrying a TP53 mutation. In addition, the frequency of frameshift mutations was doubled in squamous cell carcinomas (20.3%) compared to adenocarcinomas (9.1%). Conclusion: TP53 mutation patterns differ between the histological subgroups of lung cancers, and are also influenced by smoking history. This indicates that the histological subtypes in lung cancer are genetically different, and that smoking-induced TP53 mutations may have a different biological impact than TP53 mutations occurring in never-smokers.

Oncotarget, Jan 15, 2015

Liposarcoma (LPS) is the most common type of soft tissue sarcoma accounting for 20% of all adult ... more Liposarcoma (LPS) is the most common type of soft tissue sarcoma accounting for 20% of all adult sarcomas. Due to absence of clinically effective treatment options in inoperable situations and resistance to chemotherapeutics, a critical need exists to identify novel therapeutic targets. We analyzed LPS genomic landscape using SNP arrays, whole exome sequencing and targeted exome sequencing to uncover the genomic information for development of specific anti-cancer targets. SNP array analysis indicated known amplified genes (MDM2, CDK4, HMGA2) and important novel genes (UAP1, MIR557, LAMA4, CPM, IGF2, ERBB3, IGF1R). Carboxypeptidase M (CPM), recurrently amplified gene in well-differentiated/de-differentiated LPS was noted as a putative oncogene involved in the EGFR pathway. Notable deletions were found at chromosome 1p (RUNX3, ARID1A), chromosome 11q (ATM, CHEK1) and chromosome 13q14.2 (MIR15A, MIR16-1). Significantly and recurrently mutated genes (false discovery rate < 0.05) incl...

Journal of medical case reports, Jan 9, 2015

Renal cell carcinoma with the distinct type of t(6;11)(p21;q12) translocation (transcription fact... more Renal cell carcinoma with the distinct type of t(6;11)(p21;q12) translocation (transcription factor EB) is a rare neoplasm. In the present case study, we show for the first time an autophagy signature in a patient with transcription factor EB renal cell carcinoma. We attempted to characterize the mutational and expressional features of a t(6;11)(p21;q12) renal cell carcinoma, in an effort to address the potential for molecular guidance of personalized medical decision for a case in this renal cell carcinoma category. We report the case of a 42-year-old white man who had a late relapse of his renal cell carcinoma. The first diagnosis of clear cell renal carcinoma was derived from a histological examination; analyzing the metastasis and going back to the primary tumor it turned out to be a transcription factor EB-renal cell carcinoma. The treatment plan included local radiation and systemic therapy. As part of the multimodal approach, tumor samples for genetic assessment were obtained...