Hybridization Sequencing Research Articles

Brief Report: Clinical Response, Toxicity, and Resistance Mechanisms to Osimertinib Plus MET Inhibitors in Patients With EGFR-Mutant MET-Amplified NSCLC

IntroductionMET amplification is a known resistance mechanism to EGFR tyrosine kinase inhibitor (TKI) treatment in EGFR-mutant NSCLC. Dual EGFR-MET inhibition has been reported with success in overcoming such resistance and inducing clinical benefit. Resistance mechanisms to dual EGFR-MET inhibition require further investigation and characterization. MethodsPatients with NSCLC with both MET amplification and EGFR mutation who have received crizotinib, capmatinib, savolitinib, or tepotinib plus osimertinib (OSI) after progression on OSI at MD Anderson Cancer Center were included in this study. Molecular profiling was completed by means of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). Radiological response was assessed on the basis of Response Evaluation Criteria in Solid Tumors version 1.1. ResultsFrom March 2016 to March 2022, 23 treatments with dual MET inhibitor and osi were identified with a total of 20 patients included. Three patients received capmatinib plus OSI after progression on crizotinib plus OSI. Median age was 64 (38–89) years old and 75% were female. MET amplification was detected by FISH in 14 patients in the tissue, NGS in 10 patients, and circulating tumor DNA in three patients. Median MET gene copy number was 13.6 (6.4–20). Overall response rate was 34.8% (eight of 23). In assessable patients, tumor shrinkage was observed in 82.4% (14 of 17). Median time on treatment was 27 months. Two of three patients responded to capmatinib plus OSI after progression on crizotinib plus OSI. Dual EGFR-MET inhibition was overall well tolerated. Two patients on crizotinib plus OSI and one pt on capmatinib plus OSI discontinued therapy due to pneumonitis. One pt discontinued crizotinib plus OSI due to gastrointestinal toxicity. Six patients were still on double TKI treatment. At disease progression to dual EGFR-MET inhibition, FISH and NGS on tumor and plasma were completed in six patients. Notable resistance mechanisms observed include acquired MET D1246H (n = 1), acquired EGFR C797S (n = 2), FGFR2 fusion (n = 1, concurrent with C797S), and EGFR G796S (n = 1, concurrent with C797S). Four patients lost MET amplification. ConclusionsDual EGFR and MET inhibition yielded high clinical response rate after progression on OSI. Resistance mechanisms to EGFR-MET double TKI inhibition include MET secondary mutation, EGFR secondary mutation, or loss of MET amplification.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: The ataxia–telangiectasia mutated (ATM) gene plays an important role in the cellular response to DNA damage. Whether different types of ATM aberrations (mutation and/or deletion of (del)11q) differentially affect survival in mantle cell lymphoma (MCL) has not yet been reported. Methods: We analyzed 160 consecutive MCL patients (pts) treated in two Czech university centers (Prague, Olomouc) from 11/2006 to 12/2021. Both fluorescence in situ hybridization (FISH) and next generation sequencing (NGS, Illumina), were performed in all pts to identify ATM and TP53 gene aberrations on tumoral tissue (peripheral blood, bone marrow); the cutoff value for ATM and TP53 mutation was 3% variant allele frequency. Peripheral blood and/or bone marrow had at least 5% MCL involvement as analyzed by flow cytometry. Variables were compared by chi-squared test. Kaplan-Meier method was used to calculate the probabilities of progression and overall survival (PFS, OS); the log rank test was used for univariate comparisons of survival curves. PFS and OS were calculated from the date of diagnosis. Results: The median age at diagnosis was 67 (30-87) years. All pts had advanced disease (IV). MIPI score was low, intermediate and high in 15.6%, 26.3% and 58.1% pts, respectively. Treatment approaches used were as follows: watch and wait in 5.0%, R-CHOP/R-CHOP-like in 50.6%, intensive R-HDAC-containing in 34.4% and non-anthracycline/palliative regimen in 10.0% of pts. Complete and partial response was achieved in 56.3% and 21.9% of pts, respectively. Stable and progressive disease was observed in 13.1% of pts. Almost 29% of pts underwent autologous stem cell transplant. Sixty percent of pts received rituximab maintenance. Overall, 71 (44.4%) and 68 (42.5%) pts had ATM and TP53 aberration(s) (del/mut/del+mut), respectively. Of these, 20 (12.5%) pts had aberrations in both genes. Disruption of ATM gene was related to B-symptoms (p≤0.005). Age, sex, ECOG PS, cytomorphology, Ki67 index, MIPI and treatment response did not correlate with ATM aberration. With a median follow-up of 3.8 years, 2-year PFS and 2-year OS in all pts was 58.1% and 74.6%, respectively. Median PFS in pts with ATM mutation, ATM deletion and ATM+TP53 aberration was not reached, 3.3 and 0.7 years, respectively (p ≤ 0.005). Median OS in pts with ATM mutation, ATM deletion and ATM+TP53 aberration was not reached, 5.6 and 0.9 years, respectively (p ≤ 0.005). No significant difference in OS was found between ATM mutated and wild-type pts. (Survival graph below) Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, indolent non-Hodgkin lymphoma The research was funded by: IGA_LF_2023_005, MH CZ DRO-VFN64165, IGA_LF_2023_010, MH_CZ-DRO (FNOL, 00098892) No conflicts of interests pertinent to the abstract.

“Evaluation of ROS1 expression and rearrangements in a large cohort of early-stage lung cancer”

BackgroundROS1 fusion is an infrequent, but attractive target for therapy in patients with metastatic non- small-cell lung cancer. In studies on mainly late-stage disease, the prevalence of ROS1 fusions is about 1–3%. In early-stage lung cancer ROS1 might also provide a fruitful target for neoadjuvant or adjuvant therapy. In the present study, we investigated the prevalence of ROS1 fusion in a Norwegian cohort of early-stage lung cancer. We also explored whether positive ROS1 immunohistochemical (IHC) stain was associated with certain mutations, clinical characteristics and outcomes.MethodsThe study was performed using biobank material from 921 lung cancer patients including 542 patients with adenocarcinoma surgically resected during 2006–2018. Initially, we screened the samples with two different IHC clones (D4D6 and SP384) targeting ROS1. All samples that showed more than weak or focal staining, as well as a subgroup of negative samples, were analyzed with ROS1 fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) with a comprehensive NGS DNA and RNA panel. Positive ROS1-fusion was defined as those samples positive in at least two of the three methods (IHC, FISH, NGS).ResultsFifty cases were IHC positive. Of these, three samples were both NGS and FISH-positive and considered positive for ROS1 fusion. Two more samples were FISH positive only, and whilst IHC and NGS were negative. These were also negative with Reverse Transcription quantitative real time Polymerase Chain Reaction (RT-qPCR). The prevalence of ROS1 fusion in adenocarcinomas was 0.6%. All cases with ROS1 fusion had TP53 mutations. IHC-positivity was associated with adenocarcinoma. Among SP384-IHC positive cases we also found an association with never smoking status. There was no association between positive IHC and overall survival, time to relapse, age, stage, sex or pack-year of smoking.ConclusionsROS1 seems to be less frequent in early-stage disease than in advanced stages. IHC is a sensitive, but less specific method and the results need to be confirmed with another method like FISH or NGS.

DESIGNED ORF PROTEIN FOR SUSTAINABLE THIRD GENERATION BIOFUEL PRODUCTION

The increasing global energy demands and depletion of fossil fuels urge us to develop alternative energy resources. In recent years, algae has gained much attention as a third-generation biofuel feedstock. Among Algae, microalgae are considered a good source of biofuels because of their relatively more oil concentration and rapid production of biomass. Microalgae have the ability to produce a wide variety of biofuels including bio-oil, bio-diesel, bio-syngas, and bio-hydrogen. Commercial production of biofuels from microalgae is presently not in use because of the high expense of production. This research was designed to produce a hybrid ORF that might enhance the oil yielding ability of the microalgae by its translation into a working protein. Ban y using an intensive literature survey, 6 genes from 3 microalgae species were selected. Further, ORFs of selected genes were identified and Hybrid ORF was constructed by combining those ORFs. After that restriction enzyme analysis and thermodynamic analysis of the hybrid nucleotide sequence was carried out, and this showed that the hybrid sequence was stable. The Hybrid ORF nucleotide sequence was translated into the protein sequence. This protein sequence was used for the homology modeling of Hybrid ORF proteins. The protein conformation predicted by homology modeling was further verified by Ramachandran plot. The metabolic pathways were analyzed by KEGG, which showed that all selected genes were functioning in the biosynthesis of lipids. This showed that the Hybrid ORF constructed could be used in a way to enhance the oil content of microalgae for producing biofuel, particularly biodiesel. These results can be validated by further in vitro analysis and can be used for the construction of genetically engineered microalgae.

Anaplastic lymphoma kinase expression in PDGFRA-mutated gastrointestinal stromal tumors probably correlates with poor prognosis

BackgroundAnaplastic lymphoma kinase (ALK) overexpression and gene alterations have been detected in several mesenchymal tumors, with significant implications for diagnosis, therapy and prognosis. However, few studies have investigated the correlation between ALK expression status and clinicopathological characteristics in patients with gastrointestinal stromal tumors (GISTs).MethodsA total of 506 GIST patients were enrolled. Sanger sequencing was employed to detect c-KIT and PDGFRA gene mutations. The tissue microarray (TMA) technique and immunohistochemistry were employed to identify the ALK (clone: 1A4 and D5F3) expression status in the tumor tissues. The ALK gene variants of IHC-positive cases were analyzed by fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). The clinicopathological data were analyzed using SPSS Statistics 26.0.ResultsAmong the 506 GIST patients, the c-KIT mutation accounted for 84.2% (426/506), followed by PDGFRA mutation (10.3%, 52/506), while the wild-type accounted for the least (5.5%, 28/506). ALK-positive expression was detected in PDGFRA-mutant GISTs (7.7%, 4/52) but negative for c-KIT-mutant or wild-type GISTs by IHC. Four ALK IHC-positive patients were all male. The tumors all occurred outside the stomach. The predominant patterns of growth were epithelioid (2/4), spindle (1/4), and mixed type (1/4). They were all identified as high-risk classification according to the National Institutes of Health (NIH) classification. Aberrant ALK mutations were not identified by DNA-based NGS except in one of the 4 cases with amplification by FISH.ConclusionOur study revealed 7.7% (4/52) of ALK expression in PDGFRA-mutant GISTs, indicating that molecular tests were required to rule out the possibility of PDGFRA-mutant GISTs when encountering ALK-positive mesenchymal tumors with CD117-negative or weakly positive in immunohistochemical staining.

The challenges of the genome-based identification of antifungal resistance in the clinical routine.

The increasing number of chronic and life-threatening infections caused by antimicrobial resistant fungal isolates is of critical concern. Low DNA sequencing cost may facilitate the identification of the genomic profile leading to resistance, the resistome, to rationally optimize the design of antifungal therapies. However, compared to bacteria, initiatives for resistome detection in eukaryotic pathogens are underdeveloped. Firstly, reported mutations in antifungal targets leading to reduced susceptibility must be extensively collected from the literature to generate comprehensive databases. This information should be complemented with specific laboratory screenings to detect the highest number possible of relevant genetic changes in primary targets and associations between resistance and other genomic markers. Strikingly, some drug resistant strains experience high-level genetic changes such as ploidy variation as much as duplications and reorganizations of specific chromosomes. Such variations involve allelic dominance, gene dosage increments and target expression regime effects that should be explicitly parameterized in antifungal resistome prediction algorithms. Clinical data indicate that predictors need to consider the precise pathogen species and drug levels of detail, instead of just genus and drug class. The concomitant needs for mutation accuracy and assembly quality assurance suggest hybrid sequencing approaches involving third-generation methods will be utilized. Moreover, fatal fast infections, like fungemia and meningitis, will further require both sequencing and analysis facilities are available in-house. Altogether, the complex nature of antifungal resistance demands extensive sequencing, data acquisition and processing, bioinformatic analysis pipelines, and standard protocols to be accomplished prior to genome-based protocols are applied in the clinical setting.

Comparative genomic analysis of Colistin resistant Escherichia coli isolated from pigs, a human and wastewater on colistin withdrawn pig farm

In this study, genomic and plasmid characteristics of Escherichia coli were determined with the aim of deducing how mcr genes may have spread on a colistin withdrawn pig farm. Whole genome hybrid sequencing was applied to six mcr-positive E. coli (MCRPE) strains isolated from pigs, a farmworker and wastewater collected between 2017 and 2019. Among these, mcr-1.1 genes were identified on IncI2 plasmids from a pig and wastewater, and on IncX4 from the human isolate, whereas mcr-3 genes were found on plasmids IncFII and IncHI2 in two porcine strains. The MCRPE isolates exhibited genotypic and phenotypic multidrug resistance (MDR) traits as well as heavy metal and antiseptic resistance genes. The mcr-1.1-IncI2 and IncX4 plasmids carried only colistin resistance genes. Whereas, the mcr-3.5-IncHI2 plasmid presented MDR region, with several mobile genetic elements. Despite the MCRPE strains belonged to different E. coli lineages, mcr-carrying plasmids with high similarities were found in isolates from pigs and wastewater recovered in different years. This study highlighted that several factors, including the resistomic profile of the host bacteria, co-selection via adjunct antibiotic resistance genes, antiseptics, and/or disinfectants, and plasmid-host fitness adaptation may encourage the maintenance of plasmids carrying mcr genes in E. coli.

The Role of cMET in Gastric Cancer—A Review of the Literature

Gastric cancer (GC) is an important cause of cancer worldwide with over one million new cases yearly. The vast majority of cases present in stage IV disease, and it still bears a poor prognosis. However, since 2010, progress has been made with the introduction of targeted therapies against HER2 and with checkpoint inhibitors (PDL1). More agents interfering with other targets (FGFR2B, CLDN18.2) are being investigated. cMET is a less frequent molecular target that has been studied for gastric cancer. It is a proto-oncogene that leads to activation of the MAPK pathway and the PI3K pathway, which is responsible for activating the MTOR pathway. The prevalence of cMET is strongly debated as different techniques are being used to detect MET-driven tumors. Because of the difference in diagnostic assays, selecting patients who benefit from cMET inhibitors is difficult. In this review, we discuss the pathway of cMET, its clinical significance and the different diagnostic assays that are currently used, such as immunohistochemy (IHC), fluorescence in situ hybridization (FISH), the H-score and next-generation sequencing (NGS). Next, we discuss all the current data on cMET inhibitors in gastric cancer. Since the data on cMET inhibitors are very heterogenous, it is difficult to provide a general consensus on the outcome, as inclusion criteria differ between trials. Diagnosing cMET-driven gastric tumors is difficult, and potentially the only accurate determination of cMET overexpression/amplification may be next-generation sequencing (NGS).

The ABC-D of animal linguistics: are syntax and compositionality for real?

In several animal species, an alarm call (e.g. ABC notes in the Japanese tit Parus minor) can be immediately followed by a recruitment call (e.g. D notes) to yield a complex call that triggers a third behaviour, namely mobbing. This has been taken to be an argument for animal syntax and compositionality (i.e. the property by which the meaning of a complex expression depends on the meaning of its parts and the way they are put together). Several additional discoveries were made across species. First, in some cases, animals respond with mobbing to the order alarm-recruitment but not to the order recruitment-alarm. Second, animals sometimes respond similarly to functionally analogous heterospecific calls they have never heard before, and/or to artificial hybrid sequences made of conspecific and heterospecific calls in the same order, thus adding an argument for the productivity of the relevant rules. We consider the details of these arguments for animal syntax and compositionality and argue that, with one important exception (Japanese tit ABC-D sequences), they currently remain ambiguous: there are reasonable alternatives on which each call is a separate utterance and is interpreted as such ('trivial compositionality'). More generally, we propose that future studies should argue for animal syntax and compositionality by explicitly pitting the target theory against two deflationary analyses: the 'only one expression' hypothesis posits that there is no combination in the first place, for example just a simplex ABCD call; while the 'separate utterances' hypothesis posits that there are separate expressions (e.g. ABC and D), but that they form separate utterances and are neither syntactically nor semantically combined.

Clinicopathologic characteristics, genetic features, and treatment options for acute lymphoblastic leukemia with JAK2 rearrangement—A 10-case study and literature review

JAK2 rearrangement (JAK2-R) in acute lymphoblastic leukemia (ALL) is rare and often categorized as B-ALL with BCR::ABL1-like features based on the World Health Organization classification. We report 10 patients with JAK2-R ALL, 9 males and 1 female, with a median age 40.5 years. Eight patients presented with marked leukocytosis (median WBC, 63 × 10 9/L) and hypercellular (>95%) bone marrow with increased lymphoblasts (72%-95%). There was no evidence of bone marrow fibrosis or hypereosinophilia. Immunophenotypic analysis showed 9 B-cell and 1 T-cell neoplasms. Using fluorescence in situ hybridization (FISH) and RNA sequencing analysis, JAK2 partners were identified for 7 cases and included PCM1 (n=4), ETV6 (n=2) and BCR (n=1). All patients received upfront polychemotherapy. Additionally, 2 patients received ruxolitinib, 2 received allogeneic stem cell transplant, and 1 received CAR-T therapy. The 1- and 3-year overall survival rates were 55.6% and 22.2%, respectively. A literature review identified 24 B-ALL and 4 T-ALL cases with JAK2-R reported, including 16 males, 6 females and 6 gender not stated. Many JAK2 partner-genes were reported with the most common being PAX5 (n=7), ETV6 (n=4), BCR (n=3) and PCM1 (n=2). Survival data or 13 reported cases showed 1- and 3-year overall survival rates of 41.7% and 41.7%, respectively. In summary, JAK2-R ALL occurs more often in adult males, are mostly of B-cell lineage, and associated with an aggressive clinical course. Absence of eosinophilia and bone marrow fibrosis and no evidence of preexisting/concurrent JAK2-R myeloid neoplasms distinguish JAK2-R ALL from other myeloid/lymphoid neoplasms with eosinophilia and JAK2-R.

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets.

Protein-protein interactions that involve recognition of short peptides are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are a family of peptide-binding domains located in several intracellular signaling and trafficking pathways. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had marginal effects on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell.

MicroRNA Target Identification: Revisiting Accessibility and Seed Anchoring.

By pairing to messenger RNAs (mRNAs for short), microRNAs (miRNAs) regulate gene expression in animals and plants. Accurately identifying which mRNAs interact with a given miRNA and the precise location of the interaction sites is crucial to reaching a more complete view of the regulatory network of an organism. Only a few experimental approaches, however, allow the identification of both within a single experiment. Computational predictions of miRNA-mRNA interactions thus remain generally the first step used, despite their drawback of a high rate of false-positive predictions. The major computational approaches available rely on a diversity of features, among which anchoring the miRNA seed and measuring mRNA accessibility are the key ones, with the first being universally used, while the use of the second remains controversial. Revisiting the importance of each is the aim of this paper, which uses Cross-Linking, Ligation, And Sequencing of Hybrids (CLASH) datasets to achieve this goal. Contrary to what might be expected, the results are more ambiguous regarding the use of the seed match as a feature, while accessibility appears to be a feature worth considering, indicating that, at least under some conditions, it may favour anchoring by miRNAs.

Fluorescence activated cell sorting and fermentation analysis to study rumen microbiome responses to administered live microbials and yeast cell wall derived prebiotics.

Rapid dietary changes, such as switching from high-forage to high-grain diets, can modify the rumen microbiome and initiate gastrointestinal distress, such as bloating. In such cases, feed additives, including prebiotics and live microbials, can be used to mitigate these negative consequences. Bio-Mos® is a carbohydrate-based prebiotic derived from yeast cells that is reported to increase livestock performance. Here, the responses of rumen bacterial cells to Bio-Mos® were quantified, sorted by flow cytometry using fluorescently-labeled yeast mannan, and taxonomically characterized using fluorescence in situ hybridization and 16S rRNA sequencing. Further, to evaluate the effects of bovine-adapted Bacteroides thetaiotaomicron administration as a live microbial with and without Bio-Mos® supplementation, we analyzed microbial fermentation products, changes to carbohydrate profiles, and shifts in microbial composition of an in vitro rumen community. Bio-Mos® was shown to be an effective prebiotic that significantly altered microbial diversity, composition, and fermentation; while addition of B. thetaiotaomicron had no effect on community composition and resulted in fewer significant changes to microbial fermentation. When combined with Bio-Mos®, there were notable, although not significant, changes to major bacterial taxa, along with increased significant changes in fermentation end products. These data suggest a synergistic effect is elicited by combining Bio-Mos® and B. thetaiotaomicron. This protocol provides a new in vitro methodology that could be extended to evaluate prebiotics and probiotics in more complex artificial rumen systems and live animals.

Functional Characterization of Transgenic Mice Overexpressing Human 15-Lipoxygenase-1 (ALOX15) under the Control of the aP2 Promoter.

Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory, hyperproliferative, neurodegenerative, and metabolic diseases, but the physiological function of ALOX15 still remains a matter of discussion. To contribute to this discussion, we created transgenic mice (aP2-ALOX15 mice) expressing human ALOX15 under the control of the aP2 (adipocyte fatty acid binding protein 2) promoter, which directs expression of the transgene to mesenchymal cells. Fluorescence in situ hybridization and whole-genome sequencing indicated transgene insertion into the E1-2 region of chromosome 2. The transgene was highly expressed in adipocytes, bone marrow cells, and peritoneal macrophages, and ex vivo activity assays proved the catalytic activity of the transgenic enzyme. LC-MS/MS-based plasma oxylipidome analyses of the aP2-ALOX15 mice suggested in vivo activity of the transgenic enzyme. The aP2-ALOX15 mice were viable, could reproduce normally, and did not show major phenotypic alterations when compared with wildtype control animals. However, they exhibited gender-specific differences with wildtype controls when their body-weight kinetics were evaluated during adolescence and early adulthood. The aP2-ALOX15 mice characterized here can now be used for gain-of-function studies evaluating the biological role of ALOX15 in adipose tissue and hematopoietic cells.

Direct discovery of the inner exoplanet in the HD 206893 system

Aims. HD 206893 is a nearby debris disk star that hosts a previously identified brown dwarf companion with an orbital separation of ∼10 au. Long-term precise radial velocity (RV) monitoring, as well as anomalies in the system proper motion, has suggested the presence of an additional, inner companion in the system. Methods. Using information from ongoing precision RV measurements with the HARPS spectrograph, as well as Gaia host star astrometry, we have undertaken a multi-epoch search for the purported additional planet using the VLTI/GRAVITY instrument. Results. We report a high-significance detection over three epochs of the companion HD 206893c, which shows clear evidence for Keplerian orbital motion. Our astrometry with ∼50−100 μarcsec precision afforded by GRAVITY allows us to derive a dynamical mass of 12.7$ ^{+1.2}_{-1.0} $MJup and an orbital separation of 3.53$ ^{+0.08}_{-0.06} $ au for HD 206893c. Our fits to the orbits of both companions in the system use both Gaia astrometry and RVs to also provide a precise dynamical estimate of the previously uncertain mass of the B component, and therefore allow us to derive an age of 155 ± 15 Myr for the system. We find that theoretical atmospheric and evolutionary models that incorporate deuterium burning for HD 206893c, parameterized by cloudy atmosphere models as well as a “hybrid sequence” (encompassing a transition from cloudy to cloud-free), provide a good simultaneous fit to the luminosity of both HD 206893B and c. Thus, accounting for both deuterium burning and clouds is crucial to understanding the luminosity evolution of HD 206893c. Conclusions. In addition to using long-term RV information, this effort is an early example of a direct imaging discovery of a bona fide exoplanet that was guided in part by Gaia astrometry. Utilizing Gaia astrometry is expected to be one of the primary techniques going forward for identifying and characterizing additional directly imaged planets. In addition, HD 206893c is an example of an object narrowly straddling the deuterium-burning limit but unambiguously undergoing deuterium burning. Additional discoveries like this may therefore help clarify the discrimination between a brown dwarf and an extrasolar planet. Lastly, this discovery is another example of the power of optical interferometry to directly detect and characterize extrasolar planets where they form, at ice-line orbital separations of 2−4 au.

The diagnostic yield of CGH and WES in neurodevelopmental disorders.

Neurodevelopmental disorders are a group of conditions characterized by developmental delays leading to abnormal brain functions. The methods of diagnosis and treatment of these conditions are complicated, and their treatment involves a combination of various forms of therapy. In recent years, the development of high-resolution technologies has played an important role in revealing the microdeletions, microduplications, and single-nucleotide variants of the chromosomes and how they are linked to the development of neurodevelopmental disorders. The wide implementation and application of molecular methodologies have started to shed light on the functional importance of using the appropriate methods in detecting these genetic variations that are categorized as either pathogenic or benign. The study aimed to compare the diagnostic yield of comparative hybridization (CGH) and whole exome sequencing (WES) in neurodevelopmental disorders among children attending the King Abdullah Specialist Children Hospital, Riyadh, Saudi Arabia. A retrospective study was conducted between 2015 and 2018 on 105 patients diagnosed with neurodevelopmental disorders through array-based CGH (Array-CGH) and WES. In a sample of 105 patients, 16% was the hit rate of copy number variations (CNVs). WES was requested for CNV-negative patients (n = 79), of which 30% was the hit rate of pathogenic or likely pathogenic single-nucleotide variants. There was a difference in the diagnostic yield between CGH (16%) and WES (30%). WES was a better approach than Array-CGH to detect various DNA mutations or variants. Our findings could guide clinicians, researchers, and testing laboratories select the most cost-effective and appropriate approach for diagnosing their patients.

Prevalence of IRF4 rearrangement in large B-cell lymphomas of the Waldeyer’s ring in adults

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma (B-NHL) in adults. These lymphomas are classified according to gene expression profiling (GEP) into germinal center B-cell (GCB) and activated B-cell type (ABC). Recent studies have suggested new subtypes of large B-cell lymphoma, based on genetic and molecular alterations, among them is large B-cell lymphoma with IRF4-rearrangement (LBCL-IRF4). We used fluorescence in situ hybridization (FISH), GEP (using the DLBCL COO assay by HTG Molecular Inc), and next generation sequencing (NGS) to comprehensively characterize 30 cases of LBCLs located in Waldeyer’s ring in adult patients and to identify LBCL-IRF4. FISH revealed breaks of IRF4 in 2/30 cases (6.7%), BCL2 breaks in 6/30 cases (20.0%), and IGH breaks in 13/29 cases (44.8%). GEP classified 14 cases each as GCB or ABC subtype, and 2 cases remained unclassified; this was concordant with the immunohistochemistry (IHC) in 25/30 cases (83.3%). A subgrouping, based on GEP, was performed: group 1 included 14 GCB cases with the most frequent mutations in BCL2 and EZH2 in 6/14 cases (42.8%). The two cases with IRF4 rearrangement were assigned to this group by GEP and showed IRF4 mutations, supporting the diagnosis of LBCL-IRF4. Group 2 included 14 ABC cases; the most frequent mutations were CD79B and MYD88 identified in 5/14 patients (35.7%). Group 3 included 2 unclassifiable cases in which no molecular patterns were detected. Overall, LBCLs of Waldeyer’s ring in adult patients are a heterogeneous group, including LBCL-IRF4, which shares several features with cases in the pediatric population.

Leiomyoma-like Morphology in Metastatic Uterine Inflammatory Myofibroblastic Tumors

Uterine inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal neoplasms that frequently harbor ALK gene rearrangements and have a low risk of metastasis. We reported 3 of these tumors mimicking the appearance of leiomyoma in their recurrence. These patients were 34, 43, and 45 years old. Two uterine tumors demonstrated classic morphology, with combined myxoid, compact fascicular, and hyalinized patterns and spindled cells with bipolar cytoplasmic processes, moderate atypia, and lymphoplasmacytic inflammatory infiltrates. The third had a “leiomyoma-like” appearance, with fascicles of plump spindled cells and a sparse lymphoplasmacytic infiltrate. ALK immunohistochemistry was positive in all the tumors, and all demonstrated ALK rearrangements using fluorescence in situ hybridization (n = 2) and/or RNA sequencing (n = 2). Two classic IMTs recurred at 3 and 50 months in the lung and abdomen, respectively, and recurrent tumors had a “leiomyoma-like” appearance, with 0 and 1 mitosis per 10 high-power fields, no inflammation in 1, and a sparse lymphocytic infiltrate in the other. ALK was positive in both tumors; 1 with available tissue showed an IGFBP5::ALK fusion using RNA sequencing. The third patient, who had a “leiomyoma-like” uterine tumor, experienced multiple recurrences, first in the abdomen at 100 months showing a similar appearance. Subsequent recurrence at 105 months showed transmural invasion of the sigmoid colon and a similar microscopic appearance but with the addition of infiltrative borders, moderate cellularity, mild-to-moderate atypia, and 10 mitoses per 10 high-power fields. Both recurrences were positive for ALK, and RNA sequencing revealed the same ACTG2::ALK fusion transcript identified in the primary tumor. The patient was treated with crizotinib, resulting in prolonged clinical remission, with no evidence of disease at 168 months from the initial surgery. Although “leiomyoma-like” uterine IMTs have been recently described, to our knowledge, this is the first report of recurrence of these tumors and the first report of a “leiomyoma-like” appearance in the recurrences of conventional uterine IMTs. A low threshold for performing ALK immunohistochemistry on recurrent uterine tumors can identify patients who may benefit from tyrosine kinase inhibitors.

Inflammatory and Nested Testicular Sex Cord Tumor: A Novel Neoplasm With Aggressive Clinical Behavior and Frequent EWSR1::ATF1 Gene Fusions.

A subset of malignant testicular sex cord tumors (TSCTs), heretofore interpreted as Sertoli cell tumors, not otherwise specified, exhibits distinctive morphologic features that partially overlap with those of seminoma. In this study, we evaluated the clinicopathologic and molecular characteristics of 13 such tumors. The patients were 20 to 73 years old (median, 36y), and all with available data presented with testicular masses (median size, 3cm), with 2 having synchronous retroperitoneal metastases. All 11 patients with available follow-up developed metastases to retroperitoneal lymph nodes, nonretroperitoneal lymph nodes, bone, contralateral testis, and/or lung. Microscopically, the tumors showed solid nests and sheets of epithelioid cells with granular, eosinophilic to clear/vacuolated cytoplasm, admixed in most (12/13) cases with variable proportions of lymphocytes, plasma cells, eosinophils, and neutrophils. Additional features included intracytoplasmic hyaline inclusions and a prominent collagenous, sometimes hyalinized stroma. Mitotic activity was relatively low (median, 1 mitosis/10HPF), but tumor necrosis was frequent (11/13). Local invasion of adjacent structures and lymphovascular invasion were noted in some tumors (4/9 cases with available data for each feature). All were α-inhibin-positive and lacked nuclear reactivity for β-catenin. In addition, all tested cases were positive for epithelial membrane antigen (9/9) and steroidogenic factor-1 (8/8), and 8/10 expressed CD30. Two "index" cases were initially analyzed using a DNA sequencing panel, which identified EWSR1::ATF1 fusions in both. Subsequently, EWSR1::ATF1 fusions were demonstrated in 8 of the remaining 11 cases using fluorescence in situ hybridization or DNA sequencing. One of the 3 cases that were negative for EWSR1::ATF1 harbored ATF1 amplification. This study, therefore, shows that a group of malignant TSCTs resembling seminoma is characterized by α-inhibin and steroidogenic factor-1 positivity, no expression of nuclear β-catenin, frequent CD30 positivity and recurrent EWSR1::ATF1 fusions. We have descriptively termed these neoplasms "inflammatory and nested TSCT." Importantly, inflammatory and nested TSCTs show significant differences in morphology, immunoprofile, molecular biology, and, likely, clinical behavior from Sertoli cell tumors, not otherwise specified and should be classified separately.

Comparative analysis of histopathological parameters, genome-wide copy number alterations, and variants in genes involved in cell cycle regulation in chordomas of the skull base and sacrum.

Chordomas are rare tumors of the axial skeleton that are refractory to conventional therapy. Few studies have compared the morphological and molecular characteristics of chordomas according to the skull base and sacral locations. Histopathological data and changes revealed by array comparative genomic hybridization (CGH) and next-generation sequencing (NGS) of cell cycle regulation genes were analyzed for 28 skull base (SBCs) and 15 sacral (SC) chordomas. All cases were conventional chordomas. SBCs were significantly more frequent in patients aged <40 years and SCs predominated in patients aged >60 years. Mitotic indices ≥2 mitoses/10 high-power fields were correlated with high degrees of nuclear atypia and Ki67 labeling indices ≥6%. We identified 321 genomic positions, and copy number variation losses were more frequent than gain. Moreover, we report a panel of 85 genetic variants of cell cycle genes and the presence of molecular clusters for chordoma as well in CGH as in NGS. These new data strengthen the view that the chordoma should not be considered as a single molecular entity.