Deafness causing neuroplastin missense variants fail to promote plasma membrane Ca2+-ATPase levels and Ca2+ transient regulation in brain neurons.

Hearing, the ability to sense sounds, and the processing of auditory information are important for perception of the world. Mice lacking expression of neuroplastin (Np), a type-1 transmembrane glycoprotein, display deafness, multiple cognitive deficiencies, and reduced expression of plasma membrane calcium (Ca2+) ATPases (PMCAs) in cochlear hair cells and brain neurons. In this study, we transferred the deafness causing missense mutations pitch (C315S) and audio-1 (I122N) into human Np (hNp) constructs and investigated their effects at the molecular and cellular levels. Computational molecular dynamics show that loss of the disulfide bridge in hNppitch causes structural destabilization of immunoglobulin-like domain (Ig) III and that the novel asparagine in hNpaudio-1 results in steric constraints and an additional N-glycosylation site in IgII. Additional N-glycosylation of hNpaudio-1 was confirmed by PNGaseF treatment. In comparison to hNpWT, transfection of hNppitch and hNpaudio-1 into HEK293T cells resulted in normal mRNA levels but reduced the Np protein levels and their cell surface expression due to proteasomal/lysosomal degradation. Furthermore, hNppitch and hNpaudio-1 failed to promote exogenous PMCA levels in HEK293T cells. In hippocampal neurons, expression of additional hNppitch or hNpaudio-1 was less efficient than hNpWT to elevate endogenous PMCA levels and to accelerate the restoration of basal Ca2+ levels after electrically evoked Ca2+ transients. We propose that mutations leading to pathological Np variants, as exemplified here by the deafness causing Np mutants, can affect Np-dependent Ca2+ regulatory mechanisms and may potentially cause intellectual and cognitive deficits in humans.

Photophysical Analysis of Aggregation-Induced Emission (AIE) Luminogens Based on Triphenylamine and Thiophene: Insights into Emission Behavior in Solution and PMMA Films.

Aggregation-Induced Emission (AIE) luminogens have garnered significant interest due to their distinctive applications in different applications. Among the diverse molecular architectures, those based on triphenylamine and thiophene hold prominence. However, a comprehensive understanding of the deactivation mechanism both in solution and films remains lacking. In this study, we synthesized and characterized spectroscopically two AIE luminogens: 5-(4-(bis(4-methoxyphenyl)amino)phenyl)thiophene-2-carbaldehyde (TTY) and 5'-(4-(bis(4-methoxyphenyl)amino)phenyl)-[2,2'-bithiophene]-5-carbaldehyde (TTO). Photophysical and theoretical analyses were conducted in both solution and PMMA films to understand the deactivation mechanism of TTY and TTO. In diluted solutions, the emission behavior of TTY and TTO is influenced by the solvent, and the deactivation of the excited state can occur via locally excited (LE) or twisted intramolecular charge transfer (TICT) state. In PMMA films, rotational and translational movements are constrained, necessitating emission solely from the LE state. Nevertheless, in the PMMA film, excimers-like structures form, resulting in the emergence of a longer wavelength band and a reduction in emission intensity. The zenith of emission intensity occurs when molecules are dispersed at higher concentrations within PMMA, effectively diminishing the likelihood of excimer-like formations. Luminescent Solar Concentrators (LSC) were fabricated to validate these findings, and the optical efficiency was studied at varying concentrations of luminogen and PMMA.

International initiative for a curated SDHB variant database improving the diagnosis of hereditary paraganglioma and pheochromocytoma.

BACKGROUND: SDHB is one of the major genes predisposing to paraganglioma/pheochromocytoma (PPGL). Identifying pathogenic SDHB variants in patients with PPGL is essential to the management of patients and relatives due to the increased risk of recurrences, metastases and the emergence of non-PPGL tumours. In this context, the 'NGS and PPGL (NGSnPPGL) Study Group' initiated an international effort to collect, annotate and classify SDHB variants and to provide an accurate, expert-curated and freely available SDHB variant database. METHODS: A total of 223 distinct SDHB variants from 737 patients were collected worldwide. Using multiple criteria, each variant was first classified according to a 5-tier grouping based on American College of Medical Genetics and NGSnPPGL standardised recommendations and was then manually reviewed by a panel of experts in the field. RESULTS: This multistep process resulted in 23 benign/likely benign, 149 pathogenic/likely pathogenic variants and 51 variants of unknown significance (VUS). Expert curation reduced by half the number of variants initially classified as VUS. Variant classifications are publicly accessible via the Leiden Open Variation Database system (https://databases.lovd.nl/shared/genes/SDHB). CONCLUSION: This international initiative by a panel of experts allowed us to establish a consensus classification for 223 SDHB variants that should be used as a routine tool by geneticists in charge of PPGL laboratory diagnosis. This accurate classification of SDHB genetic variants will help to clarify the diagnosis of hereditary PPGL and to improve the clinical care of patients and relatives with PPGL.

On the role of water in the hydrogen bond network in DESs: an ab initio molecular dynamics and quantum mechanical study on the urea-betaine system.

We herein report an ab initio molecular dynamics study on a natural DES composed of urea and betaine in a 3 : 2 ratio, as a test case for evaluating the water effect. The article deals with a theoretical study using both ab initio molecular dynamics and quantum chemistry computations in order to unravel the role of water in the nanostructure of a urea-betaine mixture. Preliminary molecular dynamics outcomes (both radial and spatial distribution functions) suggest that water promotes the association between urea and betaine by increasing the hydrogen bond network and precluding the aggregation of urea molecules. In other words, the presence of water allows a less restrictive hydrogen bond network, presenting a regimen where the strong hydrogen bond interactions are replaced by a wide variety of weaker hydrogen bond interactions. On the other hand, in a water free DES there is a regimen where strong urea-betaine interactions are dominant. It is shown that second order perturbation theory energy analysis provides cogent insights into charge spreading and hydrogen bond patterns. A vibrational analysis (both IR and power spectrum) over the ab initio molecular dynamics trajectories in the water free DES as well as in the urea-betaine-water systems reveals that our results are consistent with the second order perturbation theory analysis and with the hydrogen bond network pattern.

Unraveling the Selectivity Patterns in Phosphine-Catalyzed Annulations of Azomethine Imines and Allenoates.

The mechanism and selectivity of phosphine-catalyzed [3 + 2] and [3 + 3] annulations of azomethine imines and allenoates have been computationally studied. Exploration of the potential energy surface reveals that the cyclization step is a key step controlling the selectivity of the process. This contrasts with previous studies on related transformations where the initial nucleophilic addition involving the activated allenoate was found to exclusively control the regioselectivity of the transformation. Among the possible reaction pathways, the energetically low-lying reaction channel involves an intramolecular Michael addition leading to the experimentally observed [3 + 2] product. The factors controlling the observed regioselectivity have been quantitatively rationalized by means of state-of-the-art computational methods, namely, the activation strain model of reactivity in combination with the energy decomposition analysis.

Meisenheimer complexes as hidden intermediates in the aza-SNAr mechanism.

In this work we report a computational study about the aza-SNAr mechanism in fluorine- and chlorine-containing azines with the aim to unravel the physical factors that determine the reactivity patterns in these heterocycles towards propylamine. The nature of the reaction intermediate was analyzed in terms of its electronic structure based on a topological analysis framework in some non-stationary points along the reaction coordinate. The mechanistic dichotomy of a concerted or a stepwise pathway is interpreted in terms of the qualitative Diabatic Model of Intermediate Stabilization (DMIS) approach, providing a general mechanistic picture for the SNAr process involving both activated benzenes and nitrogen-containing heterocycles. With the information collected, a unified vision of the Meisenheimer complexes as transition state, hidden intermediate or real intermediate was proposed.

Theoretical insights into the E1cB/E2 mechanistic dichotomy of elimination reactions.

E1cB and E2 eliminations have been described as competing mechanisms that can even share a common pathway when the E1cB/E2 borderline mechanism operates. A suitable case study evincing such a mechanistic dichotomy corresponds to the elimination reaction of ß-phenylmercaptoethyl phenolate, since its mechanism has been thought to be an E2 elimination. Nonetheless, according to the computational assessment of the substituents on the leaving group, we demonstrate that the reaction proceeds via a borderline E1cB mechanism. Stabilization of the carbanion was provided not only by substituent effects tuning the nucleofugality of the leaving group, but also by a base, since distortion/interaction-activation strain and Natural Bond Order (NBO) analyses suggest a stabilizing interaction between the base and Cß of the E1cB intermediate. In order to gain insights into these results in a more general context, we have rationalized them with a qualitative picture of the E1cB/E2 mechanistic dichotomy using simple relationships between diabatic parabolas modeling the potential wells of reactants, intermediates, and products. In this Diabatic Model of Intermediate Stabilization (DMIS), the borderline E1cB mechanism for the elimination reaction of ß-phenylmercaptoethyl phenolate was discussed in terms of bonding and dynamic stepwise processes. The conceptual model presented herein should be useful for the analysis of any reaction comprising competing one- and two-step mechanisms.

Outcomes of adrenal-sparing surgery or total adrenalectomy in phaeochromocytoma associated with multiple endocrine neoplasia type 2: an international retrospective population-based study.

BACKGROUND: The prevention of medullary thyroid cancer in patients with multiple endocrine neoplasia type 2 syndrome has demonstrated the ability of molecular diagnosis and prophylactic surgery to improve patient outcomes. However, the other major neoplasia associated with multiple endocrine neoplasia type 2, phaeochromocytoma, is not as well characterised in terms of occurrence and treatment outcomes. In this study, we aimed to systematically characterise the outcomes of management of phaeochromocytoma associated with multiple endocrine neoplasia type 2. METHODS: This multinational observational retrospective population-based study compiled data on patients with multiple endocrine neoplasia type 2 from 30 academic medical centres across Europe, the Americas, and Asia. Patients were included if they were carriers of germline pathogenic mutations of the RET gene, or were first-degree relatives with histologically proven medullary thyroid cancer and phaeochromocytoma. We gathered clinical information about patients'RET genotype, type of treatment for phaeochromocytoma (ie, unilateral or bilateral operations as adrenalectomy or adrenal-sparing surgery, and as open or endoscopic operations), and postoperative outcomes (adrenal function, malignancy, and death). The type of surgery was decided by each investigator and the timing of surgery was patient driven. The primary aim of our analysis was to compare disease-free survival after either adrenal-sparing surgery or adrenalectomy. FINDINGS: 1210 patients with multiple endocrine neoplasia type 2 were included in our database, 563 of whom had phaeochromocytoma. Treatment was adrenalectomy in 438 (79%) of 552 operated patients, and adrenal-sparing surgery in 114 (21%). Phaeochromocytoma recurrence occurred in four (3%) of 153 of the operated glands after adrenal-sparing surgery after 6-13 years, compared with 11 (2%) of 717 glands operated by adrenalectomy (p=0.57). Postoperative adrenal insufficiency or steroid dependency developed in 292 (86%) of 339 patients with bilateral phaeochromocytoma who underwent surgery. However, 47 (57%) of 82 patients with bilateral phaeochromocytoma who underwent adrenal-sparing surgery did not become steroid dependent. INTERPRETATION: The treatment of multiple endocrine neoplasia type 2-related phaeochromocytoma continues to rely on adrenalectomies with their associated Addisonian-like complications and consequent lifelong dependency on steroids. Adrenal-sparing surgery, a highly successful treatment option in experienced centres, should be the surgical approach of choice to reduce these complications.

The Simulation of Solvent Polarizabilities and Dipolarities with Polarizable Continuum Model.

The ability of polarizable continuum models (PCM) to simulate nonspecific solvent effects (dipolarity and polarizability) was evaluated by calculating the transition energies of 1,1,10,10-tetrabutyldecanonaene (ttbp9) and 2-N,N-dimethylamino-7-nitrofluorene (DMANF), basis of Catalán's polarizability (SP) and dipolarity (SdP) solvent scales, respectively. Time-dependent density-functional theory (TD-DFT) calculations were performed at different levels of theory, employing four basis sets in 10 different solvents, covering the full range of the normalized SP and SdP scales. Transition energies were calculated using linear response (LR) and corrected linear response (cLR2) schemes. Although these methods yielded variable mean absolute errors, the LR-PCM calculations reproduced medium polarizability and dipolarity trends. While calculated ttbp9 transition energies correlated with SP and Laurence's dispersion-induced (DI) scales, the DMANF transition energies correlated poorly with SdP or Laurence's ES dipolarity scales. This result agrees with the fact that DMANF solvatochromism is "contaminated" by solvent polarizability and HB acidity. The incorporation of SP or DI contributions led to much better (r2 > 0.95) correlations with the DMANF-calculated transitions. The results offer a clearer picture of the limitations of continuum models in simulating the behavior of solvatochromic dyes in solution by pointing out their poor performance when specific solvent effects, such as hydrogen-bond interactions, play a significant role in their solvatochromism.

Cell type signatures in cell-free DNA fragmentation profiles reveal disease biology.

Circulating cell-free DNA (cfDNA) fragments have characteristics that are specific to the cell types that release them. Current methods for cfDNA deconvolution typically use disease tailored marker selection in a limited number of bulk tissues or cell lines. Here, we utilize single cell transcriptome data as a comprehensive cellular reference set for disease-agnostic cfDNA cell-of-origin analysis. We correlate cfDNA-inferred nucleosome spacing with gene expression to rank the relative contribution of over 490 cell types to plasma cfDNA. In 744 healthy individuals and patients, we uncover cell type signatures in support of emerging disease paradigms in oncology and prenatal care. We train predictive models that can differentiate patients with colorectal cancer (84.7%), early-stage breast cancer (90.1%), multiple myeloma (AUC 95.0%), and preeclampsia (88.3%) from matched controls. Importantly, our approach performs well in ultra-low coverage cfDNA datasets and can be readily transferred to diverse clinical settings for the expansion of liquid biopsy.

Weakly Supervised Deep Learning Predicts Immunotherapy Response in Solid Tumors Based on PD-L1 Expression.

Programmed death-ligand 1 (PD-L1) IHC is the most commonly used biomarker for immunotherapy response. However, quantification of PD-L1 status in pathology slides is challenging. Neither manual quantification nor a computer-based mimicking of manual readouts is perfectly reproducible, and the predictive performance of both approaches regarding immunotherapy response is limited. In this study, we developed a deep learning (DL) method to predict PD-L1 status directly from raw IHC image data, without explicit intermediary steps such as cell detection or pigment quantification. We trained the weakly supervised model on PD-L1-stained slides from the non-small cell lung cancer (NSCLC)-Memorial Sloan Kettering (MSK) cohort (N = 233) and validated it on the pan-cancer-Vall d'Hebron Institute of Oncology (VHIO) cohort (N = 108). We also investigated the performance of the model to predict response to immune checkpoint inhibitors (ICI) in terms of progression-free survival. In the pan-cancer-VHIO cohort, the performance was compared with tumor proportion score (TPS) and combined positive score (CPS). The DL model showed good performance in predicting PD-L1 expression (TPS ≥ 1%) in both NSCLC-MSK and pan-cancer-VHIO cohort (AUC 0.88 ± 0.06 and 0.80 ± 0.03, respectively). The predicted PD-L1 status showed an improved association with response to ICIs [HR: 1.5 (95% confidence interval: 1-2.3), P = 0.049] compared with TPS [HR: 1.4 (0.96-2.2), P = 0.082] and CPS [HR: 1.2 (0.79-1.9), P = 0.386]. Notably, our explainability analysis showed that the model does not just look at the amount of brown pigment in the IHC slides, but also considers morphologic factors such as lymphocyte conglomerates. Overall, end-to-end weakly supervised DL shows potential for improving patient stratification for cancer immunotherapy by analyzing PD-L1 IHC, holistically integrating morphology and PD-L1 staining intensity. SIGNIFICANCE: The weakly supervised DL model to predict PD-L1 status from raw IHC data, integrating tumor staining intensity and morphology, enables enhanced patient stratification in cancer immunotherapy compared with traditional pathologist assessment.

Identification of Precise 3D CT Radiomics for Habitat Computation by Machine Learning in Cancer.

Purpose To identify precise three-dimensional radiomics features in CT images that enable computation of stable and biologically meaningful habitats with machine learning for cancer heterogeneity assessment. Materials and Methods This retrospective study included 2436 liver or lung lesions from 605 CT scans (November 2010-December 2021) in 331 patients with cancer (mean age, 64.5 years ± 10.1 [SD]; 185 male patients). Three-dimensional radiomics were computed from original and perturbed (simulated retest) images with different combinations of feature computation kernel radius and bin size. The lower 95% confidence limit (LCL) of the intraclass correlation coefficient (ICC) was used to measure repeatability and reproducibility. Precise features were identified by combining repeatability and reproducibility results (LCL of ICC ≥ 0.50). Habitats were obtained with Gaussian mixture models in original and perturbed data using precise radiomics features and compared with habitats obtained using all features. The Dice similarity coefficient (DSC) was used to assess habitat stability. Biologic correlates of CT habitats were explored in a case study, with a cohort of 13 patients with CT, multiparametric MRI, and tumor biopsies. Results Three-dimensional radiomics showed poor repeatability (LCL of ICC: median [IQR], 0.442 [0.312-0.516]) and poor reproducibility against kernel radius (LCL of ICC: median [IQR], 0.440 [0.33-0.526]) but excellent reproducibility against bin size (LCL of ICC: median [IQR], 0.929 [0.853-0.988]). Twenty-six radiomics features were precise, differing in lung and liver lesions. Habitats obtained with precise features (DSC: median [IQR], 0.601 [0.494-0.712] and 0.651 [0.52-0.784] for lung and liver lesions, respectively) were more stable than those obtained with all features (DSC: median [IQR], 0.532 [0.424-0.637] and 0.587 [0.465-0.703] for lung and liver lesions, respectively; P < .001). In the case study, CT habitats correlated quantitatively and qualitatively with heterogeneity observed in multiparametric MRI habitats and histology. Conclusion Precise three-dimensional radiomics features were identified on CT images that enabled tumor heterogeneity assessment through stable tumor habitat computation. Keywords: CT, Diffusion-weighted Imaging, Dynamic Contrast-enhanced MRI, MRI, Radiomics, Unsupervised Learning, Oncology, Liver, Lung Supplemental material is available for this article. © RSNA, 2024 See also the commentary by Sagreiya in this issue.

Reactivity indices profile: a companion tool of the potential energy surface for the analysis of reaction mechanisms. Nucleophilic aromatic substitution reactions as test case.

We herein report on the usefulness of the reactivity indices profiles along a reaction coordinate. The model is tested to fully describe the reaction mechanism of the title reactions. Group nucleophilicity and electrophilicity profiles help describe the bond-breaking/bond-formation processes and the intramolecular electron density reorganization. The reactivity indices' profile analysis is consistently complemented with hydrogen bonding (HB) effects along the reaction coordinate: the final outcome of the reaction is determined by the stage at which the HB complex can be formed. Transition-state structures located for six reactions studied, including the charged nucleophile thiocyanate, show that the main stabilizing interaction is that formed between the hydrogen atom of the nucleophile and the o-NO(2) group. This result discards the role of HB interaction between the nucleophile and the leaving group previously proposed in the literature.

Specific nucleophile-electrophile interactions in nucleophilic aromatic substitutions.

We herein report results obtained from an integrated experimental and theoretical study on aromatic nucleophilic substitution (S(N)Ar) reactions of a series of amines towards 1-fluoro-2,4-dinitrobenzene in water. Specific nucleophile-electrophile interactions in the title reactions have been kinetically evaluated. The whole series undergoes S(N)Ar reactions where the formation of the Meisenheimer complex is rate determining. Theoretical studies concerning specific interactions are discussed in detail. It is found that H-bonding effects along the intrinsic reaction coordinate profile promote the activation of both the electrophile and the nucleophile. Using these results, it is possible to establish a hierarchy of reactivity that is in agreement with the experimental data. Second order energy perturbation energy analysis highlights the strong interaction between the ortho-nitro group and the acidic hydrogen atom of the amine. The present study strongly suggests that any theoretical analysis must be performed at the activated transition state structure, because the static model developed around the reactant states hides most of the relevant specific interactions that characterize the aromatic substitution process.

Hypoxia-Inducible Factor 2 Alpha (HIF2α) Inhibitors: Targeting Genetically Driven Tumor Hypoxia.

Tumors driven by deficiency of the VHL gene product, which is involved in degradation of the hypoxia-inducible factor subunit 2 alpha (HIF2α), are natural candidates for targeted inhibition of this pathway. Belzutifan, a highly specific and well-tolerated HIF2α inhibitor, recently received FDA approval for the treatment of nonmetastatic renal cell carcinomas, pancreatic neuroendocrine tumors, and central nervous system hemangioblastomas from patients with von Hippel-Lindau disease, who carry VHL germline mutations. Such approval is a milestone in oncology; however, the full potential, and limitations, of HIF2α inhibition in the clinic are just starting to be explored. Here we briefly recapitulate the molecular rationale for HIF2α blockade in tumors and review available preclinical and clinical data, elaborating on mutations that might be particularly sensitive to this approach. We also outline some emerging mechanisms of intrinsic and acquired resistance to HIF2α inhibitors, including acquired mutations of the gatekeeper pocket of HIF2α and its interacting partner ARNT. Lastly, we propose that the high efficacy of belzutifan observed in tumors with genetically driven hypoxia caused by VHL mutations suggests that a focus on other mutations that similarly lead to HIF2α stabilization, such as those occurring in neuroendocrine tumors with disruptions in the tricarboxylic acid cycle (SDHA/B/C/D, FH, MDH2, IDH2), HIF hydroxylases (EGLN/PHDs), and the HIF2α-encoding gene, EPAS1, are warranted.

Targeting patient blood management's first pillar: A multicentric retrospective study on preoperative anemia.

INTRODUCTION: Anemia is a common issue in surgical patients and has been associated with worse clinical outcomes, such as a higher probability of transfusions and longer hospital stay. Therefore, Patient Blood Management programs are actively aiming to achieve early identification and treatment of anemia, previous to the surgery. METHODS AND MATERIALS: In this study, preoperative hemoglobin within the Blood Order Schedule (BOS) at 16 blood centers in several Brazilian regions were retrospectively evaluated. Data regarding hemoglobin, age, gender and Brazilian regions were further analyzed. RESULTS: From the 20,201 BOSs evaluated, the mean age was 55.65 ± 23.52 years old, with an overall prevalence of preoperative anemia of 60.9%. Women had a lower mean preoperative hemoglobin (11.74 ± 2.84 for women and 12.27 ± 3.06 for men) and higher prevalence of anemia than men (66% of females and 52.2% of males). The individuals over 65 years old and under 18 were the most affected by preoperative anemia. All regions had a high prevalence of preoperative anemia, without any direct association with the Human Development Index. CONCLUSION: In summary, upon evaluating the BOS, our study showed a high prevalence of preoperative anemia in all Brazilian regions, regardless of the gender and age group, but that women and individuals less than 18 or over 65 years old have an even higher prevalence of preoperative anemia. This information can identify the institutions in which preoperative anemia is a critical issue and in which new strategies, such as preoperative screening clinics, might be helpful.

ACT-Discover: identifying karyotype heterogeneity in pancreatic cancer evolution using ctDNA.

BACKGROUND: Liquid biopsies and the dynamic tracking of somatic mutations within circulating tumour DNA (ctDNA) can provide insight into the dynamics of cancer evolution and the intra-tumour heterogeneity that fuels treatment resistance. However, identifying and tracking dynamic changes in somatic copy number alterations (SCNAs), which have been associated with poor outcome and metastasis, using ctDNA is challenging. Pancreatic adenocarcinoma is a disease which has been considered to harbour early punctuated events in its evolution, leading to an early fitness peak, with minimal further subclonal evolution. METHODS: To interrogate the role of SCNAs in pancreatic adenocarcinoma cancer evolution, we applied whole-exome sequencing of 55 longitudinal cell-free DNA (cfDNA) samples taken from 24 patients (including 8 from whom a patient-derived xenograft (PDX) was derived) with metastatic disease prospectively recruited into a clinical trial. We developed a method, Aneuploidy in Circulating Tumour DNA (ACT-Discover), that leverages haplotype phasing of paired tumour biopsies or PDXs to identify SCNAs in cfDNA with greater sensitivity. RESULTS: SCNAs were observed within 28 of 47 evaluable cfDNA samples. Of these events, 30% could only be identified by harnessing the haplotype-aware approach leveraged in ACT-Discover. The exceptional purity of PDX tumours enabled near-complete phasing of genomic regions in allelic imbalance, highlighting an important auxiliary function of PDXs. Finally, although the classical model of pancreatic cancer evolution emphasises the importance of early, homogenous somatic events as a key requirement for cancer development, ACT-Discover identified substantial heterogeneity of SCNAs, including parallel focal and arm-level events, affecting different parental alleles within individual tumours. Indeed, ongoing acquisition of SCNAs was identified within tumours throughout the disease course, including within an untreated metastatic tumour. CONCLUSIONS: This work demonstrates the power of haplotype phasing to study genomic variation in cfDNA samples and reveals undiscovered intra-tumour heterogeneity with important scientific and clinical implications. Implementation of ACT-Discover could lead to important insights from existing cohorts or underpin future prospective studies seeking to characterise the landscape of tumour evolution through liquid biopsy.

Predictive Impact of RNF43 Mutations in Patients With Proficient Mismatch Repair/Microsatellite Stable BRAFV600E-Mutated Metastatic Colorectal Cancer Treated With Target Therapy or Chemotherapy.

PURPOSE: Target therapy (TT) with encorafenib plus cetuximab is a standard option in patients with BRAFV600E-mutated (mut) pretreated metastatic colorectal cancer (mCRC). Recently, mutations in RNF43, encoding a negative regulator of the WNT pathway, were associated with longer progression-free survival (PFS) and overall survival (OS) in patients with proficient mismatch repair/microsatellite stable (pMMR/MSS) BRAFV600E-mut mCRC treated with TT. Here, we explored the effect of RNF43 mutations on the efficacy of second-line TT versus standard chemotherapy (CT). METHODS: A retrospective cohort of patients with pMMR/MSS BRAFV600E-mut tumors, available RNF43 mutational status, and treated with second-line TT or oxaliplatin- and/or irinotecan-based CT was analyzed. RESULTS: One hundred thirty-two patients with pMMR/MSS BRAFV600E-mut mCRC were included. RNF43 was found mut in 34 (26%) cases. Among RNF43 mutants, TT was associated with longer PFS (7.7 v 3.0 months; P = .002) and higher overall response rate (ORR; 45% v 0%; P = .009) compared with CT. Conversely, among RNF43 wild-type (wt) patients, only a trend for longer PFS (4.5 v 3.7 months; P = .064) favoring TT, with no differences in ORR (P = .14), was observed. After excluding 36 patients receiving TT in third line or beyond, a longer OS (19.4 v 10.1 months; P = .022) and a numerically OS advantage (10.6 v 6.6 months; P = .068) were reported for TT both in the RNF43-mut and in the RNF43 wt groups. However, no interaction effect was reported between RNF43 mutational status and treatment in ORR (Pinteraction = .96), PFS (Pinteraction = .13), and OS (Pinteraction = .44). CONCLUSION: Patients with pMMR/MSS BRAFV600E-mut mCRC achieve benefit from TT versus CT independently of RNF43 mutational status, although a higher magnitude of benefit from TT is observed in RNF43-mut tumors. These findings deserve confirmation in concluded and ongoing randomized trials.