Technical Artifacts Research Articles

The Impact of Sample Size and Population History on Observed Mutational Spectra: A Case Study in Human and Chimpanzee Populations.

Recent studies have highlighted variation in the mutational spectra among human populations as well as closely related hominoids-yet little remains known about the genetic and nongenetic factors driving these rate changes across the genome. Pinpointing the root causes of these differences is an important endeavor that requires careful comparative analyses of population-specific mutational landscapes at both broad and fine genomic scales. However, several factors can confound such analyses. Although previous studies have shown that technical artifacts, such as sequencing errors and batch effects, can contribute to observed mutational shifts, other potentially confounding parameters have received less attention thus far. Using population genetic simulations of human and chimpanzee populations as an illustrative example, we here show that the sample size required for robust inference of mutational spectra depends on the population-specific demographic history. As a consequence, the power to detect rate changes is high in certain hominoid populations while, for others, currently available sample sizes preclude analyses at fine genomic scales.

Sensitive Detection of Cell-Free Tumour DNA Using Optimised Targeted Sequencing Can Predict Prognosis in Gastro-Oesophageal Cancer.

In this longitudinal study, cell-free tumour DNA (a liquid biopsy) from plasma was explored as a prognostic biomarker for gastro-oesophageal cancer. Both tumour-informed and tumour-agnostic approaches for plasma variant filtering were evaluated in 47 participants. This was possible through sequencing of DNA from tissue biopsies from all participants and cell-free DNA from plasma sampled before and after surgery (n = 42), as well as DNA from white blood cells (n = 21) using a custom gene panel with and without unique molecular identifiers (UMIs). A subset of the plasma samples (n = 12) was also assayed with targeted droplet digital PCR (ddPCR). In 17/31 (55%) diagnostic plasma samples, tissue-verified cancer-associated variants could be detected by the gene panel. In the tumour-agnostic approach, 26 participants (59%) had cancer-associated variants, and UMIs were necessary to filter the true variants from the technical artefacts. Additionally, clonal haematopoietic variants could be excluded using the matched white blood cells or follow-up plasma samples. ddPCR detected its targets in 10/12 (83%) and provided an ultra-sensitive method for follow-up. Detectable cancer-associated variants in plasma correlated to a shorter overall survival and shorter time to progression, with a significant correlation for the tumour-informed approaches. In summary, liquid biopsy gene panel sequencing using a tumour-agnostic approach can be applied to all patients regardless of the presence of a tissue biopsy, although this requires UMIs and the exclusion of clonal haematopoietic variants. However, if sequencing data from tumour biopsies are available, a tumour-informed approach improves the value of cell-free tumour DNA as a negative prognostic biomarker in gastro-oesophageal cancer patients.

Low-tech approaches for sustainability: key principles from the literature and practice

Low-tech approaches have come to the fore in the last few years, mainly in opposition to the techno-optimistic model proposed to solve current and future environmental crises. However, low-tech thinking is multifaceted, making the concept potentially rich but also vague. This article develops a seven-principle framework to categorize low-tech concepts based on an abductive approach which included a literature review and interviews with low-tech actors. Principle occurrence was assessed among the authors and interviewees. The results demonstrate that the low-tech movement entails more than a shift to robust and less-consumptive technical artifacts. While resource efficiency and material reuse are important traits of low-tech approaches, technical appropriation is the most frequently cited key principle in the literature and by the actors. This delineation into several principles can help to differentiate low-tech from other sustainability concepts related to resource conservation such as frugal innovations and circular economy. We aim in this article to open a discussion about the ways low-tech proponents are seeking to introduce transformative development pathways to sustainability.

Deduplication Improves Cost-Efficiency and Yields of De Novo Assembly and Binning of Shotgun Metagenomes in Microbiome Research.

In the last decade, metagenomics has greatly revolutionized the study of microbial communities. However, the presence of artificial duplicate reads raised mainly from the preparation of metagenomic DNA sequencing libraries and their impacts on metagenomic assembly and binning have never been brought to attention. Here, we explicitly investigated the effects of duplicate reads on metagenomic assemblies and binning based on analyses of five groups of representative metagenomes with distinct microbiome complexities. Our results showed that deduplication considerably increased the binning yields (by 3.5% to 80%) for most of the metagenomic data sets examined thanks to the improved contig length and coverage profiling of metagenome-assembled contigs, whereas it slightly decreased the binning yields of metagenomes with low complexity (e.g., human gut metagenomes). Specifically, 411 versus 397, 331 versus 317, 104 versus 88, and 9 versus 5 metagenome-assembled genomes (MAGs) were recovered from MEGAHIT assemblies of bioreactor sludge, surface water, lake sediment, and forest soil metagenomes, respectively. Noticeably, deduplication significantly reduced the computational costs of the metagenomic assembly, including the elapsed time (9.0% to 29.9%) and the maximum memory requirement (4.3% to 37.1%). Collectively, we recommend the removal of duplicate reads in metagenomes with high complexity before assembly and binning analyses, for example, the forest soil metagenomes examined in this study. IMPORTANCE Duplicated reads in shotgun metagenomes are usually considered technical artifacts. Their presence in metagenomes would theoretically not only introduce bias into the quantitative analysis but also result in mistakes in the coverage profile, leading to adverse effects on or even failures in metagenomic assembly and binning, as the widely used metagenome assemblers and binners all need coverage information for graph partitioning and assembly binning, respectively. However, this issue was seldom noticed, and its impacts on downstream essential bioinformatic procedures (e.g., assembly and binning) remained unclear. In this study, we comprehensively evaluated for the first time the implications of duplicate reads for the de novo assembly and binning of real metagenomic data sets by comparing the assembly qualities, binning yields, and requirements for computational resources with and without the removal of duplicate reads. It was revealed that deduplication considerably increased the binning yields of metagenomes with high complexity and significantly reduced the computational costs, including the elapsed time and the maximum memory requirement, for most of the metagenomes studied. These results provide empirical references for more cost-efficient metagenomic analyses in microbiome research.

Knowledge and knowledge gaps in semantic memory of technical artifacts

Two hypotheses have been considered regarding the relation between knowledge and semantic knowledge gaps: a “knowledge clash” hypothesis predicting more awareness of knowledge gaps with increasing knowledge, and a “knowledge deficit” hypothesis whereby the relation is the opposite. In order to examine these hypotheses, graduate and undergraduate students were asked to state what they knew and what they did not know about a sample of familiar and unfamiliar artifacts. None of the above hypotheses accounted for the results. Instead, knowledge was found to be differently related to various types of unknown features: increasing knowledge was related to less unknown features of the artifacts' function and to more unknown features about contingency relations. Unknown features of the artifacts’ behaviors were frequently reported in the two knowledge conditions. The results suggest new strands of research on the metacognition of “not knowing.”

Defective E2 electrode lead gives low-amplitude compound muscle action potential.

Low-amplitude compound muscle action potential (CMAP) suggests a neuromuscular pathology. Low amplitude will also result from a defective E1 electrode or its lead, that is, a technical artifact. The aim of this study was to investigate the effect of a defective E2 electrode lead on the CMAP. The CMAP was recorded using standard nerve conduction methodology and all electrode leads connected properly. Signals were then recorded when either the E1 or the E2 electrode lead was disconnected from the amplifier. This simulated a defective electrode lead. Studies were performed in four nerves of a healthy subject. CMAP amplitude was reduced as expected when E1 was disconnected. Surprisingly, the amplitude fell by more than 65% when the E2 lead was disconnected, although E1 was properly connected. E1 and E2 electrodes contribute to the CMAP. A defective recording electrode lead to E1 or E2 results in a low-amplitude CMAP. The amplitude drop observed with a disconnected E2 lead was far greater than the signal recorded by the E2 electrode. This occurs due to the amplifier's inherent property to reduce the voltage difference between the E1 and E2 inputs. When E2 lead is defective, the CMAP will be an attenuated version of the signal recorded by the E1 electrode, and vice versa. When low-amplitude CMAP amplitude is observed in all conduction studies, technical artifact should be considered before exploring the pathological basis for the abnormal results.

Single-cell RNA sequencing reveals transcriptional changes of human choroidal and retinal pigment epithelium cells during fetal development, in healthy adult and intermediate age-related macular degeneration.

Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the developed world. Vision loss in the advanced stages of the disease is caused by atrophy of retinal photoreceptors, overlying retinal pigment epithelium (RPE) and choroidal endothelial cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression to intermediate and advanced stages AMD are not yet fully understood. We performed single-cell RNA-sequencing (RNA-Seq) of human fetal and adult RPE-choroidal tissues, profiling in detail all the cell types and elucidating cell type-specific proliferation, differentiation and immunomodulation events that occur up to midgestation. Our data demonstrate that progression from the fetal to adult state is characterized by an increase in expression of genes involved in the oxidative stress response and detoxification from heavy metals, suggesting a better defence against oxidative stress in the adult RPE-choroid tissue. Single-cell comparative transcriptional analysis between a patient with intermediate AMD and an unaffected subject revealed a reduction in the number of RPE cells and melanocytes in the macular region of the AMD patient. Together these findings may suggest a macular loss of RPE cells and melanocytes in the AMD patients, but given the complex processing of tissues required for single-cell RNA-Seq that is prone to technical artefacts, these findings need to be validated by additional techniques in a larger number of AMD patients and controls.

High performance clean versus artifact dry electrode EEG data classification using Convolutional Neural Network transfer learning

ObjectiveConvolutional Neural Networks (CNNs) are promising for artifact detection in electroencephalography (EEG) data, but require large amounts of data. Despite increasing use of dry electrodes for EEG data acquisition, dry electrode EEG datasets are sparse. We aim to develop an algorithm for clean versus artifact dry electrode EEG data classification using transfer learning. MethodsDry electrode EEG data were acquired in 13 subjects while physiological and technical artifacts were induced. Data were per 2-second segment labeled as clean or artifact and split in an 80% train and 20% test set. With the train set, we fine-tuned a pre-trained CNN for clean versus artifact wet electrode EEG data classification using 3-fold cross validation. The three fine-tuned CNNs were combined in one final clean versus artifact classification algorithm, in which the majority vote was used for classification. We calculated accuracy, F1-score, precision, and recall of the pre-trained CNN and fine-tuned algorithm when applied to unseen test data. ResultsThe algorithm was trained on 0.40 million and tested on 0.17 million overlapping EEG segments. The pre-trained CNN had a test accuracy of 65.6%. The fine-tuned clean versus artifact classification algorithm had an improved test accuracy of 90.7%, F1-score of 90.2%, precision of 89.1% and recall of 91.2%. ConclusionsDespite a relatively small dry electrode EEG dataset, transfer learning enabled development of a high performing CNN-based algorithm for clean versus artifact classification. SignificanceDevelopment of CNNs for classification of dry electrode EEG data is challenging as dry electrode EEG datasets are sparse. Here, we show that transfer learning can be used to overcome this problem.

Немое кино как медиа-археология: подходы к конструированию магистерского курса

The article presents a methodological elaboration of a syllabus for a university course. The purpose of the course and this presentation is to offer a new focus for teaching the theory and history of silent film within the framework of New Film History concepts. The article is addressed to the expert community of historians, film critics, media researchers, archivists and future specialists in these fields. As a consequence of its rediscovery, early cinema is today an important construct of historical and film studies knowledge. And early cinema itself, with its non-linear development, forgotten histories of color and “talking” films, and huge screens, requires new historiographical models. The dossier of early cinema pushes to investigate the filming schemes, screen technologies, and aspects of music, noise, and verbal accompaniment, as well as draws attention to the potential of film tape restoration. In addition to the traditional teaching methods including lectures and seminars with discussion of theoretical and historiographical texts, the course engages students in doing research using little-studied written sources and technical artifacts available in archives and museum collections. In the cases of Perm, these include materials deposited at the Perm Museum of Local Lore, the Perm State Archive, and the Perm Cinematheque. Students get acquainted with the latest methods of attribution of film archival documents, celluloid, samples of film equipment and film carriers, the vocabulary of archivists and film historians. As a result, students are introduced to current research practices, new horizons of studying the history of cinema through the prism of technical advances and technological experiments. The course attunes the future specialists to the values of the obsolete, the forgotten, and, thanks to new cultural histories, sets a precedent for a conceptual and practical exercise in identifying the facets of contemporary media.

A Naturalistic View of the Technical Artifacts

This paper suggests revising the notion of in consideration of the naturalist position. I analyze whether the characterizations of technical artifacts proposed by the philosophy of technology can be extended to include the technical creations of other organisms. This will be done using the theories of “niche construction” and “organisms as ecosystem engineers.” Those theories would allow us to understand human technical creations within what human beings do naturally and in gradual continuity with what other species do.

Information systems, sociomaterial practices and the emergence of environmental management infrastructures

Information systems are an integral part of environmental management infrastructures – complex assemblages of social and technical artifacts, human actors and sociomaterial routines enacted in the pursuit of environmental sustainability outcomes. We analyse the case of an environmental management infrastructure developed around a ‘vessel management system’ by a South Australian regulator between 2004 and 2013. The goal of this infrastructure was to sustain water quality by controlling the discharge of ‘greywater’ from vessels on South Australia’s inland waters. We conceptualise environmental management infrastructure, and the information systems they encompass, as the temporally emergent outcome of human agents’ attempts to extend their environmental management practice in a particular direction, and how that trajectory of emergence is shaped by the intersection of human agencies, material performances and disciplinary practices.

From tool to tool-making: Reflections on authorship in social media research software

Social media research software has come to play increasingly important roles in processes of knowledge production. While epistemological, logistical, legal, and ethical concerns put the spotlight on the software tools researchers are relying on, little attention is paid to the role of the ‘toolmaker’ beyond a vague idea of the ‘power’ wielded by those who design, develop, and maintain these technical artifacts. This paper seeks to address this role, both conceptually and with attention to practical concerns, as a form of hybrid and relational authorship. We thereby shift the focus from tool to tool-making, from artifact to practice, in an attempt to produce a different kind of ‘unblackboxing’ of tools than the somewhat overused tropes of open source code or open data. Our contribution proceeds in three steps. We first address tools and tool-making from a theoretical perspective, suggesting that their epistemological orientation reaches more deeply into the networks of research practice than words like ‘bias’ admit and proposing to consider the specific kind of hybrid authorship that emerges in this context. Calling on our own experiences as toolmakers, we then reflect on a cluster of issues where this authorial function becomes particularly visible. Here, we examine how motivations and commitments orient what a piece of software does and how it does it and discuss tool-making from the perspectives of co-development, maintenance and care, and ethics by design. We conclude by arguing that the most pressing concerns for tool-making lie in institutional arrangements that are crucial for the life of research software.

THE ROLE OF MAGNETIC RESONANCE IMAGING IN THE EVALUATION OF TRAUMATIC INJURIES OF ANKLE JOINT

Background: Because of its wide array of advantages, MRI has gained popularity in evaluation of the musculoskeletal system. The advantages include non-invasiveness, lack of ionizing radiations, superior soft-tissue imaging and direct multiplanar imaging. Excellent visualization of cartilage, ligaments, tendons and bones can be achieved by the high resolution images of MRI. MRI can provide a comprehensive evaluation of ankle, including soft tissue and bone pathology. The soft tissue contrast resolution of MRI is superior to that of CT, and MRI is as good as or better than CT for most bone pathology. MRI is more sensitive than CT in the detection of stress fractures and trabecular bone injury.[4] Aim & Objective: 1.To study the normal anatomy of ankle joint on MRI.2 To assess acute traumatic injury patterns of ankle joint.3.To study the role of MRI in the evaluation of traumatic injuries of ankle joint..4 To study the chronic sequelae of ankle injuries. Methods: Study setting: Cross sectional study Tertiary care center. Study duration: 2 years ( from October 2019 to March 2021). Study population: The study population included all the cases with traumatic ankle injury referred to the Dept. of Radiodiagnosis our institute for MRI scan with or without X-ray ndings admitted at a tertiary care center such cases were included in the study. 50 Majority of cases found in 20-30 years age group 18( 36% Sample size: Results: ) followed by 40-50 group 12 cases (24%), 9 cases in 30-40, 7 cases in >50 and 4 cases in 10-20. Males contributing 34 (68%) and females 16 (32%). Deltoid ligament injuries are observed in 12 patients. However, isolated injuries are not seen. Deltoid ligament injuries are associated with bone, other ligamentous and tendon injuries. Anterior compartment tendon injuries were not observed in this study. 16 patients had a history of acute inversion injury. MRI plays an important role Conclusions: for planning further management and to decide whether the patient will require conservative or surgical management. Even though anatomical variants and technical artefacts can mimic a tear on MRI, it is still considered the primary imaging modality for optimal depiction of internal derangement in ankle injury. Common injuries observed in our study are - anterior talo- bular ligament injury, deltoid ligament injury, tenosynovitis, bone contusions and joint effusion.

Evidence of somatic hypermutation in the antigen binding sites of patients with CLL harboring IGHV genes with 100% germline identity.

Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a 'truly unmutated' CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3'IGHV, IGHD, 5'IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.

The Circulation of Morphological Knowledge: Understanding “Form” across Disciplines in the Twentieth and Twenty-First Centuries

This essay pushes the history of a scientific discipline, morphology, toward a broader philosophically informed and cross-disciplinarily engaged history of knowledge. It shows that by looking at how knowledge and practices circulated between scientific disciplines (such as biology) and technoscientific ones (like architecture and design) we can better understand how (morphological) knowledge was produced. By doing so, the analysis contributes to the study of the mechanisms of knowledge exchange between the organic and the technical worlds and, more broadly, to the study of the dynamics of knowledge circulation. First, the essay analyzes the engineering studies of organic form conducted by the Austro-Hungarian botanist Raoul Heinrich Francé (1874–1943) and the Scottish polymath D’Arcy Wentworth Thompson (1860–1948). Subsequently, the essay moves to twenty-first-century morphogenesis. It examines the transfer of the notion of morphospace, a paleontological practice of data visualization, into biology-inspired architectural design. The conclusion deals with the broader features of knowledge transfer and circulation. Twentieth- and twenty-first-century morphologists successfully translate and transfer morphogenetic processes from nature into technical artifacts to achieve technical solutions to given problems. In the translation of organic morphogenesis into a technical process, evolution’s intrinsic historical contingency was deliberately left behind.

HRD-related morphology discovery in breast cancer by controlling for confounding factors

Lazard etal.1 predict homologous recombination deficiency from hematoxylin and eosin-stained slides of breast cancer tissue using deep learning. By controlling for technical artifacts on a curated dataset, the model puts forward novel HRD-related morphologies in luminal breast cancers.

Regulatory Artifacts: Prescribing, Constituting, Steering

Generally, when thinking of artifacts, one imagines “technical artifacts”. Technical artifacts are those artifacts that perform a mere causal function. Their purpose is to instrumentally help and support an action, not to change behaviour. However, technical artifacts do not exhaust the set of artifacts. Alongside technical artifacts there are also artifacts that we can call “cognitive artifacts”. Cognitive artifacts are all those artifacts that operate upon information in order to improve human cognitive performances. Artifacts of a further, different kind are what we may call “regulatory artifacts”; that is, material artifacts devised and made to regulate behaviour. Consider a roundabout, a traffic light or a speed bump. These artifacts do not make us stronger, faster, or more intelligent. They are placed on the road surface to regulate traffic. This article investigates artifacts of this third kind and, especially, the functions that they perform.

A machine learning framework for scRNA-seq UMI threshold optimization and accurate classification of cell types.

Recent advances in single cell RNA sequencing (scRNA-seq) technologies have been invaluable in the study of the diversity of cancer cells and the tumor microenvironment. While scRNA-seq platforms allow processing of a high number of cells, uneven read quality and technical artifacts hinder the ability to identify and classify biologically relevant cells into correct subtypes. This obstructs the analysis of cancer and normal cell diversity, while rare and low expression cell populations may be lost by setting arbitrary high cutoffs for UMIs when filtering out low quality cells. To address these issues, we have developed a novel machine-learning framework that: 1. Trains cell lineage and subtype classifier using a gold standard dataset validated using marker genes 2. Systematically assess the lowest UMI threshold that can be used in a given dataset to accurately classify cells 3. Assign accurate cell lineage and subtype labels to the lower read depth cells recovered by setting the optimal threshold. We demonstrate the application of this framework in a well-curated scRNA-seq dataset of breast cancer patients and two external datasets. We show that the minimum UMI threshold for the breast cancer dataset could be lowered from the original 1500 to 450, thereby increasing the total number of recovered cells by 49%, while achieving a classification accuracy of >0.9. Our framework provides a roadmap for future scRNA-seq studies to determine optimal UMI threshold and accurately classify cells for downstream analyses.

Genomic Landscaping of Post-Transplant Lymphoproliferative Disorders Using Circulating Tumor DNA

Introduction: Identification of post-transplant lymphoproliferative disorder (PTLD) is a diagnostic challenge. The current diagnostic framework usually require analysis on tissue and imaging through PET/CT scans. Yet, these methods have diagnostic limitations. For instance due to missing information on tumor heterogeneity and relatively low sensitivity of PET/CT scans. Measuring cell free DNA (cfDNA) represents a novel, minimally-invasive method for tumor detection and genotyping. In this study we explored the feasibility of plasma cfDNA tumor genotyping with targeted deep sequencing to detect copy number variation (CNV), single nucleotide variants (SNVs) and Epstein Bar Virus (EBV) sequences, using a new in-house variant calling pipeline. Methods: In this multi-center observational cohort study of 18 patients diagnosed with monomorphic PTLD, we performed deep targeted NGS for SNV detection and low-coverage whole genome sequencing (lcWGS) for CNV detection. NGS was targeted on a panel of genes (n = 39) known to be recurrently mutated in B-cell lymphomas (244 kbp). All patients gave written informed consent. The R package CNAclinic was used to identify genome wide CNAs and fraction of the genome altered (FGA). The ichorCNA package was used to determine the ctDNA fraction in cfDNA samples. SNVs were called using a pipeline, combining 4 (cfDNA) SNV calling tools: Mutect2, LoFreq, VarDict & SiNVICT. Variants were filtered on presumed functional effect (Synonymous-, UTR-, Intron-, 2 kb upstream-, 2 kb downstream variants were removed). A "panel of normals” (variant blacklist) was generated from variants found in cfDNA from healthy controls, which was used to produce a variant blacklist to filter out technical artifacts and SNPs. Finally, variants with a variant allele frequency (VAF) < 1% and variants with an allele frequency (AF) of > 1% in 1000Genomes and/or gnomAD were filtered out. Results: The cohort included 18 PTLD patients. The majority of patients had stage IV disease (n=13, 76%). All of the PTLD cases were histologically proven, of diffuse large B cell lymphoma type and 10/18 patients (55%) were considered to have an EBV-negative tumor. EBV copy number in plasma was performed by qPCR in 12 patients and was considered to be elevated in four patients (>5000 copies/mL). The fraction of reads mapping to the EBV genome was compared between controls (n=8), EBV-negative PTLD (n=10) and EBV-positive PTLD (n=8). The median fraction of EBV reads for controls and EBV-negative PTLD were similar with a fraction of 1.5x10-4 (Q1-Q3: 1.3x10-4-1.6x10-4) and 1.8x10-4 (Q1-Q3: 1.4x10-4-3.9x10-4), respectively (p>0.05, Kruskal-Wallis). In EBV-positive tumors, the median fraction of EBV reads was 3-log higher, 5.3x10-1 (Q1-Q3: 7.3 x10-2-2) (p≤0.05, Kruskal-Wallis) CNVs were detected in cfDNA of 11/18 (61%) patients. Two of the patients in whom no CNVs were detected had initiated therapy at the time of plasma sampling. Median fraction of genome altered was higher in EBV-negative tumors (0.03, Q1-Q3: 2.5x10-4-0.12) than in EBV-positive tumors (5x10-4, Q1-Q3: 0-0.03), but this difference was not significant (p>0.05, Mann-Whitney U) (Figure 1). In the top 15 recurrently mutated genes (described below), across all 18 samples, 180 SNVs were found in total. The median amount of SNVs found in each sample was 11 (range 6-25). The most frequently mutated gene was CREPPB (12/18, 67%), in which the most prevalent mutation was p.K1497T (8/18, 44%). ARID1A was mutated in 10/18 cases (56%), followed by KMT2D (9/18, 50%). TET2, TP53, MEF2B & TNFAIP3 were all mutated in 7/18 cases (39%). SPEN & IRF2BP2 were mutated in 6/18 cases (33%). PIM1, NOTCH1, ATM, CARD11, DDX3X, IGLL5 were all mutated in 5/18 cases (28%) (Figure 2). Few of the mutations that are called in the top 15 recurrently mutated genes (8/180, 0.04%) lay close to 50% VAF, which might imply that these are SNPs not filtered out by the pipeline. Our SNV findings are comparable with previous literature, in which KMT2D, TET2 & TP53 are also amongst top-mutated genes (Menter et al., 2017), highlighting disruption of epigenetic regulation, NFkB pathway, and DNA damage response as drivers of PTLD. Conclusion: lcWGS in combination deep targeted sequencing allows for detection of CNV an SNV in diagnostic samples of patients with monomorphic PTLD. These data can potentially be used at screening, diagnosis and response monitoring, which is part of the ongoing prospective study NTR 7402. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RUNX1 Mutated AML and MDS: Similarities, Differences and Molecular Factors Leading to Disease Progression

Background: Mutations in the myeloid transcription factor RUNX1 are frequently detected in AML and MDS. Differences have been described regarding additional cytogenetic and molecular genetic abnormalities, however the molecular factors responsible for progression of RUNX1 mutated MDS to AML are yet not fully elucidated. Aim: Analysis of genetic (1) similarities and (2) differences of RUNX1 mutated AML and MDS cases, (3) identification of factors potentially causing disease progression from RUNX1 mutated MDS to AML. Methods: Whole-genome sequencing (WGS) was performed for 773 AML and 747 MDS patients (median coverage 100x). 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). As no sample specific normal tissue was available, a so-called Tumor/Unmatched normal (TUN) workflow was used to reduce technical artefacts and germline calls. All reported p-values are two-sided and were considered significant at p<0.05. Results: Mutations in RUNX1 (RUNX1mut) were detected at comparable frequencies in AML and MDS (AML: 97/773 cases, 13%; MDS: 71/747, 10%). In MDS, an association of the RUNX1mut frequency with higher blast counts was observed (0-5% blasts: 15/419 with RUNX1mut, 4%; ≥5,<10%: 16/175, 9%; ≥10,<20%: 40/153, 26%). However, blast count did not influence overall survival (OS) in RUNX1mut subgroups (0-5% blasts: 18 months (m); ≥5,<10%: 25 m; ≥10,<20%: 18 m; AML: 21 m). The variant allele frequency (VAF) of RUNX1mut differed significantly dependent on the blast count (median VAF in cases <5% blasts: 0.22; ≥5%: 0.42; p<0.001). Regarding cytogenetic abnormalities, RUNX1mut MDS showed a higher frequency of normal karyotype (NK) compared to AML (66% vs. 38%, p<0.001) but similar frequencies of trisomies (18% vs. 22%) and complex karyotype (3% vs. 5%), although differences regarding the trisomy type were detected (MDS: only trisomy 8; AML: trisomies 8, 11 and 13). In 10% of AML cases with RUNX1mut a rearrangement involving MECOM was detected (5% GATA::MECOM, 5% other MECOM rearrangements), which was absent in MDS with RUNX1mut. Similarly, KMT2A-PTD and FLT3-ITD alterations were detected in AML but not in MDS with RUNX1mut (KMT2A-PTD: 7% in AML vs. 0% in MDS, p=0.021; FLT3-ITD: 13% vs. 0%, p=0.001). By contrast, TP53mut were found at comparably low frequencies (MDS: 4/71, 6%; AML: 3/97, 3%). For both AML and MDS, a strong association of RUNX1mut with mutations in spliceosome genes (SF3B1, SRSF2, U2AF1, ZRSR2) was detected (AML: 58/97 = 60% in RUNX1mut vs. 139/676 = 21% in RUNX1wild type (wt); p<0.001; MDS: 52/71 = 73% vs. 346/676 = 51%, p<0.001). However, while SRSF2mut in MDS were associated with increasing blast counts similar to RUNX1mut (0-5% blasts: 25/419 with SRSF2mut, 6%; ≥5,<10%: 130/175, 17%; ≥10,<20%: 50/153, 32%), SF3B1mut were negatively correlated to blast counts and thus to RUNX1mut (0-5% blasts: 179/419, 43%; ≥5,<10%: 28/175, 16%; ≥10,<20%: 11/153, 7%). In the total AML cohort, in 24/773 of cases a CN-LOH involving RUNX1 was detected, 21 of these also showed a RUNX1mut (22% (21/97) of RUNX1mut cases). By contrast, in the total MDS cohort in only 2/747 cases a CN-LOH covering RUNX1 was found, none of them harboring a RUNX1mut. The VAF of cases with RUNX1mut + CN-LOH (RUNX1mutLOH) was >0.5 for all cases and was significantly higher than the VAF of RUNX1mut cases without CN-LOH (0.89 (range 0.54-0.97) vs. 0.42 (range 0.05-0.85), p<0.001). Moreover, cases with RUNX1mutLOH were found to be significantly associated with FLT3-ITD (RUNX1mutLOH, 6/21 = 29%; RUNX1mut without CN-LOH, 7/76 = 9%; p=0.032). Conclusions: (1) RUNX1mut occur at similar frequencies in AML and MDS and genetically show an MDS signature, reflected e.g. by association with mutations in spliceosome genes. OS is similar in all subgroups independent of blast counts. (2) In MDS, RUNX1mut are associated with higher blast counts, thus potentially functioning as driver towards progression to AML. (3) Additional cytogenetic/molecular genetic alterations that might function as progression factors from RUNX1mut MDS to AML include: (i) karyotype, especially MECOM rearrangements, trisomy 11 and trisomy 13 (ii) KMT2A-PTD, (iv) FLT3-ITD, (v) and CN-LOH covering RUNX1 (Figure). (4) By contrast, TP53mut do not seem to play a role in progression of RUNX1 mutated MDS to AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal