Hybridization Sequencing Research Articles

Malakoplakia with aberrant ALK expression by immunohistochemistry: a case report

BackgroundMalakoplakia is a rare inflammatory disease of the urogenital tract. There have been no reports of malakoplakia expressing anaplastic lymphoma kinase (ALK) to date. Here, we present one case of malakoplakia with aberrant ALK expression by immunohistochemistry and discuss the clinical significance.Case presentationA 65-year-old Chinese woman with a history of diabetes presented with solid masses in the liver and kidney and elevated lesions on the mucosal surface of the colon. Right nephrectomy and partial liver resection were performed. Microscopically, sheets of histiocytes with poor intercellular adhesion were seen, with Michaelis–Gutmann bodies present in both the intracellular and extracellular interstitium. CD10-, CD68-, and CD163-positive cells were present, with Michaelis–Gutmann bodies confirmed by staining with Alcian blue, periodic acid-Schiff (PAS), periodic acid-Schiff with diastase, Von Kossa, and Prussian blue. Aberrant ALK1 and ALK (D5F3) expression was observed in the cytoplasm and nucleus of cells. However, ALK gene mutation was not detected by fluorescence in situ hybridization or whole exome next-generation sequencing. NGS revealed nine individual somatic gene mutations: GOT1L1, GLIS2, SPOUT1, TMEM97, MUC3A, NSD2, SFXN5, ADAD1 and RAD50. The significance of the somatic gene mutations detected in this study is not clear, and the relationship between them and malakoplakia cannot be clarified by existing scientific studies. The pathological diagnosis was malakoplakia with aberrant ALK expression by immunohistochemistry. The antibiotics imipenem and vancomycin were started based on the results of drug sensitivity analysis and the patient was subsequently discharged. She experienced no discomfort during 30 months of follow-up.ConclusionThis is the first reported case of malakoplakia with aberrant ALK expression, it should be differentiated from ALK-positive histiocytosis to avoid misdiagnosis.

Understanding the Hybridization of Everyday Activities from a Time-Geographic Perspective

In contrast to the dichotomous distinction between offline and online activity in much previous research, we argue for hybridization as a key feature of the digitalized postpandemic society, shaping and constraining the everyday lives and activities of individuals in new, unforeseen ways. The current understanding of the complex ways in which hybridization plays out and reorders everyday life is limited, partly due to a lack of relevant conceptualizations and methodological tools. The aim of this article is to further develop the time-geographic approach, as theory and method, to understand and visualize the hybridization of everyday activities. The article contributes to previous literature on everyday life digitalization in several important respects. The notions of hybrid activities, grounds, and sequences are proposed for an enhanced theoretical understanding of hybridization. Moreover, we argue that interrelated hybrid constraints shape the spatiotemporal organization of everyday activities. The concept of pocket of mediated order is proposed as a new domain of everyday activities in the hybrid era, supporting the accomplishment of everyday projects, yet also transforming the local pockets of order. Finally, drawing on a real family case, we refine the time-geographical notation system to capture and visualize the full complexity of hybridization in the time–space setting of daily life.

Isolation and characterization of Enterococcus faecalis isolate VT-H1: A highly efficient hydrogen-producing bacterium from palm oil mill effluent (POME)

Facultative hydrogen-producing bacteria hold immense potential for fermentative hydrogen and valuable metabolite production. In this study, a mesophilic hydrogen-producing bacterium was isolated from palm oil mill effluent (POME) and identified as Enterococcus faecalis isolate VT-H1 using hybrid sequencing technologies. Our investigation encompassed the growth of the isolate and its capacity for hydrogen and volatile fatty acid (VFA) production. The results revealed that optimal growth occurred when glucose was utilized at 37 °C and initial pH of 7.0, resulting in a final OD600 of 2.58. The isolate demonstrated exceptional hydrogen production capabilities, achieving a maximum cumulative hydrogen yield of 2.22 mol-H2/mol-glucose at 30 °C and pH 7.0, outperforming previously reported E. faecalis strains by 5.84-fold. Kinetic analysis revealed favorable parameters for hydrogen production at 30 °C, with maximum hydrogen production (Hm = 2.63 mol-H2/mol-glucose), maximum hydrogen production rate (Rm = 0.03 mol-H2/mol-glucose·h), and lag-phase time (LPT = 18.34 h). Additionally, E. faecalis isolate VT-H1 exhibited significant butyric acid production (63.0 mM) alongside acetic acid (7.0 mM). Genome analysis unveiled the presence of key enzymes associated with hydrogen production, including formate hydrogen lyase (FHL) complex with formate dehydrogenase subunit alpha (FDH-α), bidirectional [NiFe] hydrogenase, NADH-dependent oxidoreductase subunit E, and NADH-quinone oxidoreductase subunit F. Moreover, genes associated with butyric acid production were also identified. These findings highlight the potential of E. faecalis isolate VT-H1 as a promising candidate for sustainable hydrogen and VFA production. Its unique characteristics, distinct genetic profile, and exceptional hydrogen and VFA production capabilities bring exciting opportunities for future biotechnological applications and further research to optimize and scale-up its potential for industrial use.

Development of DNA markers using next-generation sequencing approach for molecular authentication of Boerhavia diffusa L. and Tinospora cordifolia (Willd.) Miers

The adulteration of plants and their materials used in herbal formulations poses a severe health concern. Hence, there is a need to establish a reliable, cost-effective, and robust molecular biomarker to distinguish among species and identify herbal plants and raw drugs from adulterants. The present study used suppressive subtractive hybridization and next-generation sequencing technology to identify novel DNA markers for Boerhavia diffusa L. and Tinospora cordifolia (Willd.) Miers. We identified two primer sets for B. diffusa and one for T. cordifolia. The DNA markers were validated in different accessions of B. diffusa and T. cordifolia and their common adulterants to determine the sensitivity and specificity of developed DNA markers. The designed DNA markers showed 100% sensitivity and specificity in detecting B. diffusa and T. cordifolia from their adulterants. The strategy described here can be extrapolated for developing DNA markers to authenticate other plant species.

The ABCB1 C3435T Polymorphism is Associated with Triglyceride Reduction in Atorvastatin-treated Uygur Patients with Coronary Heart Disease and Dyslipidemia: An Observational Study.

The morbidity of coronary heart disease (CHD) and dyslipidemia in the Uygur population of Xinjiang is higher than the national average. Interindividual variability of the response to atorvastatin is a major clinical problem; generally, statins shed less impressive benefits for females than males. Nevertheless, it is unclear whether ABCB1 genes and sex modify the efficacy of atorvastatin in Uygur patients. To determine the impact of ABCB1 gene polymorphisms on the therapeutic response to atorvastatin in a Uygur population with dyslipidemia. Patients with dyslipidemia were treated with 20 mg/d or 40 mg/d atorvastatin for two to six months. TC, LDL-C, HDL-C, TG, APOB, APOE, LP(a), and APOA1 levels were measured before and after atorvastatin administration. We performed genotyping of ABCB1 C3435T and G2677T variants using hybridization sequencing. The association of variants between the percentage of change in TG levels was examined using multiple linear regression analysis. We enrolled 193 Uygur patients. Atorvastatin reduced TG, LDL-C, TC, APOB, and APOE levels (P < 0.05), whereas LP(a) and APOA1 levels increased (P < 0.05). In multiple linear regression analysis, baseline TG level (beta 0.204; 95% confidence interval (CI): 1.980-10.493; P = 0.004) and TT genotype of ABCB1 C3435T (beta 0.162; 95% CI: 2.517-23.406; P = 0.023) predicted TG reduction with atorvastatin therapy in overall patients. Baseline TG level (beta 0.346; 95% CI: 4.374 -13.34; P < 0.001) with the TT genotype of ABCB1 C3435T (beta 0.401; 95% CI: 4.053-28.356; P = 0.021) was associated with a significant reduction in TG levels in men. Only baseline TG level predicted TG reduction within six months of atorvastatin therapy for females (beta 0.61; 95% CI: 3.204-20.557; P = 0.041). In patients with the ABCB1 C3435T TT genotype, atorvastatin more effectively lowered TG than other polymorphisms. This investigation may provide insights into effective individualized therapies for CHD and dyslipidemia in the Uygur population.

Detecting intragenic trans-splicing events from non-co-linearly spliced junctions by hybrid sequencing.

Trans-spliced RNAs (ts-RNAs) are a type of non-co-linear (NCL) transcripts that consist of exons in an order topologically inconsistent with the corresponding DNA template. Detecting ts-RNAs is often interfered by experimental artifacts, circular RNAs (circRNAs) and genetic rearrangements. Particularly, intragenic ts-RNAs, which are derived from separate precursor mRNA molecules of the same gene, are often mistaken for circRNAs through analyses of RNA-seq data. Here we developed a bioinformatics pipeline (NCLscan-hybrid), which integrated short and long RNA-seq reads to minimize false positives and proposed out-of-circle and rolling-circle long reads to distinguish between intragenic ts-RNAs and circRNAs. Combining NCLscan-hybrid screening and multiple experimental validation steps successfully confirmed that four NCL events, which were previously regarded as circRNAs in databases, originated from trans-splicing. CRISPR-based endogenous genome modification experiments further showed that flanking intronic complementary sequences can significantly contribute to ts-RNA formation, providing an efficient/specific method to deplete ts-RNAs. We also experimentally validated that one ts-RNA (ts-ARFGEF1) played an important role for p53-mediated apoptosis through affecting the PERK/eIF2a/ATF4/CHOP signaling pathway in breast cancer cells. This study thus describedboth bioinformatics procedures and experimental validation steps for rigorous characterization of ts-RNAs, expanding future studies for identification, biogenesis, and function of these important but understudied transcripts.

Effect of hybridization and stacking sequences on mechanical properties and thermal stability of aloe vera‐roselle‐glass fiber reinforced polymer composites

AbstractEnvironment friendly polymer composites made of plant based natural fibers such as bamboo fiber, roselle fiber (RF), aloe vera fiber (AF) oil, or kenaf fiber are cardinal of the current world toward sustainability. They offer lower carbon footprint, higher biodegradability, higher specific strength, higher thermal, and acoustic characteristics. On the other hand, properties of synthetic glass fibers (GFs) such as high specific strength to weight ratio, great resistance during impact, and high durability extend their application perspective for various engineering materials. As such, in the present study, mechanical properties and thermal stability of hybrid laminate comprising of natural fiber (i.e., AF and RF) and synthetic fiber (i.e., GF) fabricated using hand layup process were investigated. Experimental findings reveal that sequencing of the fibers significantly affects properties of the composite. Introducing a layer of AF between RF increased the tensile strength by 30.2% respectively while hybrid laminates composed of three successive layers of RF exhibited 12.6% higher impact strength compared to hybrid laminate composed of AF. Besides, the thermal stability of hybrid laminates was higher (i.e., minimal weight loss of &lt;6.1% when heated up to 800°) compared to neat polymer or laminates with single reinforcement. The enhanced thermal stability, mechanical properties, and tribological properties of the “greener” hybrid laminates can be employed in various structural or lightweight industrial applications.Highlights Hybridization using natural fibers and synthetic fibers are attempted and investigated. Fiber sequencing significantly affects mechanical properties of the composite. Higher thermal stability of hybrid laminates, that is, minimal weight loss of &lt;6.1% when heated up to 800°. Laminated composed of AF in between RF exhibited lowest frictional coefficient. “Green” hybrid polymer laminate suitable for various structural and lightweight industrial applications is identified.

Significance of YAP1–MAML2 rearrangement and GTF2I mutation in the diagnosis and differential diagnosis of metaplastic thymoma

Background Metaplastic thymoma (MT) is a very uncommon thymoma type, with biphasic differentiation as one of its histological characteristics. This histological pattern, however, can also be mistaken for type A thymoma and the A component in type AB thymoma. Methods Postoperative specimens were collected from five MT and four type A thymomas with a retrospective analysis involving immunohistochemistry, fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). Results The histological morphology of the MT overlapped with that of the type A thymoma. With immunostains, the former’s spindle cell components expressed vimentin and EMA, but not CD20. In MT, 3/5 cases had the nuclear expression of YAP1. The spindle cell component of the type A thymoma was found to express CD20. In all five cases of MT, FISH detection revealed YAP1–MAML2 fusion, which was not found in any type A thymoma cases. NGS sequencing confirmed YAP1–MAML2 rearrangement in all five cases of MT, and mutations in POLE and HRAS were also found in two cases, respectively. GTF2I c.74146970 T > A mutations were found in all cases of type A thymoma, and HRAS and NRAS mutations were found in two cases, but no YAP1–MAML2 rearrangement was evident. Conclusions For the diagnosis and differential diagnosis of challenging cases, the YAP1–MAML2 rearrangement and GTF2I mutation were both significant molecular events specific to MT and type A thymoma, respectively.

Importance of PLD2 in an IL-23 driven psoriasiform dermatitis model and potential link to human psoriasis.

Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.

Bipolar Biogeographical Distribution of Parafrancisella Bacteria Carried by the Ciliate Euplotes.

Parafrancisella adeliensis, a Francisella-like endosymbiont, was found to reside in the cytoplasm of an Antarctic strain of the bipolar ciliate species, Euplotes petzi. To inquire whether Euplotes cells collected from distant Arctic and peri-Antarctic sites host Parafrancisella bacteria, wild-type strains of the congeneric bipolar species, E. nobilii, were screened for Parafrancisella by in situ hybridization and 16S gene amplification and sequencing. Results indicate that all Euplotes strains analyzed contained endosymbiotic bacteria with 16S nucleotide sequences closely similar to the P. adeliensis 16S gene sequence. This finding suggests that Parafrancisella/Euplotes associations are not endemic to Antarctica, but are common in both the Antarctic and Arctic regions.

An ovarian phenotype of alpha 7 nicotinic receptor knockout mice.

Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is expressed by various murine ovarian cells. Morphological and molecular investigations, including a proteomic study of adult Chrna7 knockout (KO) mouse ovaries, reveal the roles of these receptors in the local regulation of the ovary. Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is involved in cellular functions ranging from synaptic transmission in neurons to regulation of inflammation, cell growth and metabolism to cell death in other cells. Our qPCR results and other studies indicated that nAChRa7 is expressed in the adult mouse ovary, while in situ hybridization and single-cell sequencing data suggested this expression may be shared by several ovarian cells, including fibroblast-like and steroidogenic stroma cells, macrophages and oocytes of small follicles. To explore a possible involvement of nAChRa7 in ovarian functions, we evaluated ovarian morphology of Chrna7-null mutant adult mice (KO) and wildtype mice (WT; 3 months, metestrus) by performing immunohistochemistry, qPCR studies, measurements of serum progesterone and proteomic analyses. The evaluation of serial sections indicated fewer primordial follicles but similar numbers of primary, secondary and tertiary follicles, as well as corpora lutea in KO and WT mice. Atresia was unchanged. Serum progesterone and mRNA levels of proliferation and most apoptosis markers were not changed, yet two typical macrophage markers were elevated. Furthermore, the proteomes of KO ovaries were significantly altered with 96 proteins increased and 32 decreased in abundance in KOs compared to WTs. Among the elevated proteins were markers for stroma cells. Hence, the lack of nAChRa7 causes changes in small follicle counts and alterations of the ovarian stroma cells. The ovarian phenotype of Chrna7 mutant mice links this channel protein to the local regulation of ovarian cells, including stroma cells.

Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5′-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Virus impacted community adaptation in oligotrophic groundwater environment revealed by Hi-C coupled metagenomic and viromic study

Viruses play a crucial role in microbial mortality, diversity and biogeochemical cycles. Groundwater is the largest global freshwater and one of the most oligotrophic aquatic systems on Earth, but how microbial and viral communities are shaped in this special habitat is largely unexplored. In this study, we collected groundwater samples from 23 to 60 m aquifers at Yinchuan Plain, China. In total, 1920 non-reductant viral contigs were retrieved from metagenomes and viromes constructed by Illumina and Nanopore hybrid sequencing. Only 3% of them could be clustered with known viruses, most of which were Caudoviricetes. Coupling 1.2 Tb Hi-C sequencing with CRISPR matching and homology search, we connected 469 viruses with their hosts while some viral clusters presented a broad-host-range trait. Meanwhile, a large proportion of biosynthesis related auxiliary metabolism genes were identified. Those characteristics might benefit viruses for a better survival in this special oligotrophic environment. Additionally, the groundwater virome showed genomic features distinct from those of the open ocean and wastewater treatment facilities in GC distribution and unannotated gene compositions. This paper expands the current knowledge of the global viromic records and serves as a foundation for a more thorough understanding of viruses in groundwater.

Clinical, pathological, and comprehensive molecular analysis of the uterine clear cell carcinoma: a retrospective national study from TMRG and GINECO network

BackgroundUterine clear cell carcinomas (CCC) represent less than 5% of uterine cancers. Their biological characteristics and clinical management remain uncertain. A multicenter study to explore both clinical and molecular features of these rare tumors was conducted.MethodsThis multicenter retrospective national study was performed within the French TMRG (Rare Gynecologic Malignant Tumors) network. Clinical data and, when available, FFPE blocks were collected. Clinical features, treatments, and outcome (progression-free survival (PFS) and overall survival (OS)) were analyzed and correlated to the protein (tissue micro-array), RNA (Nanostring nCounter® technology), and DNA (array-Comparative Genomic hybridization and target-next generation sequencing) levels using the tumor samples available.ResultsSixty-eight patients with uterine CCC were enrolled, 61 from endometrial localization and 5 with cervix localization. Median age at diagnosis was 68.9 years old (range 19–89.7). Most tumors were diagnosed at an early stage (78% FIGO stage I–II). Hysterectomy (performed in 90%) and lymph node dissection (80%) were the most frequent surgical treatment. More than 70% of patients received external beam radiotherapy and 57% received brachytherapy. Nearly half (46%) of the patients received chemotherapy. After a median follow-up of 24.7 months, median PFS was 64.8 months (95 CI [5.3–124.4]) and median OS was 79.7 (IC95 [31.0–128.4]). Low hormone receptor expression (13% estrogen-receptor positive), frequent PI3K pathway alterations (58% PTEN loss, 50% PIK3CA mutations), and P53 abnormalities (41%) were observed. Mismatch repair deficiency was identified in 20%. P16 expression was associated with shorter PFS (HR = 5.88, 95 CI [1.56–25], p = 0.009). Transcriptomic analyzes revealed a specific transcriptomic profile notably with a high expression of immune response-associated genes in uterine CCC displaying a very good overall prognosis.ConclusionsUterine CCC reported to be potentially MSI high, hormone receptors negative, and sometimes TP53 mutated. However, some patients with immune response-associated features and better prognosis may be candidate to treatment de-escalation and immunotherapy.

Genome sequence analysis provides insights into the mode of 2n egg formation in Solanum malmeanum.

Our genomic investigation confirms the mechanism of 2n eggs formation in S. malmeanum and aid in optimizing the use of wild germplasm. Wild potatoes are a valuable source of agronomic traits. However, substantial reproductive barriers limit gene flow into cultivated species. 2n gametes are instrumental in preventing endosperm abortion caused by genetic imbalances in the endosperm. However, little is known about the molecular mechanisms underlying the formation of 2n gametes. Here, the wild species Solanum malmeanum Bitter (2x, 1EBN, endosperm balance number) was used in inter- and intrapoloid crosses with other Solanum species, with viable seeds being produced only when S. malmeanum was used as the female parent to cross the 2EBN Solanum genus and with the likely involvement of 2n gametes. Subsequently, we substantiated the formation of 2n eggs in S. malmeanum using fluorescence in situ hybridization (FISH) and genomic sequencing technology. Additionally, the transmission rate of maternal heterozygous polymorphism sites was assessed from a genomic perspective to analyze the mode of 2n egg formation in S. malmeanum × S. tuberosum and S. malmeanum × S. chacoense crosses; each cross acquired an average of 31.12% and 22.79% maternal sites, respectively. This confirmed that 2n egg formation in S. malmeanum attributed to second-division restitution (SDR) coupled with the occurrence of exchange events. The high-throughput sequencing technology used in this study has strong advantages over traditional cytological analyses. Furthermore, S. malmeanum, which has a variety of excellent traits not available from present cultivated potato genepool, has received little research attention and has successfully achieved gene flow in cultivated species in the current study. These findings will facilitate the understanding and optimization of wild germplasm utilization in potatoes.

The ndrg2 Gene Regulates Hair Cell Morphogenesis and Auditory Function during Zebrafish Development.

Damages of sensory hair cells (HCs) are mainly responsible for sensorineural hearing loss, however, its pathological mechanism is not yet fully understood due to the fact that many potential deafness genes remain unidentified. N-myc downstream-regulated gene 2 (ndrg2) is commonly regarded as a tumor suppressor and a cell stress-responsive gene extensively involved in cell proliferation, differentiation, apoptosis and invasion, while its roles in zebrafish HC morphogenesis and hearing remains unclear. Results of this study suggested that ndrg2 was highly expressed in the HCs of the otic vesicle and neuromasts via in situ hybridization and single-cell RNA sequencing. Ndrg2 loss-of-function larvae showed decreased crista HCs, shortened cilia, and reduced neuromasts and functional HCs, which could be rescued by the microinjection of ndrg2 mRNA. Moreover, ndrg2 deficiency induced attenuated startle response behaviors to sound vibration stimuli. Mechanistically, there were no detectable HC apoptosis and supporting cell changes in the ndrg2 mutants, and HCs were capable of recovering by blocking the Notch signaling pathway, suggesting that ndrg2 was implicated in HC differentiation mediated by Notch. Overall, our study demonstrates that ndrg2 plays crucial roles in HC development and auditory sensory function utilizing the zebrafish model, which provides new insights into the identification of potential deafness genes and regulation mechanism of HC development.

Hybrid-seq deciphers the complex transcriptional profile of the human BRCA1 DNA repair associated gene

ABSTRACT Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants (BRCA1 sv.7 – sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

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.