Publications by authors named "Michael R Speicher"

123 Publications

Biallelic truncating variants in cause a novel neurodevelopmental disorder involving postnatal microcephaly and failure to thrive.

J Med Genet 2021 Jun 18. Epub 2021 Jun 18.

Institute of Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany

Background: Genes implicated in the Golgi and endosomal trafficking machinery are crucial for brain development, and mutations in them are particularly associated with postnatal microcephaly (POM).

Methods: Exome sequencing was performed in three affected individuals from two unrelated consanguineous families presenting with delayed neurodevelopment, intellectual disability of variable degree, POM and failure to thrive. Patient-derived fibroblasts were tested for functional effects of the variants.

Results: We detected homozygous truncating variants in A. While the variant in family A is predicted to result in an early premature termination codon, the variant in family B affects a canonical splice site. Both variants lead to a substantial reduction of mRNA expression. It has been shown previously that ATP9A localises to early and recycling endosomes, whereas its depletion leads to altered gene expression of components from this compartment. Consistent with previous findings, we also observed overexpression of and , genes strongly interacting with .

Conclusion: In aggregate, our findings show that pathogenic variants in cause a novel autosomal recessive neurodevelopmental disorder with POM. While the physiological function of endogenous ATP9A is still largely elusive, our results underline a crucial role of this gene in endosomal transport in brain tissue.
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http://dx.doi.org/10.1136/jmedgenet-2021-107843DOI Listing
June 2021

Evolutionary conservation in noncoding genomic regions.

Trends Genet 2021 10 5;37(10):903-918. Epub 2021 Jul 5.

Institute of Human Genetics, Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, 8010 Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Humans may share more genomic commonalities with other species than previously thought. According to current estimates, ~5% of the human genome is functionally constrained, which is a much larger fraction than the ~1.5% occupied by annotated protein-coding genes. Hence, ~3.5% of the human genome comprises likely functional conserved noncoding elements (CNEs) preserved among organisms, whose common ancestors existed throughout hundreds of millions of years of evolution. As whole-genome sequencing emerges as a standard procedure in genetic analyses, interpretation of variations in CNEs, including the elucidation of mechanistic and functional roles, becomes a necessity. Here, we discuss the phenomenon of noncoding conservation via four dimensions (sequence, regulatory conservation, spatiotemporal expression, and structure) and the potential significance of CNEs in phenotype variation and disease.
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http://dx.doi.org/10.1016/j.tig.2021.06.007DOI Listing
October 2021

Profiling of circulating tumor DNA and tumor tissue for treatment selection in patients with advanced and refractory carcinoma: a prospective, two-stage phase II Individualized Cancer Treatment trial.

Ther Adv Med Oncol 2021 27;13:1758835920987658. Epub 2021 Feb 27.

Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria.

Background: Molecular profiling (MP) represents an opportunity to match patients to a targeted therapy and when tumor tissue is unavailable, circulating tumor deoxyribonucleic acid (ctDNA) can be harnessed as a non-invasive analyte for this purpose. We evaluated the success of a targeted therapy selected by profiling of ctDNA and tissue in patients with advanced and refractory carcinoma.

Patients And Methods: A blood draw as well as an optional tissue biopsy were obtained for MP. Whole-genome sequencing and a cancer hotspot panel were performed, and publicly available databases were used to match the molecular profile to targeted treatments. The primary endpoint was the progression-free survival (PFS) ratio (PFS on MP-guided therapy/PFS on the last evidence-based therapy), whereas the success of the targeted therapy was defined as a PFS ratio ⩾1.2. To test the impact of molecular profile-treatment matching strategies, we retrospectively analyzed selected cases the CureMatch PreciGENE™ decision support algorithm.

Results: Interim analysis of 24 patients yielded informative results from 20 patients (83%). A potential tumor-specific drug could be matched in 11 patients (46%) and eight (33%) received a matched treatment. Median PFS in the matched treatment group was 61.5 days [interquartile range (IQR) 49.8-71.0] compared with 81.5 days (IQR 68.5-117.8) for the last evidence-based treatment, resulting in a median PFS ratio of 0.7 (IQR 0.6-0.9). Hence, as no patient experienced a PFS ratio ⩾1.2, the study was terminated. Except for one case, the CureMatch analysis identified either a two-drug or three-drug combination option.

Conclusions: Our study employed a histotype-agnostic approach to harness molecular profiling data from both ctDNA and metastatic tumor tissue. The outcome results indicate that more innovative approaches to study design and matching algorithms are necessary to achieve improved patient outcomes. EudraCT: 2014-005341-44.
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http://dx.doi.org/10.1177/1758835920987658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923987PMC
February 2021

On-treatment measurements of circulating tumor DNA during FOLFOX therapy in patients with colorectal cancer.

NPJ Precis Oncol 2020 Nov 13;4(1):30. Epub 2020 Nov 13.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

We addressed a significant unknown feature of circulating tumor DNA (ctDNA), i.e., how ctDNA levels change during chemotherapy, by serially monitoring ctDNA in patients with colorectal cancer during the 48-h application of FOLFOX. Surprisingly, we did not observe a spike in ctDNA as a sign of a responsive tumor, but instead ctDNA levels initially decreased and remained low in patients with stable disease or partial response. Our observations reveal further insights into cell destruction during chemotherapy with important implications for the management of patients.
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http://dx.doi.org/10.1038/s41698-020-00134-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666126PMC
November 2020

Comparison of three commercial decision support platforms for matching of next-generation sequencing results with therapies in patients with cancer.

ESMO Open 2020 09;5(5):e000872

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Objective: Precision oncology depends on translating molecular data into therapy recommendations. However, with the growing complexity of next-generation sequencing-based tests, clinical interpretation of somatic genomic mutations has evolved into a formidable task. Here, we compared the performance of three commercial clinical decision support tools, that is, NAVIFY Mutation Profiler (NAVIFY; Roche), QIAGEN Clinical Insight (QCI) Interpret (QIAGEN) and CureMatch Bionov (CureMatch).

Methods: In order to obtain the current status of the respective tumour genome, we analysed cell-free DNA from patients with metastatic breast, colorectal or non-small cell lung cancer. We evaluated somatic copy number alterations and in parallel applied a 77-gene panel (AVENIO ctDNA Expanded Panel). We then assessed the concordance of tier classification approaches between NAVIFY and QCI and compared the strategies to determine actionability among all three platforms. Finally, we quantified the alignment of treatment suggestions across all decision tools.

Results: Each platform varied in its mode of variant classification and strategy for identifying druggable targets and clinical trials, which resulted in major discrepancies. Even the frequency of concordant actionable events for tier I-A or tier I-B classifications was only 4.3%, 9.5% and 28.4% when comparing NAVIFY with QCI, NAVIFY with CureMatch and CureMatch with QCI, respectively, and the obtained treatment recommendations differed drastically.

Conclusions: Treatment decisions based on molecular markers appear at present to be arbitrary and dependent on the chosen strategy. As a consequence, tumours with identical molecular profiles would be differently treated, which challenges the promising concepts of genome-informed medicine.
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http://dx.doi.org/10.1136/esmoopen-2020-000872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513637PMC
September 2020

Childhood-onset epileptic encephalopathy due to exon 1-4 tandem duplication.

Neurol Genet 2020 Oct 17;6(5):e494. Epub 2020 Jul 17.

Institute of Human Genetics (S.V., M.R.S., B.R.), Diagnostic and Research Center for MolecularBioMedicine, Medical University of Graz; and Department of Pediatrics and Adolescent Medicine (B.P.), Division of General Pediatrics, Medical University of Graz, Austria.

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http://dx.doi.org/10.1212/NXG.0000000000000494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371371PMC
October 2020

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

EMBO Mol Med 2020 09 15;12(9):e11908. Epub 2020 Jul 15.

Laboratory of Rare Human Circulating Cells (LCCRH), University Medical Centre, Montpellier, France.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.
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http://dx.doi.org/10.15252/emmm.201911908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507517PMC
September 2020

Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling.

Cancers (Basel) 2020 Jun 16;12(6). Epub 2020 Jun 16.

Institute of Human Genetics, Diagnostic & Research Center for Molecular BioMedicine, Medical University of Graz, 8010 Graz, Austria.

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.
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http://dx.doi.org/10.3390/cancers12061588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352370PMC
June 2020

Shallow Whole-Genome Sequencing from Plasma Identifies FGFR1 Amplified Breast Cancers and Predicts Overall Survival.

Cancers (Basel) 2020 Jun 6;12(6). Epub 2020 Jun 6.

Division of Biotechnology, Servier Research Institute, 125, Chemin de ronde, 78290 Croissy Sur-seine, France.

Focal amplification of fibroblast growth factor receptor 1 () defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for amplification by FISH, and positive cases were confirmed with a microarray platform (Oncoscan). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the status. Tissue-based analyses identified amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% ( = 12) showed concordance for status between tissue and cfDNA. In one case, we were able to detect a high-level amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Screening for amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials.
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http://dx.doi.org/10.3390/cancers12061481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353062PMC
June 2020

Cell-Free DNA and Apoptosis: How Dead Cells Inform About the Living.

Trends Mol Med 2020 05 17;26(5):519-528. Epub 2020 Feb 17.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Cell-free DNA (cfDNA) is evolving into a widely used prognostic and predictive biomarker, particularly in oncology. However, its versatile clinical use precedes a profound understanding of the underlying biology of cfDNA release. There is much evidence to suggest that cfDNA is mainly derived from dying (i.e., apoptotic) cells. However, numerous cancer studies have shown that cfDNA is informative about acquired resistance to given therapies, which is present in living, proliferating tumor subclones. To explain this contradiction, we review current insights regarding cfDNA release, in particular the interplay between apoptosis and proliferation. We describe how improved knowledge about cfDNA biology could be used for novel therapeutic strategies and how this may affect patient management.
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http://dx.doi.org/10.1016/j.molmed.2020.01.012DOI Listing
May 2020

Publisher Correction: Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection.

Nat Commun 2020 04 20;11(1):1965. Epub 2020 Apr 20.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-15799-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170910PMC
April 2020

Cell-free DNA analysis reveals POLR1D-mediated resistance to bevacizumab in colorectal cancer.

Genome Med 2020 02 22;12(1):20. Epub 2020 Feb 22.

Institute of Human Genetics, Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, Graz, Austria.

Background: Bevacizumab, a monoclonal antibody against soluble VEGFA, is an approved and commonly administered anti-angiogenic drug in patients with metastasized colorectal cancer (mCRC). The survival benefit of anti-VEGF therapy in mCRC patients is limited to a few months, and acquired resistance mechanisms are largely unknown. Here, we employed whole-genome sequencing of plasma DNA to evaluate the tumor genome of patients undergoing treatment with bevacizumab to determine novel aberrations associated with resistance.

Methods: Using longitudinal plasma analyses, we studied the evolution of tumor genomes in a mCRC cohort (n = 150) and conducted analyses of CRC cases from The Cancer Genome Atlas (TCGA) database (n = 619) to identify associations between genomic aberrations and clinical features. We employed whole-genome sequencing to identify the most frequently occurring focal somatic copy number alterations (SCNAs). Using the TCGA data as a comparative and supporting dataset, we defined the minimally amplified overlapping region and studied the mechanistic consequences of copy number gain of the involved genes in this segment. In addition, we established an in vitro cell model and conducted downstream gene expression and cell viability assays to confirm our findings from the patient dataset.

Results: We observed a recurrent focal amplification (8.7% of cases) on chromosome 13q12.2. Analysis of CRC cases from the TCGA database suggested that this amplicon is associated with more advanced stages. We confirmed that this 13q12.2 amplicon frequently emerges later during the clinical course of disease. After defining the minimally amplified region, we observed that the amplification and expression of one gene, POLR1D, impacted cell proliferation and resulted in upregulation of VEGFA, an important regulator of angiogenesis which has been implicated in the resistance to bevacizumab treatment. In fact, in several patients, we observed the emergence of this 13q12.2 amplicon under bevacizumab treatment, which was invariably associated with therapy resistance.

Conclusions: Non-invasive analyses of cell-free DNA from patients undergoing treatment with bevacizumab enabled the tracking of evolving tumor genomes and helped identify a recurrent focal SCNA of clinical relevance. Here, we describe a novel resistance mechanism against a widely applied treatment in patients with mCRC which will impact the clinical management of patients.
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http://dx.doi.org/10.1186/s13073-020-0719-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036260PMC
February 2020

Novel phenotypes observed in patients with -linked leukaemia/familial thrombocytopenia syndrome and a biallelic risk allele as leukaemogenic cofactor.

J Med Genet 2020 06 8;57(6):427-433. Epub 2019 Nov 8.

Division of Pediatric Hemato-Oncology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Graz, Austria

The phenotypes of patients with the recently discovered, dominant, -linked leukaemia predisposition and familial thrombocytopenia syndrome are variable, and the exact mechanism of leukaemogenesis remains unclear. Here, we present novel clinical and laboratory phenotypes of seven individuals from three families with germline mutations and a refined genetic analysis of one child with additional high-hyperdiploid acute lymphoblastic leukaemia (HD-ALL), aiming to elucidate second oncogenic hits. Four individuals from two pedigrees harboured one novel or one previously described variant in the central domain of (c.592C>T, p.Gln198* or c.641C>T, p.Pro241Leu, respectively). Neutropenia was an accompanying feature in one of these families that also harboured a variant in (c.1098_1103dup, p.Ile366_Gly367dup), while in the other, an autism-spectrum disorder was observed. In the third family, the index patient suffered from HD-ALL and life-threatening pulmonary mucor mycosis, and had a positive family history of 'immune' thrombocytopenia. Genetic analyses revealed a novel heterozygous mutation in the ETS domain of (c.1136T>C, p.Leu379Pro) along with absence of heterozygosity of chromosome (10)(q21.2q21.3), yielding a biallelic leukaemia risk allele in (rs7090445-C). The neutrophil function was normal in all individuals tested, and the platelet immune histochemistry of all three pedigrees showed delta-storage-pool defect-like features and cytoskeletal defects. Our clinical observations and results of high-resolution genetic analyses extend the spectrum of possible phenotypes cosegregating with germline mutations. Further, we propose as potential leukaemogenic cofactor in patients with -linked leukaemia predisposition and familial thrombocytopenia syndrome.
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http://dx.doi.org/10.1136/jmedgenet-2019-106339DOI Listing
June 2020

Genome-Wide Analysis of the Nucleosome Landscape in Individuals with Coffin-Siris Syndrome.

Cytogenet Genome Res 2019 26;159(1):1-11. Epub 2019 Oct 26.

The switch/sucrose non-fermenting (SWI/SNF) complex is an ATP-dependent chromatin remodeller that regulates the spacing of nucleosomes and thereby controls gene expression. Heterozygous mutations in genes encoding subunits of the SWI/SNF complex have been reported in individuals with Coffin-Siris syndrome (CSS), with the majority of the mutations in ARID1B. CSS is a rare congenital disorder characterized by facial dysmorphisms, digital anomalies, and variable intellectual disability. We hypothesized that mutations in genes encoding subunits of the ubiquitously expressed SWI/SNF complex may lead to alterations of the nucleosome profiles in different cell types. We performed the first study on CSS-patient samples and investigated the nucleosome landscapes of cell-free DNA (cfDNA) isolated from blood plasma by whole-genome sequencing. In addition, we studied the nucleosome landscapes of CD14+ monocytes from CSS-affected individuals by nucleosome occupancy and methylome-sequencing (NOMe-seq) as well as their expression profiles. In cfDNA of CSS-affected individuals with heterozygous ARID1B mutations, we did not observe major changes in the nucleosome profile around transcription start sites. In CD14+ monocytes, we found few genomic regions with different nucleosome occupancy when compared to controls. RNA-seq analysis of CD14+ monocytes of these individuals detected only few differentially expressed genes, which were not in proximity to any of the identified differential nucleosome-depleted regions. In conclusion, we show that heterozygous mutations in the human SWI/SNF subunit ARID1B do not have a major impact on the nucleosome landscape or gene expression in blood cells. This might be due to functional redundancy, cell-type specificity, or alternative functions of ARID1B.
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http://dx.doi.org/10.1159/000503266DOI Listing
December 2019

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

Clin Chem 2020 01;66(1):149-160

QIAGEN GmbH, Hilden, Germany.

Background: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making.

Methods: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites.

Results: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT.

Conclusions: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.
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http://dx.doi.org/10.1373/clinchem.2019.306837DOI Listing
January 2020

Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection.

Nat Commun 2019 10 11;10(1):4666. Epub 2019 Oct 11.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

Deregulation of transcription factors (TFs) is an important driver of tumorigenesis, but non-invasive assays for assessing transcription factor activity are lacking. Here we develop and validate a minimally invasive method for assessing TF activity based on cell-free DNA sequencing and nucleosome footprint analysis. We analyze whole genome sequencing data for >1,000 cell-free DNA samples from cancer patients and healthy controls using a bioinformatics pipeline developed by us that infers accessibility of TF binding sites from cell-free DNA fragmentation patterns. We observe patient-specific as well as tumor-specific patterns, including accurate prediction of tumor subtypes in prostate cancer, with important clinical implications for the management of patients. Furthermore, we show that cell-free DNA TF profiling is capable of detection of early-stage colorectal carcinomas. Our approach for mapping tumor-specific transcription factor binding in vivo based on blood samples makes a key part of the noncoding genome amenable to clinical analysis.
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http://dx.doi.org/10.1038/s41467-019-12714-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789008PMC
October 2019

Detection and Characterization of Circulating Tumor Cells in Patients with Merkel Cell Carcinoma.

Clin Chem 2019 03 9;65(3):462-472. Epub 2019 Jan 9.

Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with increasing incidence and high mortality rates. MCC has recently become the subject of immune checkpoint therapy, but reliable biomarkers for estimating prognosis, risk stratification, and prediction of response are missing.

Methods: Circulating tumor cells (CTCs) were detected in peripheral blood from patients with MCC by use of the CellSearch system. Moreover, CTCs of selected cases were characterized for Merkel cell polyomavirus (MCPyV), chromosomal aberrations, and programed death ligand 1 (PD-L1) production.

Results: Fifty-one patients were tested at first blood draw (baseline), and 16 patients had 2 or 3 consecutive measurements to detect CTCs. At baseline, ≥1 CTC (range, 1-790), >1, or ≥5 CTCs/7.5 mL were detected in 21 (41%), 17 (33%), and 6 (12%) patients, respectively. After a median follow-up of 21.1 months for 50 patients, detection of CTCs correlated with overall survival (≥1, = 0.030; >1, < 0.020; and ≥5 CTCs/7.5 mL, < 0.0001). In multivariate Cox regression analysis, the detection of ≥5 CTCs/7.5 mL adjusted to age and sex compared to that of <5 was associated with a reduced overall survival ( = 0.001, hazard ratio = 17.8; 95% CI, 4.0-93.0). MCPyV DNA and genomic aberrations frequently found in MCC tissues could also be detected in single CTCs. Analyzed CTCs were PD-L1 negative or only weakly positive.

Conclusions: The presence of CTCs is a prognostic factor of impaired clinical outcome, with the potential to monitor the progression of the disease in real time. Molecular characterization of CTCs might provide new insights into the biology of MCC.
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http://dx.doi.org/10.1373/clinchem.2018.297028DOI Listing
March 2019

Single tube liquid biopsy for advanced non-small cell lung cancer.

Int J Cancer 2019 06 28;144(12):3127-3137. Epub 2019 Jan 28.

Department of Pulmonary Diseases, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands.

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM circulating tumor cells (CTC), EpCAM CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAM CTC, 15% had ≥2 EpCAM CTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.
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http://dx.doi.org/10.1002/ijc.32056DOI Listing
June 2019

Comprehensive Study of the Clinical Phenotype of Germline BAP1 Variant-Carrying Families Worldwide.

J Natl Cancer Inst 2018 12;110(12):1328-1341

Medical Oncology Unit, Ospedale Policlinico San Martino, Genoa, Italy.

Background: The BRCA1-associated protein-1 (BAP1) tumor predisposition syndrome (BAP1-TPDS) is a hereditary tumor syndrome caused by germline pathogenic variants in BAP1 encoding a tumor suppressor associated with uveal melanoma, mesothelioma, cutaneous melanoma, renal cell carcinoma, and cutaneous BAP1-inactivated melanocytic tumors. However, the full spectrum of tumors associated with the syndrome is yet to be determined. Improved understanding of the BAP1-TPDS is crucial for appropriate clinical management of BAP1 germline variant carriers and their families, including genetic counseling and surveillance for new tumors.

Methods: We collated germline variant status, tumor diagnoses, and information on BAP1 immunohistochemistry or loss of somatic heterozygosity on 106 published and 75 unpublished BAP1 germline variant-positive families worldwide to better characterize the genotypes and phenotypes associated with the BAP1-TPDS. Tumor spectrum and ages of onset were compared between missense and null variants. All statistical tests were two-sided.

Results: The 181 families carried 140 unique BAP1 germline variants. The collated data confirmed the core tumor spectrum associated with the BAP1-TPDS and showed that some families carrying missense variants can exhibit this phenotype. A variety of noncore BAP1-TPDS -associated tumors were found in families of variant carriers. Median ages of onset of core tumor types were lower in null than missense variant carriers for all tumors combined (P < .001), mesothelioma (P < .001), cutaneous melanoma (P < .001), and nonmelanoma skin cancer (P < .001).

Conclusions: This analysis substantially increases the number of pathogenic BAP1 germline variants and refines the phenotype. It highlights the need for a curated registry of germline variant carriers for proper assessment of the clinical phenotype of the BAP1-TPDS and pathogenicity of new variants, thus guiding management of patients and informing areas requiring further research.
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http://dx.doi.org/10.1093/jnci/djy171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292796PMC
December 2018

Current and future perspectives of liquid biopsies in genomics-driven oncology.

Nat Rev Genet 2019 02;20(2):71-88

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

Precision oncology seeks to leverage molecular information about cancer to improve patient outcomes. Tissue biopsy samples are widely used to characterize tumours but are limited by constraints on sampling frequency and their incomplete representation of the entire tumour bulk. Now, attention is turning to minimally invasive liquid biopsies, which enable analysis of tumour components (including circulating tumour cells and circulating tumour DNA) in bodily fluids such as blood. The potential of liquid biopsies is highlighted by studies that show they can track the evolutionary dynamics and heterogeneity of tumours and can detect very early emergence of therapy resistance, residual disease and recurrence. However, the analytical validity and clinical utility of liquid biopsies must be rigorously demonstrated before this potential can be realized.
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http://dx.doi.org/10.1038/s41576-018-0071-5DOI Listing
February 2019

One size does not fit all: Size-based plasma DNA diagnostics.

Sci Transl Med 2018 11;10(466)

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, A-8010 Graz, Austria.

Understanding the biological properties of plasma cell-free DNA may expand its applications in oncology (Mouliere , this issue).
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http://dx.doi.org/10.1126/scitranslmed.aav3873DOI Listing
November 2018

The potential of liquid biopsies for the early detection of cancer.

NPJ Precis Oncol 2017 17;1(1):36. Epub 2017 Oct 17.

1Institute of Human Genetics, Medical University of Graz, Neue Stiftingtalstraße 6, A-8010 Graz, Austria.

Precision medicine refers to the choosing of targeted therapies based on genetic data. Due to the increasing availability of data from large-scale tumor genome sequencing projects, genome-driven oncology may have enormous potential to change the clinical management of patients with cancer. To this end, components of tumors, which are shed into the circulation, i.e., circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or extracellular vesicles, are increasingly being used for monitoring tumor genomes. A growing number of publications have documented that these "liquid biopsies" are informative regarding response to given therapies, are capable of detecting relapse with lead time compared to standard measures, and reveal mechanisms of resistance. However, the majority of published studies relate to advanced tumor stages and the use of liquid biopsies for detection of very early malignant disease stages is less well documented. In early disease stages, strategies for analysis are in principle relatively similar to advanced stages. However, at these early stages, several factors pose particular difficulties and challenges, including the lower frequency and volume of aberrations, potentially confounding phenomena such as clonal expansions of non-tumorous tissues or the accumulation of cancer-associated mutations with age, and the incomplete insight into driver alterations. Here we discuss biology, technical complexities and clinical significance for early cancer detection and their impact on precision oncology.
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http://dx.doi.org/10.1038/s41698-017-0039-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871864PMC
October 2017

Genomic alterations in plasma DNA from patients with metastasized prostate cancer receiving abiraterone or enzalutamide.

Int J Cancer 2018 09 10;143(5):1236-1248. Epub 2018 Apr 10.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Neue Stiftingtalstraße 6, A-8010, Graz, Austria.

In patients with metastatic castrate-resistant prostate cancer (mCRPC), circulating tumor DNA (ctDNA) analysis offers novel opportunities for the development of non-invasive biomarkers informative of treatment response with novel agents targeting the androgen-receptor (AR) pathway, such as abiraterone or enzalutamide. However, the relationship between ctDNA abundance, detectable somatic genomic alterations and clinical progression of mCRPC remains unexplored. Our study aimed to investigate changes in plasma DNA during disease progression and their associations with clinical variables in mCRPC patients. We analyzed ctDNA in two cohorts including 94 plasma samples from 25 treatment courses (23 patients) and 334 plasma samples from 125 patients, respectively. We conducted whole-genome sequencing (plasma-Seq) for genome-wide profiling of somatic copy number alterations and targeted sequencing of 31 prostate cancer-associated genes. The combination of plasma-Seq with targeted AR analyses identified prostate cancer-related genomic alterations in 16 of 25 (64%) treatment courses in the first cohort, in which we demonstrated that AR amplification does not always correlate with poor abiraterone and enzalutamide therapy outcome. As we observed a wide variability of ctDNA levels, we evaluated ctDNA levels and their association with clinical parameters and included the second, larger cohort for these analyses. Employing altogether 428 longitudinal plasma samples from 148 patients, we identified the presence of bone metastases, increased lactate dehydrogenase and prostate-specific antigen (PSA) as having the strongest association with high ctDNA levels. In summary, ctDNA alterations are observable in the majority of patients with mCRPC and may eventually be useful to guide clinical decision-making in this setting.
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http://dx.doi.org/10.1002/ijc.31397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6099279PMC
September 2018

Digital Circulating Tumor Cell Analyses for Prostate Cancer Precision Oncology.

Cancer Discov 2018 03;8(3):269-271

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

In this issue of , Miyamoto and colleagues adapted their microfluidic CTC-iChip isolation platform with a digital RNA-PCR readout for eight prostate-specific transcripts and two assays for the androgen receptor mRNA splice variant and the translocation transcript. In patients with metastatic castrate-resistant prostate cancer at initiating abiraterone therapy in a first-line setting, the resulting RNA-based digital circulating tumor cell signatures identified patients with a shorter overall survival, and in patients with clinically localized disease, the signatures identified those with seminal vesicle invasion and pelvic lymph node involvement. .
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http://dx.doi.org/10.1158/2159-8290.CD-18-0075DOI Listing
March 2018

Single-Stranded DNA Library Preparation Does Not Preferentially Enrich Circulating Tumor DNA.

Clin Chem 2017 10 14;63(10):1656-1659. Epub 2017 Aug 14.

Institute of Human Genetics Medical University of Graz Graz, Austria

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http://dx.doi.org/10.1373/clinchem.2017.277988DOI Listing
October 2017

Searching for cancer vulnerabilities amid genetic chaos.

Genome Biol 2017 08 3;18(1):147. Epub 2017 Aug 3.

Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, A-8010, Graz, Austria.

A meeting report on the Third European Association for Cancer Research Conference on Cancer Genomics, held at Churchill College, Cambridge, UK, 25-28 June 2017.
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http://dx.doi.org/10.1186/s13059-017-1283-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541741PMC
August 2017

Patient monitoring through liquid biopsies using circulating tumor DNA.

Int J Cancer 2017 09 25;141(5):887-896. Epub 2017 May 25.

Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, Graz, A-8010, Austria.

Tumors release components such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and tumor-derived extracellular vesicles into the circulation. Multiple studies have demonstrated that molecular information about tumors and metastases can be extracted from these factors, which are therefore frequently referred to as "liquid biopsies." Liquid biopsies allow the longitudinal monitoring of tumor genomes non-invasively and may hence ensure that patients receive appropriate treatments that target the molecular features of their disease. Accordingly, the number of studies employing liquid biopsy based assays has been skyrocketing in the last few years. Here, we focus on three important issues, which are of high relevance for monitoring tumor genomes. First, we analyze the relation between the allele frequency of somatic tumor-specific mutations and the tumor fraction within plasma DNA. Second, we ask how well current tumor evolution models correlate with findings in longitudinal liquid biopsy studies. And, finally, as sensitivity is one of the key challenges of mutation detection, we address the challenge of detecting mutations occurring at very low allele frequencies in plasma DNA.
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http://dx.doi.org/10.1002/ijc.30759DOI Listing
September 2017

Emerging concepts in liquid biopsies.

BMC Med 2017 04 6;15(1):75. Epub 2017 Apr 6.

Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, A-8010, Graz, Austria.

Characterizing and monitoring tumor genomes with blood samples could achieve significant improvements in precision medicine. As tumors shed parts of themselves into the circulation, analyses of circulating tumor cells, circulating tumor DNA, and tumor-derived exosomes, often referred to as "liquid biopsies", may enable tumor genome characterization by minimally invasive means. Indeed, multiple studies have described how molecular information about parent tumors can be extracted from these components. Here, we briefly summarize current technologies and then elaborate on emerging novel concepts that may further propel the field. We address normal and detectable mutation levels in the context of our current knowledge regarding the gradual accumulation of mutations during aging and in light of technological limitations. Finally, we discuss whether liquid biopsies are ready to be used in routine clinical practice.
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http://dx.doi.org/10.1186/s12916-017-0840-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382440PMC
April 2017
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