Longitudinal Multi-omics Analyses Identify Responses of Megakaryocytes, Erythroid Cells, and Plasmablasts as Hallmarks of Severe COVID-19.

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.

Carbonate-rich crust subduction drives the deep carbon and chlorine cycles.

The flux balances of carbon and chlorine between subduction into the deep mantle and volcanic emissions into the atmosphere are crucial for the habitability of our planet1,2. However, pervasive loss of fluids from subducting slabs has been thought to cut off the delivery of both carbon and chlorine to the deep mantle owing to their high mobility under hydrous conditions3,4. Our new high-pressure experiments show that most carbonates (>75 wt%) in carbonate-rich crustal rocks-one of the main subducting carbon reservoirs-survive devolatilization and hydrous melting in cold and warm subduction zones, indicating that their subduction has driven the deep carbon cycle since the Mesoproterozoic. We found that KCl and NaCl, respectively, become stable phases crystallizing from hydrous carbonatite melts with low chlorine solubility in warm and hot subduction zones, resulting in the sequestration of chlorine in the solid residue in downwelling slabs. Accordingly, the subduction of carbonate-rich rocks facilitated highly effective recycling of both chlorine and carbon into the deep mantle at intermediate stages of Earth's history and led to declining atmospheric pCO2 and the formation of carbon-rich and chlorine-rich mantle reservoirs since the Mesoproterozoic. This period of optimal carbon and chlorine subduction may explain the ages of eclogitic diamonds and the formation of the HIMU mantle source.

Pharmacologic fibroblast reprogramming into photoreceptors restores vision.

Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision.

A critical spotlight on the paradigms of FFPE-DNA sequencing.

In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.

Phylogenetic network analysis of SARS-CoV-2 genomes.

In a phylogenetic network analysis of 160 complete human severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) genomes, we find three central variants distinguished by amino acid changes, which we have named A, B, and C, with A being the ancestral type according to the bat outgroup coronavirus. The A and C types are found in significant proportions outside East Asia, that is, in Europeans and Americans. In contrast, the B type is the most common type in East Asia, and its ancestral genome appears not to have spread outside East Asia without first mutating into derived B types, pointing to founder effects or immunological or environmental resistance against this type outside Asia. The network faithfully traces routes of infections for documented coronavirus disease 2019 (COVID-19) cases, indicating that phylogenetic networks can likewise be successfully used to help trace undocumented COVID-19 infection sources, which can then be quarantined to prevent recurrent spread of the disease worldwide.

GSK3α Regulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells.

Amino acid availability is crucial for cancer cells' survivability. Leukemia and colorectal cancer cells have been shown to resist asparagine depletion by utilizing GSK3-dependent proteasomal degradation, termed the Wnt-dependent stabilization of proteins (Wnt/STOP), to replenish their amino acid pool. The inhibition of GSK3α halts the sourcing of amino acids, which subsequently leads to cancer cell vulnerability toward asparaginase therapy. However, resistance toward GSK3α-mediated protein breakdown can occur, whose underlying mechanism is poorly understood. Here, we set out to define the mechanisms driving dependence toward this degradation machinery upon asparagine starvation in cancer cells. We show the independence of known stress response pathways including the integrated stress response mediated with GCN2. Additionally, we demonstrate the independence of changes in cell cycle progression and expression levels of the asparagine-synthesizing enzyme ASNS. Instead, RNA sequencing revealed that GSK3α inhibition and asparagine starvation leads to the temporally dynamic downregulation of distinct ribosomal proteins, which have been shown to display anti-proliferative functions. Using a CRISPR/Cas9 viability screen, we demonstrate that the downregulation of these specific ribosomal proteins can rescue cell death upon GSK3α inhibition and asparagine starvation. Thus, our findings suggest the vital role of the previously unrecognized regulation of ribosomal proteins in bridging GSK3α activity and tolerance of asparagine starvation.

Assessing the Phenotype of a Homologous Recombination Deficiency Using High Resolution Array-Based Comparative Genome Hybridization in Ovarian Cancer.

Ovarian cancer (OC) cells with homologous recombination deficiency (HRD) accumulate genomic scars (LST, TAI, and LOH) over a value of 42 in sum. PARP inhibitors can treat OC with HRD. The detection of HRD can be done directly by imaging these genomic scars, or indirectly by detecting mutations in the genes involved in HR. We show that HRD detection is also possible using high-resolution aCGH. A total of 30 OCs were analyzed retrospectively with high-resolution arrays as a test set and 19 OCs prospectively as a validation set. Mutation analysis was performed by HBOC TruRisk V2 panel to detect HR-relevant mutations. CNVs were clustered with respect to the involved HR genes versus the OC cases. In prospective validation, the HRD status determined by aCGH was compared with external HRD assessments. Two BRCA mutation carriers did not have HRD. OC could approximately differentiate into two groups with characteristic CNV patterns with different survival rates. Mutation frequencies have a linear regression on the HRD score. Mutations in individual HR-relevant genes do not always indicate HRD. This may depend on the mutation frequency in tumor cells. The aCGH shows the genomic scars of an HRD inexpensively and directly.

Combining Solid and Liquid Biopsy for Therapy Monitoring in Esophageal Cancer.

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53, SMAD4, TSHZ3, and SETBP1, with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.

Thin Polymer Films by Oxidative or Reductive Electropolymerization and Their Application in Electrochromic Windows and Thin-Film Sensors.

Electrically conducting and semiconducting polymers represent a special and still very attractive class of functional chromophores, especially due to their unique optical and electronic properties and their broad device application potential. They are potentially suitable as materials for several applications of high future relevance, for example flexible photovoltaic modules, components of displays/screens and batteries, electrochromic windows, or photocatalysts. Therefore, their synthesis and structure elucidation are still intensely investigated. This article will demonstrate the very fruitful interplay of current electropolymerization research and its exploitation for science education issues. Experiments involving the synthesis of conducting polymers and their assembly into functional devices can be used to teach basic chemical and physical principles as well as to motivate students for an innovative and interdisciplinary field of chemistry.

The Influence of the Side Chain Structure on the Photostability of Low Band Gap Polymers.

Side chains play an important role in the photo-oxidation process of low band gap (LBG) polymers. For example, it has been shown that their photostability can be increased by the introduction of aromatic-oxy-alkyl links. We studied the photostability of prototypical LBG polymers with alkyl and oxyalkyl side chains during irradiation with white light (AM 1.5 conditions) in dry air using UV/vis and IR spectroscopy. Though its degradation kinetics were distinctly affected by the presence or absence of oxygen in the structure of the side chains, in particular cases, the stability was more affected by the presence of linear or branched side chains. Moreover, we showed that the exact position of the alkyl/oxyalkyl side chain at the polymer backbone could be crucial. Although minor effects of chemical modifications on the electronic parameters (ionization potential and gap) were observed, the molecular orientation, determined by polarization modulation-infrared reflection-absorption spectroscopy (PMIRRAS), could be affected. The aggregation and crystallinity of these polymers may distinctly affect their stability.

Influence of Backbone Ladderization and Side Chain Variation on the Orientation of Diketopyrrolopyrrole-Based Donor-Acceptor Copolymers.

Ladder polymers with poly(diketopyrrolopyrrole) (DPP) moieties have recently attracted enormous interest for a large variety of opto-electronic applications. Since the rigidity of the backbone increases with ladderization, a strong influence on the self-organization of thin films is expected. We study the molecular orientation of DPP-based ladder polymers in about 50 nm thin films using polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS). Exemplarily, for one polymer, the orientation in thicker films is qualitatively investigated by infrared spectroscopy in transmission. Further, this method allows us to rule out the effects of a possible azimuthal ordering, which would affect the analysis of the orientation by PM-IRRAS. For all polymers, the long axis of the polymer backbone is preferentially oriented parallel to the substrate surface, pointing to a high degree of ordering. It is suggested that the choice of the side chains might be a promising way to tune for face-on and edge-on orientations. The exemplarily performed investigation of interface properties on substrates with different work functions suggests that the choice of the side chains has a minor effect on the interfacial electronic interface structure.

Modelling Green Volume Using Sentinel-1, -2, PALSAR-2 Satellite Data and Machine Learning for Urban and Semi-Urban Areas in Germany.

Urban Green Infrastructure (UGI) provides ecosystem services such as cooling of temperatures and is majorly important for climate change adaptation. Green Volume (GV) describes the 3-D space occupied by vegetation and is highly useful for the assessment of UGI. This research uses Sentinel-2 (S-2) optical data, vegetation indices (VIs), Sentinel-1 (S-1) and PALSAR-2 (P-2) radar data to build machine learning models for yearly GV estimation on large scales. Our study compares random and stratified sampling of reference data, assesses the performance of different machine learning algorithms and tests model transferability by independent validation. The results indicate that stratified sampling of training data leads to improved accuracies when compared to random sampling. While the Gradient Tree Boost (GTB) and Random Forest (RF) algorithms show generally similar performance, Support Vector Machine (SVM) exhibits considerably greater model error. The results suggest RF to be the most robust classifier overall, achieving highest accuracies for independent and inter-annual validation. Furthermore, modelling GV based on S-2 features considerably outperforms using only S-1 or P-2 based features. Moreover, the study finds that underestimation of large GV magnitudes in urban forests constitutes the biggest source of model error. Overall, modelled GV explains around 79% of the variability in reference GV at 10 m resolution and over 90% when aggregated to 100 m resolution. The research shows that accurately modelling GV is possible using openly available satellite data. Resulting GV predictions can be useful for environmental management by providing valuable information for climate change adaptation, environmental monitoring and change detection.

Behavioral pharmacology of five novel synthetic cannabinoids.

Newly emerging synthetic cannabinoid compounds continue to be found in the designer drug market. They are often targeted as a 'legal high' alternative to traditional cannabinoids via 'darknet' markets and their increased potency and efficacy are becoming a growing concern internationally. The purpose of this study was to determine whether 4-CN-CUMYL-BUTINACA, 4F-MDMB-BINACA, 5F-AEB, 5F-CUMYL-P7AICA and EMB-FUBINACA exhibited similar behavioral effects as Δ9-tetrahydrocannabinol (Δ9-THC). Locomotor activity was assessed in an open-field assay using Swiss-Webster mice. Male Sprague-Dawley rats were trained to discriminate between intraperitoneal injections of Δ9-THC (3 mg/kg) and vehicle. Following successful training, substitution tests for 4-CN-CUMYL-BUTINACA, 4F-MDMB-BINACA, 5F-AEB, 5F-CUMYL-P7AICA and EMB-FUBINACA were conducted. All of the test compounds decreased locomotor activity. 4-CN-CUMYL-BUTINACA (ED50 = 0.26 mg/kg), 4F-MDMB-BINACA (ED50 = 0.019 mg/kg), 5F-CUMYL-P7AICA (ED50 = 0.13 mg/kg) and EMB-FUBINACA (ED50 = 0.13 mg/kg) each fully substituted for the discriminative stimulus effects of the training dose of Δ9-THC, whereas 5F-AEB produced only a maximum of 67% drug-appropriate responding at 0.5 mg/kg. Higher doses produced piloerection, exophthalmos and convulsions. 4-CN-CUMYL-BUTINACA, 4F-MDMB-BINACA, 5F-CUMYL-P7AICA and EMB-FUBINACA are likely to produce similar subjective effects in humans as those produced by abused synthetic cannabinoids, and may therefore share similar abuse liability. In contrast, 5F-AEB may have a reduced abuse liability given its weaker THC-like discriminative stimulus effects but maybe more dangerous due to the adverse effects observed at doses needed to produce discriminative stimulus effects.

Modelling Fagus sylvatica stem growth along a wide thermal gradient in Italy by incorporating dendroclimatic classification and land surface phenology metrics.

Calibrating land surface phenology (LSP) with tree rings is important to model spatio-temporal variations in forest productivity. We used MODIS (resolution: 250 m) NDVI, WDRVI and EVI series 2000-2014 to derive LSP metrics quantifying phenophase timing and canopy photosynthetic rates of 26 European beech forests covering a large thermal gradient (5-16 °C) in Italy. Average phenophase timing changed greatly with site temperature (e.g. growing season 70 days longer at low- than high-elevation); average VI values were affected by precipitation. An annual temperature about 12 °C (c. 1100 m asl) represented a bioclimatic threshold dividing warm from cold beech forests, distinguished by different phenology-BAI (basal area increment) relationships and LSP trends. Cold forests showed decreasing VI values (browning) and delayed phenophases and had negative BAI slopes. Warmer forests tended to increase VI (greening), and positive BAI slopes. NDVI peak, commonly used in global trend assessments, changed with elevation in agreement with changes in wood production. A cross-validation modelling approach demonstrated the ability of LSP to predict average BAI and its interannual variability. Merging sites into bioclimatic groups improved models by amplifying the signal in growth or LSP. NDVI had highest performances when informing on BAI trends; WDRVI and EVI were mostly selected for modelling mean and interannual BAI. WDRVI association with tree rings, tested in this study for the first time, showed that this VI is highly promising for studying forest dynamics. MODIS LSP can quantify forest functioning changes across landscapes and model interannual spatial variations and trends in productivity dynamics under climate change.

Estimating soil moisture content under grassland with hyperspectral data using radiative transfer modelling and machine learning.

The monitoring of soil moisture content (SMC) at very high spatial resolution (<10m) using unmanned aerial systems (UAS) is of high interest for precision agriculture and the validation of large scale SMC products. Data-driven approaches are the most common method to retrieve SMC with UAS-borne data at water limited sites over non-disturbed agricultural crops. A major disadvantage of data-driven algorithms is the limited transferability in space and time and the need of a high number of ground reference samples. Physically-based approaches are less dependent on the amount of samples and are transferable in space and time. This study explores the potential of (1) a hybrid method targeting the soil brightness factor of the PROSAIL model using a variational heteroscedastic Gaussian Processes regression (VHGPR) algorithm, and (2) a data-driven method employing VHGPR for the retrieval of SMC over three grassland sites based on UAS-borne VIS-NIR (399-1001 nm) hyperspectral data. The sites were managed by mowing (Fendt), grazing (Grosses Bruch) and irrigation (Marquardt). With these distinct local pre-conditions we aimed to identify factors that favor and limit the retrieval of SMC. The hybrid approach presented encouraging results in Marquardt (RMSE = 1.5 Vol_%, R2 = 0.2). At the permanent grassland sites (Fendt, Grosses Bruch) the thatch layer jeopardized the application of the hybrid model. We identified the complex canopy structure of grassland as the main factor impacting the hybrid SMC retrieval. The data-driven approach showed high accuracy for Fendt (R2 = 0.84, RMSE = 8.66) and Marquardt (R2 = 0.4, RMSE = 10.52). All data-driven models build on the LAI-SMC relationship. However, this relationship was hampered by mowing (Fendt), leading to a lack of transferability in time. The alteration of plant traits by grazing prevents finding a relationship with SMC in Grosses Bruch. In Marquardt, we identified the timelag between changes in SMC and plant response as the main reason of decrease in model accuracy. Yet, the model performance is accurate in undisturbed and water-limited areas (Marquardt). The analysis points to challenges that need to be tackled in future research and opens the discussion for the development of robust models to retrieve high resolution SMC from UAS-borne remote sensing observations.

Genome-wide analysis of 944 133 individuals provides insights into the etiology of haemorrhoidal disease.

OBJECTIVE: Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date. DESIGN: We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry. RESULTS: We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix. CONCLUSION: HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.

Loss of MYSM1 inhibits the oncogenic activity of cMYC in B cell lymphoma.

MYSM1 is a chromatin-binding protein, widely investigated for its functions in haematopoiesis in human and mouse; however, its role in haematologic malignancies remains unexplored. Here, we investigate the cross-talk between MYSM1 and oncogenic cMYC in the transcriptional regulation of genes encoding ribosomal proteins, and the implications of these mechanisms for cMYC-driven carcinogenesis. We demonstrate that in cMYC-driven B cell lymphoma in mouse models, MYSM1-loss represses ribosomal protein gene expression and protein synthesis. Importantly, the loss of MYSM1 also strongly inhibits cMYC oncogenic activity and protects against B cell lymphoma onset and progression in the mouse models. This advances the understanding of the molecular and transcriptional mechanisms of lymphomagenesis, and suggests MYSM1 as a possible drug target for cMYC-driven malignancies.

Cationic Diazapentacenium Polymers Made in a Sequence of CN Cross Coupling Polymerization and Acid-Mediated Postpolymerization Cyclization.

Polycationic stepladder polymers containing 5,12-diazapentacenium bistriflate repeat units are made in a two-step sequence of a carbon-nitrogen cross coupling polymerization and subsequent postpolymerization cyclization. The deeply colored products show a rather weak conjugative interaction between the dicationic diazapentacenium repeat units along the polymer chains.

Behavioral effects of four novel synthetic cathinone analogs in rodents.

A new generation of novel cathinone compounds has been developed as stimulant substitutes to avoid drug control laws and detection of use by blood tests. Dipentylone, N-ethylhexedrone, 4-chloroethcathinone (4-CEC), and 4'-methyl-α-pyrrolidinohexiophenone (MPHP) were tested for in vivo psychostimulant-like effects to assess their abuse liability. Locomotor activity was assessed in an open-field assay using Swiss-Webster mice to screen for locomotor stimulant effects and to identify behaviorally-active dose ranges, times of peak effect, and durations of action. Discriminative stimulus effects were assessed in separate groups of Sprague-Dawley rats trained to discriminate cocaine or methamphetamine from vehicle. Dipentylone, N-ethylhexedrone, 4-CEC, and MPHP dose-dependently increased locomotor activity. Dipentylone, N-ethylhexedrone, and MPHP produced maximal stimulant effects similar to cocaine and methamphetamine. 4-CEC was less efficacious, producing peak stimulant effects of about 74% of that of methamphetamine. The compounds were less potent than methamphetamine and approximately equipotent with cocaine. The doses of cocaine, methamphetamine, dipentylone, and 4-CEC that produced peak effects lasted 2 to 3 h, the peak dose of N-ethylhexedrone lasted 4 h, and the peak dose of MPHP lasted 6 h. All four compounds fully substituted for the discriminative stimulus effects of methamphetamine and cocaine, although full substitution by 4-CEC occurred at doses that substantially decreased response rate. Only 4-CEC fully substituted for MDMA. These data provide evidence that the novel cathinone compounds dipentylone, N-ethylhexedrone, 4-CEC, and MPHP demonstrate potential for abuse as psychostimulants, given their ability to stimulate locomotor activity and their substitution for the discriminative stimulus effects of methamphetamine and cocaine.

Rapid response of stage IV colorectal cancer with APC/TP53/KRAS mutations to FOLFIRI and Bevacizumab combination chemotherapy: a case report of use of liquid biopsy.

BACKGROUND: Liquid biopsies of blood plasma cell free DNA can be used to monitor treatment response and potentially detect mutations that are present in resistant clones in metastatic cancer patients. CASE PRESENTATION: In our non-interventional liquid biopsy study, a male patient in his fifties diagnosed with stage IV colorectal cancer and polytope liver metastases rapidly progressed after completing chemotherapy and deceased 8 months after diagnosis. Retrospective cell free DNA testing showed that the APC/TP53/KRAS major clone responded quickly after 3 cycles of FOLFIRI + Bevacizumab. Retrospective exome sequencing of pre-chemotherapy and post-chemotherapy tissue samples including metastases confirmed that the APC/TP53/KRAS and other major clonal mutations (GPR50, SLC5A, ZIC3, SF3A1 and others) were present in all samples. After the last chemotherapy cycle, CT imaging, CEA and CA19-9 markers validated the cfDNA findings of treatment response. However, 5 weeks later, the tumour had rapidly progressed. CONCLUSION: As FOLFIRI+Bevacizumab has recently also been associated with sustained complete remission in a APC/TP53/KRAS triple-mutated patient, these driver genes should be tested and monitored in a more in-depth manner in future patients. Patients with metastatic disease should be monitored more closely during and after chemotherapy, ideally using cfDNA.