CRISPR/Cas9-edited ROS1 + non-small cell lung cancer cell lines highlight differential drug sensitivity in 2D vs 3D cultures while reflecting established resistance profiles.

INTRODUCTION: The study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting. METHODS: In the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose-response curves was the variable used to quantify the cellular response towards TKIs. RESULTS: Spheroids derived from ROS1G2032R cells were significantly more resistant to repotrectinib (AOC fold change = - 7.33), lorlatinib (AOC fold change = - 6.17), ceritinib (AOC fold change = - 2.8) and entrectinib (AOC fold change = - 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = - 2.35), entrectinib (AOC fold change = - 2.44) and ceritinib (AOC fold change = - 2.12) in targeting the ROS1 G2032R mutation. ROS1L2026M cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = - 2.19) and entrectinib (spheroid AOC fold change = - 1.98). ROS1S1986Y cells were resistant only towards crizotinib in 2D (AOC fold change = - 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose-response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells. CONCLUSION: In this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.

Rational design of a genomically humanized mouse model for dominantly inherited hearing loss, DFNA9.

DFNA9 is a dominantly inherited form of adult-onset progressive hearing impairment caused by mutations in the COCH gene. COCH encodes cochlin, a crucial extracellular matrix protein. We established a genomically humanized mouse model for the Dutch/Belgian c.151C>T founder mutation in COCH. Considering upcoming sequence-specific genetic therapies, we exchanged the genomic murine Coch exons 3-6 for the corresponding human sequence. Introducing human-specific genetic information into mouse exons can be risky. To mitigate unforeseen consequences on cochlin function resulting from the introduction of the human COCH protein-coding sequence, we converted all human-specific amino acids to mouse equivalents. We furthermore optimized the recognition of the human COCH exons by the murine splicing machinery during pre-mRNA splicing. Subsequent observations in mouse embryonic stem cells revealed correct splicing of the hybrid Coch transcript. The inner ear of the established humanized Coch mice displays correctly-spliced wild-type and mutant humanized Coch alleles. For a comprehensive study of auditory function, mice were crossbred with C57BL/6 Cdh23753A>G mice to remove the Cdh23ahl allele from the genetic background of the mice. At 9 months, all humanized Coch genotypes showed hearing thresholds comparable to wild-type C57BL/6 Cdh23753A>G mice. This indicates that both the introduction of human wildtype COCH, and correction of Cdh23ahl in the humanized Coch lines was successful. Overall, our approach proved beneficial in eliminating potential adverse events of genomic humanization of mouse genes, and provides us with a model in which sequence-specific therapies directed against the human mutant COCH alle can be investigated. With the hearing and balance defects anticipated to occur late in the second year of life, a long-term follow-up study is ongoing to fully characterize the humanized Coch mouse model.

Singlet oxygen-based photoelectrochemical detection of single-point mutations in the KRAS oncogene.

Single nucleotide point mutations in the KRAS oncogene occur frequently in human cancers, rendering them intriguing targets for diagnosis, early detection and personalized treatment. Current detection methods are based on polymerase chain reaction, sometimes combined with next-generation sequencing, which can be expensive, complex and have limited availability. Here, we propose a novel singlet oxygen (1O2)-based photoelectrochemical detection methodology for single-point mutations, using KRAS mutations as a case study. This detection method combines the use of a sandwich assay, magnetic beads and robust chemical photosensitizers, that need only air and light to produce 1O2, to ensure high specificity and sensitivity. We demonstrate that hybridization of the sandwich hybrid at high temperatures enables discrimination between mutated and wild-type sequences with a detection rate of up to 93.9%. Additionally, the presence of background DNA sequences derived from human cell-line DNA, not containing the mutation of interest, did not result in a signal, highlighting the specificity of the methodology. A limit of detection as low as 112 pM (1.25 ng/mL) was achieved without employing any amplification techniques. The developed 1O2-based photoelectrochemical methodology exhibits unique features, including rapidity, ease of use, and affordability, highlighting its immense potential in the field of nucleic acid-based diagnostics.

Widely Split P Waves After Pharmacologic Cardioversion in a Patient With Atrial Fibrillation.

We report a case of a clinically asymptomatic patient with extreme P-wave separation on the electrocardiogram mimicking atrial tachycardia with atrioventricular block. The accurate examination of the patient, analysis of the electrocardiogram, and echocardiographic findings led to proper diagnosis, management, and treatment. (Level of Difficulty: Intermediate.).

The Quest for Circulating Biomarkers in Neuroendocrine Neoplasms: a Clinical Perspective.

OPINION STATEMENT: Given the considerable heterogeneity in neuroendocrine neoplasms (NENs), it appears unlikely that a sole biomarker exists capable of fully capturing all useful clinical aspects of these tumors. This is reflected in the abundant number of biomarkers presently available for the diagnosis, prognosis, and monitoring of NEN patients. Although assessment of immunohistochemical and radiological markers remains paramount and often obligatory, there has been a notable surge of interest in circulating biomarkers over the years given the numerous benefits associated with liquid biopsies. Currently, the clinic primarily relies on single-analyte assays such as the chromogranin A assay, but these are far from ideal because of limitations such as compromised sensitivity and specificity as well as a lack of standardization. Consequently, the quest for NEN biomarkers continued with the exploration of multianalyte markers, exemplified by the development of the NETest and ctDNA-based analysis. Here, an extensive panel of markers is simultaneously evaluated to identify distinct signatures that could enhance the accuracy of patient diagnosis, prognosis determination, and response to therapy prediction and monitoring. Given the promising results, the development and implementation of these multianalyte markers are expected to usher in a new era of NEN biomarkers in the clinic. In this review, we will outline both clinically implemented and more experimental circulating markers to provide an update on developments in this rapidly evolving field.

Combined Cardiac Damage Staging by Echocardiography and Cardiac Catheterization in Patients With Clinically Significant Aortic Stenosis.

BACKGROUND: Cardiac damage (CD) staging enhances risk stratification in patients with clinically significant aortic stenosis (AS). We aimed to assess the prognostic value and reclassification rate of right heart catheterization (RHC) compared with transthoracic echocardiography (TTE) in characterising CD staging at 3-year follow-up in patients with clinically significant AS, to identify patients that would benefit from RHC for prognostic stratification, and to test the prognostic value of combined CD staging. METHODS: An observational cohort study of 432 AS patients undergoing TTE and RHC were divided into moderate or asymptomatic severe (m/asAS) and symptomatic severe (ssAS) AS. Kaplan-Meier curves were used to compare survival. The accuracy in prognostic stratification was tested by area under the receiver operating characteristic curve analysis and Delong test. RESULTS: In both cohorts, TTE- and RHC-derived staging systems had prognostic value, although the agreement between them appeared moderate. A higher proportion of patients were assigned to stage 2 by TTE than by RHC. Patients in TTE-derived stage 2 had a high reclassification rate, with 40%-50% presenting with right chamber involvement (stages 3-4) according to RHC. Discordant cases were significantly older, with higher prevalence of atrial fibrillation, markedly elevated N-terminal pro-B-type natriuretic peptide, and higher indexed left atrial volume, E/e', and systolic pulmonary artery pressure vs concordant cases (P < 0.05). The combined CD staging, integrating TTE and RHC, was more accurate in predicting mortality than the TTE-derived system (P < 0.05). CONCLUSIONS: In patients with m/asAS and ssAS, the combined CD staging, derived from TTE and RHC, was more accurate in predicting mortality than TTE alone. In a subset of AS patients, the integration of RHC may significantly improve prognostic stratification.

Association of Mild-to-Moderate Aortic Regurgitation With Outcomes in Heart Failure With Preserved Ejection Fraction.

OBJECTIVE: To assess aortic regurgitation (AR) prevalence, its hemodynamic effect, and long-term prognostic implications in patients admitted with de novo or worsened heart failure with preserved ejection fraction (HFpEF). METHODS: Consecutive patients hospitalized with de novo or worsened HFpEF between 2014 and 2020 were enrolled. Patients with more than moderate aortic and/or mitral valve disease were excluded. Based on the presence and degree of AR, patients were divided into those without AR, those with mild, and those with moderate AR. Data on cardiovascular death, heart failure (HF) rehospitalization, and their composite (major adverse cardiovascular events) were collected. RESULTS: The final study population consisted of 458 HFpEF patients: 156 (34.1%) with mild-AR, 153 (33.4%) with moderate-AR, and the remaining 149 (32.5%) with no AR. Mild-to-moderate AR patients were older, with larger left atrium-left ventricle (LV) volumes, greater LV mass index, higher filling pressure, and prevalence of diastolic dysfunction compared with the no-AR group (all P<.05). During 5-year follow-up, 113 patients died of cardiovascular causes, 124 patients were rehospitalized for HF, whereas 196 experienced the composite endpoint. Mild-to-moderate AR was identified as an independent predictor of all-cause death (HR, 1.62; 95% CI, 1.14 to 1.58; P=.04) and major adverse cardiovascular event occurrence (HR, 1.48; 95% CI, 1.05 to 2.09; P=.02). A total of 126 (35.5%) of 355 patients showed progression of AR at follow-up echocardiography. CONCLUSION: Mild-to-moderate AR is common among patients hospitalized for HFpEF. It is associated with adverse LV remodeling and worse long-term outcomes. These findings warrant further prospective studies addressing the importance of AR in prognostic stratification and exploring therapeutic strategies to mitigate its hemodynamic effect on HF.

Contemporary European practice in transcatheter aortic valve implantation: results from the 2022 European TAVI Pathway Registry.

Background: A steep rise in the use of transcatheter aortic valve implantation (TAVI) for the management of symptomatic severe aortic stenosis occurred. Minimalist TAVI procedures and streamlined patient pathways within experienced Heart Valve Centres are designed to overcome the challenges of ever-increasing procedural volume. Aims: The 2022 European TAVI Pathway Survey aims to describe contemporary TAVI practice across Europe. Materials and methods: Between October and December 2022, TAVI operators from 32 European countries were invited to complete an online questionnaire regarding their current practice. Results: Responses were available from 147 TAVI centres in 26 countries. In 2021, the participating centres performed a total number of 27,223 TAVI procedures, with a mean of 185 TAVI cases per centre (median 138; IQR 77-194). Treatment strategies are usually (87%) discussed at a dedicated Heart Team meeting. Transfemoral TAVI is performed with local anaesthesia only (33%), with associated conscious sedation (60%), or under general anaesthesia (7%). Primary vascular access is percutaneous transfemoral (99%) with secondary radial access (52%). After uncomplicated TAVI, patients are transferred to a high-, medium-, or low-care unit in 28%, 52%, and 20% of cases, respectively. Time to discharge is day 1 (12%), day 2 (31%), day 3 (29%), or day 4 or more (28%). Conclusion: Reported adoption of minimalist TAVI techniques is common among European TAVI centres, but rates of next-day discharge remain low. This survey highlights the significant progress made in refining TAVI treatment and pathways in recent years and identifies possible areas for further improvement.

Epigenome-wide methylation analysis of colorectal carcinoma, adenoma and normal tissue reveals novel biomarkers addressing unmet clinical needs.

BACKGROUND: Biomarker discovery in colorectal cancer has mostly focused on methylation patterns in normal and colorectal tumor tissue, but adenomas remain understudied. Therefore, we performed the first epigenome-wide study to profile methylation of all three tissue types combined and to identify discriminatory biomarkers. RESULTS: Public methylation array data (Illumina EPIC and 450K) were collected from a total of 1 892 colorectal samples. Pairwise differential methylation analyses between tissue types were performed for both array types to "double evidence" differentially methylated probes (DE DMPs). Subsequently, the identified DMPs were filtered on methylation level and used to build a binary logistic regression prediction model. Focusing on the clinically most interesting group (adenoma vs carcinoma), we identified 13 DE DMPs that could effectively discriminate between them (AUC = 0.996). We validated this model in an in-house experimental methylation dataset of 13 adenomas and 9 carcinomas. It reached a sensitivity and specificity of 96% and 95%, respectively, with an overall accuracy of 96%. Our findings raise the possibility that the 13 DE DMPs identified in this study can be used as molecular biomarkers in the clinic. CONCLUSIONS: Our analyses show that methylation biomarkers have the potential to discriminate between normal, precursor and carcinoma tissues of the colorectum. More importantly, we highlight the power of the methylome as a source of markers for discriminating between colorectal adenomas and carcinomas, which currently remains an unmet clinical need.

Genetic Evaluation of Prelingual Hearing Impairment: Recommendations of an European Network for Genetic Hearing Impairment.

The cause of childhood hearing impairment (excluding infectious pathology of the middle ear) can be extrinsic (embryofoetopathy, meningitis, trauma, drug ototoxicity, noise trauma, etc [...].

New Developments in Treating RAS-Mutated Metastatic Colorectal Cancer.

OPINION STATEMENT: One of the great challenges in digestive oncology is choosing the optimal therapy for RAS-mutated metastatic colorectal cancer (mCRC). Even though the RAS genes and accompanying pathway were identified decades ago and extensive knowledge exists on their role in carcinogenesis, it has proven challenging to translate these insights into new therapies and clinical benefit for patients. However, recently, new drugs targeting this pathway (for example, KRASG12C inhibitors) have shown promising results in clinical trials, as monotherapy or in combination regimens. Although resistance remains an important issue, more knowledge on adaptive resistance and feedback loops in the RAS-pathway has led to strategical combination regimens to overcome this problem. In the past year, many encouraging results have been published or presented at conferences. Even though some of the data is still preliminary, these studies may bring practice-changing results and can lead to a clinical benefit for patients over the coming years. Because of these recent developments, the treatment of RAS-mutated mCRC has become a topic of great interest. Therefore, in this review, we will summarize the standard of care and discuss the most important emerging therapies for this patient population.

A Belgian Population-Based Study Reveals Subgroups of Right-sided Colorectal Cancer with a Better Prognosis Compared to Left-sided Cancer.

BACKGROUND: Patients with left-sided colorectal cancer (L-CRC) are known to have a significantly better prognosis than those with right-sided CRC (R-CRC). It has been hypothesized that RAS, BRAF mutations, or deficient mismatch repair status (MMR) might be responsible for the prognostic effect of primary tumor location (PTL). This study aims to evaluate the prognostic effect of PTL in the Belgian population and to determine the role of biomarkers (MMR, BRAF, and RAS status) in this effect. PATIENTS AND METHODS: We performed a retrospective analysis of Belgian Cancer Registry data. First, we studied the prognostic effect of PTL on 5-year relative survival of 91,946 patients diagnosed with CRC (all stages) from 2004-2015. Second, we investigated the interaction between biomarkers and the prognostic effect of PTL in 1818 patients diagnosed with stage IV CRC in 2014-2015. RESULTS: L-CRC was associated with a significantly better 5-year relative survival compared to R-CRC in all stages and ages combined (68.4%, 95% CI, 67.7-69.1% vs 65.6%, 95% CI, 64.7-66.4%). Also, when stratified by age, sex, and stage, the prognosis of L-CRC was better compared to R-CRC in most subgroups. Only in stage II and certain subgroups of elderly patients, the opposite was observed. Furthermore, our data showed that none of the biomarkers had a significant interaction with the effect of PTL on survival. CONCLUSION: This population-based study confirms that L-CRC is associated with significantly better relative survival compared to R-CRC, in all stages and ages combined. Furthermore, in stage IV L-CRC is associated with a longer survival than R-CRC, regardless of MMR, RAS, and BRAF status.

Same Day Discharge Strategy by Default in a Tertiary Catheterization Laboratory in Belgium: Value Based Healthcare-Change in Practice.

AIMS: To assess the effects on outcomes and hospital revenues (societal cost) of a by default strategy of same day discharge (SDD) in patients undergoing a cardiac catheterization procedure in a Belgian Hospital. METHODS AND RESULTS: Outcome and complete financial data were obtained in all consecutive patients with a cardiac catheterization performed in 2019 (n=5237) and in 2021 (n=5377). Patient-reported experience, patient satisfaction and Net promotor score were obtained prospectively for the SDD cohort in 2021. The proportion of patients receiving catheterization procedure in SDD increased from 28 to 44 % (p<0.001). This translates to the saving of 889 conventional hospitalizations in 2021. All-cause death and readmission rate remained unchanged (0,17% vs 0,15% (p=0,004); and 0,7% vs 1,8% (p>0,05)) in 2019 and 2021, respectively. Patients satisfaction top box score was 91% and the Net Promotor Score was 89,5. The by default SDD strategy was associated with reduction in in-hospital health care spending, on average 3206€ per procedure is saved. This means a 57% decrease in hospital revenues and translates into an important decrease in physician income. CONCLUSION: Implementing a by default SDD cardiac catheterization strategy results in a reduction of societal cost, excellent patient satisfaction and unchanged clinical outcome. Yet, in the given context this approach negatively impacts hospital and physician revenues precluding the sustainability of such protocol.

Pre-clinical modelling of ROS1+ non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a heterogeneous group of diseases which accounts for 80% of newly diagnosed lung cancers. In the previous decade, a new molecular subset of NSCLC patients (around 2%) harboring rearrangements of the c-ros oncogene 1 was defined. ROS1+ NSCLC is typically diagnosed in young, nonsmoker individuals presenting an adenocarcinoma histology. Patients can benefit from tyrosine kinase inhibitors (TKIs) such as crizotinib and entrectinib, compounds initially approved to treat ALK-, MET- or NTRK- rearranged malignancies respectively. Given the low prevalence of ROS1-rearranged tumors, the use of TKIs was authorized based on pre-clinical evidence using limited experimental models, followed by basket clinical trials. After initiating targeted therapy, disease relapse is reported in approximately 50% of cases as a result of the appearance of resistance mechanisms. The restricted availability of TKIs active against resistance events critically reduces the overall survival. In this review we discuss the pre-clinical ROS1+ NSCLC models developed up to date, highlighting their strengths and limitations with respect to the unmet clinical needs. By combining gene-editing tools and novel cell culture approaches, newly developed pre-clinical models will enhance the development of next-generation tyrosine kinase inhibitors that overcome resistant tumor cell subpopulations.

Targeted Next-Generation Sequencing in Children With Bilateral Sensorineural Hearing Loss: Diagnostic Yield and Predictors of a Genetic Cause.

OBJECTIVE: To investigate the diagnostic yield of targeted next-generation sequencing using hearing loss panels and to identify patient-related factors that are associated with a definite genetic cause. STUDY DESIGN: Retrospective chart review. SETTING: Tertiary referral center. PATIENTS: Children with congenital or late-onset, bilateral sensorineural hearing loss. INTERVENTIONS: Diagnostic. MAIN OUTCOME MEASURES: The number of patients with a definite genetic diagnosis. RESULTS: We report on 238 patients with hearing loss: 130 were male and 108 were female. About 55% had congenital hearing loss. A genetic cause was identified in 94 of the patients (39.5%), with 72.3% of these showing nonsyndromic and 27.6% showing syndromic hearing loss. The diagnostic yield was highest among North African patients (66.7%). A multiple linear regression model shows that profound hearing loss, family history of hearing loss, congenital hearing loss, and North African ethnicity are significantly related to identifying a genetic cause. CONCLUSIONS: Targeted next-generation sequencing using a panel of hearing loss genes identified a genetic diagnosis in almost 40% of children with bilateral sensorineural hearing loss. We describe the predictors of a genetic diagnosis, and this information may be used during genetic counseling.