DszA Catalyzes C-S Bond Cleavage through N5-Hydroperoxyl Formation.

Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N5OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.

Ultra-sensitive CTC-based liquid biopsy for pancreatic cancer enabled by large blood volume analysis.

The limited sensitivity of circulating tumor cell (CTC) detection in pancreatic adenocarcinoma (PDAC) stems from their extremely low concentration in the whole circulating blood, necessitating enhanced detection methodologies. This study sought to amplify assay-sensitivity by employing diagnostic leukapheresis (DLA) to screen large blood volumes. Sixty patients were subjected to DLA, with a median processed blood volume of ~ 2.8 L and approximately 5% of the resulting DLA-product analyzed using CellSearch (CS). Notably, DLA significantly increased CS-CTC detection to 44% in M0-patients and 74% in M1-patients, yielding a 60-fold increase in CS-CTC enumeration. DLA also provided sufficient CS-CTCs for genomic profiling, thereby delivering additional genomic information compared to tissue biopsy samples. DLA CS-CTCs exhibited a pronounced negative prognostic impact on overall survival (OS), evidenced by a reduction in OS from 28.6 to 8.5 months (univariate: p = 0.002; multivariable: p = 0.043). Additionally, a marked enhancement in sensitivity was achieved (by around 3-4-times) compared to peripheral blood (PB) samples, with positive predictive values for OS being preserved at around 90%. Prognostic relevance of CS-CTCs in PDAC was further validated in PB-samples from 228 PDAC patients, consolidating the established association between CTC-presence and reduced OS (8.5 vs. 19.0 months, p < 0.001). In conclusion, DLA-derived CS-CTCs may serve as a viable tool for identifying high-risk PDAC-patients and aiding the optimization of multimodal treatment strategies. Moreover, DLA enables comprehensive diagnostic profiling by providing ample CTC material, reinforcing its utility as a reliable liquid-biopsy approach. This high-volume liquid-biopsy strategy presents a potential pathway for enhancing clinical management in this malignancy.

Engineering DszC Mutants from Transition State Macrodipole Considerations and Evolutionary Sequence Analysis.

We describe an approach to identify enzyme mutants with increased turnover using the enzyme DszC as a case study. Our approach is based on recalculating the barriers of alanine mutants through single-point energy calculations at the hybrid QM/MM level in the wild-type reactant and transition state geometries. We analyze the difference in the electron density between the reactant and transition state to identify sites/residues where electrostatic interactions stabilize the transition state over the reactants. We also assess the insertion of a unit probe charge to identify positions in which the introduction of charged residues lowers the barrier.

QM/MM Study of the Reaction Mechanism of Thermophilic Glucuronoyl Esterase for Biomass Treatment.

Hydrolysis of lignocellulosic biomass, composed of a lignin-carbohydrate-complex (LCC) matrix, is critical for producing bioethanol from glucose. However, current methods for LCC processing require costly and polluting processes. The fungal Thermothelomyces thermophila glucuronoyl esterase (TtGE) is a promising thermophilic enzyme that hydrolyses LCC ester bonds. This study describes the TtGE catalytic mechanism using QM/MM methods. Two nearly-degenerate rate-determining transition states were found, with barriers of 16 and 17 kcal ⋅ mol-1 , both with a zwitterionic nature that results from a proton interplay from His346 to either the Ser213-hydroxyl or the lignin leaving group and the rehybridisation of the ester moiety of the substrate to an alkoxide. An oxyanion hole, characteristic of esterases, was provided by the conserved Arg214 through its backbone and sidechain. Our work further suggests that a mutation of Glu267 to a non-negative residue will decrease the energetic barrier in ca. -5 kcal ⋅ mol-1 , improving the catalytic rate of TtGE.

Role of Enzyme and Active Site Conformational Dynamics in the Catalysis by α-Amylase Explored with QM/MM Molecular Dynamics.

We assessed enzyme:substrate conformational dynamics and the rate-limiting glycosylation step of a human pancreatic α-amylase:maltopentose complex. Microsecond molecular dynamics simulations suggested that the distance of the catalytic Asp197 nucleophile to the anomeric carbon of the buried glucoside is responsible for most of the enzyme active site fluctuations and that both Asp197 and Asp300 interact the most with the buried glucoside unit. The buried glucoside binds either in a 4C1 chair or 2SO skew conformations, both of which can change to TS-like conformations characteristic of retaining glucosidases. Starting from four distinct enzyme:substrate complexes, umbrella sampling quantum mechanics/molecular mechanics simulations (converged within less than 1 kcal·mol-1 within a total simulation time of 1.6 ns) indicated that the reaction occurrs with a Gibbs barrier of 13.9 kcal·mol -1, in one asynchronous concerted step encompassing an acid-base reaction with Glu233 followed by a loose SN2-like nucleophilic substitution by the Asp197. The transition state is characterized by a 2H3 half-chair conformation of the buried glucoside that quickly changes to the E3 envelope conformation preceding the attack of the anomeric carbon by the Asp197 nucleophile. Thermodynamic analysis of the reaction supported that a water molecule tightly hydrogen bonded to the glycosidic oxygen of the substrate at the reactant state (∼1.6 Å) forms a short hydrogen bond with Glu233 at the transition state (∼1.7 Å) and lowers the Gibbs barrier in over 5 kcal·mol-1. The resulting Asp197-glycosyl was mostly found in the 4C1 conformation, although the more endergonic B3,O conformation was also observed. Altogether, the combination of short distances for the acid-base reaction with the Glu233 and for the nucleophilic attack by the Asp197 nucleophile and the availability of water within hydrogen bonding distance of the glycosidic oxygen provides a reliable criteria to identify reactive conformations of α-amylase complexes.

Different Enzyme Conformations Induce Different Mechanistic Traits in HIV-1 Protease.

The influence of the dynamical flexibility of enzymes on reaction mechanisms is a cornerstone in biological sciences. In this study, we aim to 1) study the convergence of the activation free energy by using the first step of the reaction catalysed by HIV-1 protease as a case study, and 2) provide further evidence for a mechanistic divergence in this enzyme, as two different reaction pathways were seen to contribute to this step. We used quantum mechanics/molecular mechanics molecular dynamics simulations, on four different initial conformations that led to different barriers in a previous study. Despite the sampling, the four activation free energies still spanned a range of 5.0 kcal ⋅ mol-1 . Furthermore, the new simulations did confirm the occurrence of an unusual mechanistic divergence, with two different mechanistic pathways displaying equivalent barriers. An active-site water molecule is proposed to influence the mechanistic pathway.

Towards the Accurate Thermodynamic Characterization of Enzyme Reaction Mechanisms.

We employed QM/MM molecular dynamics (MD) simulations to characterize the rate-limiting step of the glycosylation reaction of pancreatic α-amylase with combined DFT/molecular dynamics methods (PBE/def2-SVP : AMBER). Upon careful choice of four starting active site conformations based on thorough reactivity criteria, Gibbs energy profiles were calculated with umbrella sampling simulations within a statistical convergence of 1-2 kcal ⋅ mol-1 . Nevertheless, Gibbs activation barriers and reaction energies still varied from 11.0 to 16.8 kcal ⋅ mol-1 and -6.3 to +3.8 kcal ⋅ mol-1 depending on the starting conformations, showing that despite significant state-of-the-art QM/MM MD sampling (0.5 ns/profile) the result still depends on the starting structure. The results supported the one step dissociative mechanism of Asp197 glycosylation preceded by an acid-base reaction by the Glu233, which are qualitatively similar to those from multi-PES QM/MM studies, and thus support the use of the latter to determine enzyme reaction mechanisms.

Genetic analysis of single disseminated tumor cells in the lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma.

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker-positive cells were isolated by micromanipulation followed by single-cell whole-genome amplification and metaphase-based comparative genomic hybridization (mCGH) to determine genome-wide copy number alterations. The findings were correlated with clinical parameters and follow-up data. We detected chromosomal aberrations in KRT18- and EpCAM-positive cells from both compartments; BM-derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow-up. Genomic profiling of BM-DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker-positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM-DTCs with high-resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer.

Image-Based Identification and Genomic Analysis of Single Circulating Tumor Cells in High Grade Serous Ovarian Cancer Patients.

In Ovarian Cancer (OC), the analysis of single circulating tumor cells (sCTCs) might help to investigate genetic tumor evolution during the course of treatment. Since common CTC identification features failed to reliably detect CTCs in OC, we here present a workflow for their detection and genomic analysis. Blood of 13 high-grade serous primary OC patients was analyzed, using negative immunomagnetic enrichment, followed by immunofluorescence staining and imaging for Hoechst, ERCC1, CD45, CD11b and cytokeratin (CK) and sCTC sorting with the DEPArrayTM NxT. The whole genome of single cells was amplified and profiled for copy number variation (CNV). We detected: Type A-cells, epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKpos); Type B-cells, potentially epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKneg) and Type C-cells, potentially mesenchymal (Hoechstpos, ERCC1pos, CD45neg, CD11bneg, CKneg). In total, we identified five (38.5%) patients harboring sCTCs with an altered CN profile, which were mainly Type A-cells (80%). In addition to inter-and intra-patient genomic heterogeneity, high numbers of Type B- and C-cells were identified in every patient with their aberrant character only confirmed in 6.25% and 4.76% of cases. Further identification markers and studies in the course of treatment are under way to expand sCTC analysis for the identification of tumor evolution in OC.

Animal Fatty Acid Synthase: A Chemical Nanofactory.

Fatty acids are crucial molecules for most living beings, very well spread and conserved across species. These molecules play a role in energy storage, cell membrane architecture, and cell signaling, the latter through their derivative metabolites. De novo synthesis of fatty acids is a complex chemical process that can be achieved either by a metabolic pathway built by a sequence of individual enzymes, such as in most bacteria, or by a single, large multi-enzyme, which incorporates all the chemical capabilities of the metabolic pathway, such as in animals and fungi, and in some bacteria. Here we focus on the multi-enzymes, specifically in the animal fatty acid synthase (FAS). We start by providing a historical overview of this vast field of research. We follow by describing the extraordinary architecture of animal FAS, a homodimeric multi-enzyme with seven different active sites per dimer, including a carrier protein that carries the intermediates from one active site to the next. We then delve into this multi-enzyme's detailed chemistry and critically discuss the current knowledge on the chemical mechanism of each of the steps necessary to synthesize a single fatty acid molecule with atomic detail. In line with this, we discuss the potential and achieved FAS applications in biotechnology, as biosynthetic machines, and compare them with their homologous polyketide synthases, which are also finding wide applications in the same field. Finally, we discuss some open questions on the architecture of FAS, such as their peculiar substrate-shuttling arm, and describe possible reasons for the emergence of large megasynthases during evolution, questions that have fascinated biochemists from long ago but are still far from answered and understood.

Generating human prostate cancer organoids from leukapheresis enriched circulating tumour cells.

BACKGROUND: Circulating tumour cell (CTC)-derived organoids have the potential to provide a powerful tool for personalised cancer therapy but are restrained by low CTC numbers provided by blood samples. Here, we used diagnostic leukapheresis (DLA) to enrich CTCs from patients with metastatic prostate cancer (mPCa) and explored whether organoids provide a platform for ex vivo treatment modelling. METHODS: We prospectively screened 102 patients with mPCa and performed DLA in 40 patients with ≥5 CTCs/7.5 mL blood. We enriched CTCs from DLA using white blood cell (WBC) depletion alone or combined with EpCAM selection. The enriched CTC samples were cultured in 3D to obtain organoids and used for downstream analyses. RESULTS: The DLA procedure resulted in a median yield of 5312 CTCs as compared with 22 CTCs in 7.5 mL of blood. Using WBC depletion, we recovered 46% of the CTCs, which reduced to 12% with subsequent EpCAM selection. From the isolated and enriched CTC samples, organoid expansion succeeded in 35%. Successful organoid cultures contained significantly higher CTC numbers at initiation. Moreover, we performed treatment modelling in one organoid cell line and identified substantial tumour heterogeneity in CTCs using single cell DNA sequencing. CONCLUSIONS: DLA is an efficient method to enrich CTCs, although the modest success rate of culturing CTCs precludes large scale clinical application. Our data do suggest that DLA and subsequent processing provides a rich source of viable tumour cells. Therefore, DLA offers a promising alternative to biopsy procedures to obtain sufficient number of tumour cells to study sequential samples in patients with mPCa. TRIAL REGISTRATION NUMBER: NL6019.

Proficiency Testing to Assess Technical Performance for CTC-Processing and Detection Methods in CANCER-ID.

BACKGROUND: Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. METHODS: We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. RESULTS: We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. CONCLUSIONS: This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).

Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.

Magnetic-Based Enrichment of Rare Cells from High Concentrated Blood Samples.

Here, we tested two magnetic-bead based systems for the enrichment and detection of rare tumor cells in concentrated blood products. For that, the defined numbers of cells from three pancreatic cancer cell lines were spiked in 108 peripheral blood mononuclear cells (PBMNCs) concentrated in 1 mL, mimicking diagnostic leukapheresis (DLA) samples, and samples were processed for circulating tumor cells (CTC) enrichment with the IsoFlux or the KingFisher systems, using different types of magnetic beads from the respective technology providers. Beads were conjugated with different anti-EpCAM and MUC-1 antibodies. Recovered cells were enumerated and documented by fluorescent microscopy. For the IsoFlux system, best performance was obtained with IsoFlux CTC enrichment kit, but these beads compromised the subsequent immunofluorescence staining. For the KingFisher system, best recoveries were obtained using Dynabeads Biotin Binder beads. These beads also allowed one to capture CTCs with different antibodies and the subsequent immunofluorescence staining. KingFisher instrument allowed a single and streamlined protocol for the enrichment and staining of CTCs that further prevented cell loss at the enrichment/staining interface. Both IsoFlux and KingFisher systems allowed the enrichment of cell line cells from the mimicked-DLA samples. However, in this particular experimental setting, the recovery rates obtained with the KingFisher system were globally higher, the system was more cost-effective, and it allowed higher throughput.

Circulating biomarkers for early detection and clinical management of colorectal cancer.

New non-invasive approaches that can complement and improve on current strategies for colorectal cancer (CRC) screening and management are urgently needed. A growing number of publications have documented that components of tumors, which are shed into the circulation, can be detected in the form of liquid biopsies and can be used to detect CRC at early stages, to predict response to certain therapies and to detect CRC recurrence in a minimally invasive way. The analysis of circulating tumor DNA (ctDNA), tumor-derived cells (CTC, circulating tumor cells) or circulating microRNA (miRNA) in blood and other body fluids, have a great potential to improve different aspects of CRC management. The challenge now is to find which types of components, biofluids and detection methods would be the most suitable to be applied in the different steps of CRC detection and treatment. This chapter will provide an up to date review on ctDNA, CTCs and circulating miRNAs as new biomarkers for CRC, either for clinical management or early detection, highlighting their advantages and limitations.

Detection of circulating tumor cells in colorectal cancer patients using the GILUPI CellCollector: results from a prospective, single-center study.

The GILUPI CellCollector (CC) is a novel in vivo circulating tumor cell (CTC) detection device reported to overcome the limitations of small blood sample volumes. The aim of this prospective, blinded study was to evaluate the clinical application of the CC and to compare its performance to the CellSearch (CS) system in M0 and M1 colorectal cancer (CRC) patients. A total of 80 patients (31 M0, 49 M1) with CRC were enrolled. CTCs were simultaneously measured in the peripheral blood using CS and the CC, and the results of both assays were correlated to clinicopathological variables and overall survival. The total number of detected CTCs and CTC-positive patients did not significantly differ between both assays. In the M0 patients, the CC detected CTCs more frequently than CS. There was no significant difference in total CTC numbers detected with the CC between M0 and M1 patients. In addition, no significant correlation with clinicopathological parameters or overall survival was observed with CC CTCs. In contrast, detection of CTCs with CS was significantly correlated with Union for International Cancer Control stage and reduced overall survival. There was no correlation between CTCs detected by the CC and the CS system. Using in silico analysis, we estimate that CC screens a volume of 0.33-18 mL during in vivo application, in contrast to much higher volumes reported elsewhere. In conclusion, while being safe and easy to use, the CC did not outperform CS in terms of CTC yield or sensitivity. While CTC detection in M0 CRC patients was significantly increased with the CC, the clinical relevance of these CTCs appears inferior to the cells identified by the CS system.

Structural and mechanistic aspects of S-S bonds in the thioredoxin-like family of proteins.

Disulfide bonds play a critical role in a variety of structural and mechanistic processes associated with proteins inside the cells and in the extracellular environment. The thioredoxin family of proteins like thioredoxin (Trx), glutaredoxin (Grx) and protein disulfide isomerase, are involved in the formation, transfer or isomerization of disulfide bonds through a characteristic thiol-disulfide exchange reaction. Here, we review the structural and mechanistic determinants behind the thiol-disulfide exchange reactions for the different enzyme types within this family, rationalizing the known experimental data in light of the results from computational studies. The analysis sheds new atomic-level insight into the structural and mechanistic variations that characterize the different enzymes in the family, helping to explain the associated functional diversity. Furthermore, we review here a pattern of stabilization/destabilization of the conserved active-site cysteine residues presented beforehand, which is fully consistent with the observed roles played by the thioredoxin family of enzymes.

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting.

Diagnostic leukapheresis (DLA) is based on continuous centrifugation that collects mononuclear cells from peripheral blood with a density of 1.055-1.08 g/ml. As epithelial cells have a similar density, DLA cocollects circulating tumor cell (CTCs) along with the targeted mononuclear cells. Here, we report on our single center experience applying DLA in 40 nonmetastatic and metastatic breast cancer patients and its impact on CTC detection. We found that the use of just 5% of the DLA product (corresponding to a median peripheral blood volume of around 60 ml) in the CellSearch® assay already leads to a significant increase in CTC detection frequency and yield. The implementation of the method was unproblematic, and we did not observe any adverse events in our patient cohort. Extrapolating the CTC counts in the DLA samples to the whole DLA product indicated that enormous CTC numbers could be harvested by this approach (around 205x more CTCs than in the 7.5 ml blood sample in M1 patients). In conclusion, DLA is a clinically safe method to collect CTCs from liters of blood enabling a real liquid biopsy. Yet, further technical developments are required to process whole DLA products and exploit the full potential of this approach. As it is foreseeable that DLA will be used by several groups, and hopefully ultimately brought to the patients in a routine setting, we discuss recommendations on the minimum of required information for reporting on DLAs to allow comparison across different approaches. © 2018 International Society for Advancement of Cytometry.

Single-Cell Analyses of Prostate Cancer Liquid Biopsies Acquired by Apheresis.

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency.Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity.Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses.Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635-44. ©2018 AACR.