Fluorescence In Situ Hybridization Research Articles

An Insulin Upstream Open Reading Frame (INSU) Is Present in Skeletal Muscle Satellite Cells: Changes with Age.

Insulin resistance, stem cell dysfunction, and muscle fiber dystrophy are all age-related events in skeletal muscle (SKM). However, age-related changes in insulin isoforms and insulin receptors in myogenic progenitor satellite cells have not been studied. Since SKM is an extra-pancreatic tissue that does not express mature insulin, we investigated the levels of insulin receptors (INSRs) and a novel human insulin upstream open reading frame (INSU) at the mRNA, protein, and anatomical levels in Baltimore Longitudinal Study of Aging (BLSA) biopsied SKM samples of 27-89-year-old (yrs) participants. Using RT-qPCR and the MS-based selected reaction monitoring (SRM) assay, we found that the levels of INSR and INSU mRNAs and the proteins were positively correlated with the age of human SKM biopsies. We applied RNAscope fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to SKM cryosections and found that INSR and INSU were co-localized with PAX7-labeled satellite cells, with enhanced expression in SKM sections from an 89 yrs old compared to a 27 yrs old. We hypothesized that the SKM aging process might induce compensatory upregulation of INSR and re-expression of INSU, which might be beneficial in early embryogenesis and have deleterious effects on proliferative and myogenic satellite cells with advanced age.

Investigation of Astyanax mexicanus (Characiformes, Characidae) chromosome 1 structure reveals unmapped sequences and suggests conserved evolution.

Natural selection in the cave habitat has resulted in unique phenotypic traits (including pigmentation loss and ocular degeneration) in the Mexican tetra Astyanax mexicanus, considered a model species for evolutionary research. A. mexicanus has a karyotype of 2n = 50 chromosomes, and long-read sequencing and quantitative trait linkage maps (QTLs) have completely reconstructed the reference genome at the chromosomal level. In the current work, we performed whole chromosome isolation by microdissection and total amplification using DOP-PCR and Whole Chromosome Painting (WCP), followed by sequencing on the Illumina NextSeq platform, to investigate the microstructure of the large and conserved metacentric chromosome 1 of A. mexicanus. The sequences aligned to linkage block 3 of the reference genome, as determined by processing the reads with the DOPseq pipeline and characterizing the satellites with the TAREAN program. In addition, part of the sequences was anchored in linkage blocks that have not yet been assigned to the chromosomes. Furthermore, fluorescence in situ hybridization using WCP 1 carried out in other nearby species revealed a high degree of chromosome conservation, which allows us to hypothesize a common origin of this element. The physical mapping of the repetitive marker sequences provided a micro- and macrostructural overview and confirmed their position in chromosome pair 1. These sequences can serve as comparative tools for understanding the evolution and organization of this chromosome in other species of the family in future studies.

Genome content in the non-model ciliate Chilodonella uncinata : insights into nuclear architecture, gene-sized chromosomes among the total DNA in their somatic macronuclei during their development.

Ciliates are a model lineage for studies of genome architecture given their unusual genome structures. All ciliates have both somatic macronuclei (MAC) and germline micronuclei (MIC), both of which develop from a zygotic nucleus following sex (i.e., conjugation). Nuclear developmental stages are not as well explored among non-model ciliate genera, including Chilodonella uncinata (Class-Phyllopharyngea), the focus of our work. Here, we characterize nuclear architecture and genome dynamics in C. uncinata by combining DAPI (4',6-diamidino-2-phenylindole) staining and fluorescence in situ hybridization (FISH) techniques with confocal microscopy. We developed a telomere probe for staining alongside DAPI, which allows for the identification of fragmented somatic chromosomes among the total DNA in the nuclei. We quantify both total DNA and telomere-bound signals to explore changes in DNA content and chromosome maturation across Chilodonella 's nuclear life cycle. Specifically, we find that MAC developmental stages in the ciliate C. uncinata are different than the data reported from other ciliate species. These data provide insights into nuclear dynamics during nuclear development and enrich our understanding of genome evolution in non-model ciliates.

Sclerosing mucoepidermoid carcinoma of salivary glands.

Sclerosing mucoepidermoid carcinoma (SMEC) of the salivary glands is a rare variant of low-grade mucoepidermoid carcinoma with scanty cellular atypia characterized by marked fibrosis/sclerosis and a rich inflammatory infiltrate. Herein, we report 25 unpublished cases of SMEC, two of them with prominent eosinophilia (2/25; 8%) and three with abundant IgG4-positive plasma cells (3/25; 12%). In our series of salivary SMEC, molecular analysis using fluorescence in situ hybridization (FISH) and/or next-generation sequencing (NGS) provided evidence of MAML2 gene rearrangement in 18 cases of the 21 analyzable cases tested (86%), while this gene locus was intact in 3 cases (14%). This study focuses on the diagnostic criteria of salivary SMEC given its challenge of abundant collagenous stroma, minimal residual neoplastic areas, and inconspicuous mucous cells. Follow-up data of our cases indicate that salivary SMECs have favorable outcomes. Molecular analysis for MAML2 gene rearrangement suggests that SMECs of salivary glands represent a rare variant of conventional low-grade MECs of salivary glands. In contrast, SMECs of the thyroid gland are genetically distinct from salivary-type thyroid MECs.

Cytogenetic characterization of EPSPS gene amplification in glyphosate-resistant Hordeum glaucum and Bromus diandrus from Australia.

As a result of extensive selection, two polyploid grass weeds, Hordeum glaucum (northern barley grass; 2n = 4x = 28) and Bromus diandrus (ripgut brome; 2n = 8x = 56), have evolved resistance to glyphosate, in Australia. Previous research suggested amplification of 5-enolpyruvylshikimate-3-Phosphate synthase (EPSPS) gene confers resistance in these two weed species. The objective of this research was to investigate the genomic organization of the EPSPS gene in these two species through molecular cytogenetic analyses of fluorescence in situ hybridization (FISH) to understand possible mechanism of amplification of this gene. EPSPS copy number of H. glaucum and B. diandrus plants was estimated via quantitative polymerase chain reaction. The susceptible plants of both species had one copy of EPSPS, whereas the resistant plants of H. glaucum and B. diandrus had 14-17 and 16-32 copies, respectively. FISH analysis of glyphosate-susceptible (Hg-RWS) H. glaucum, revealed four faint signals of the EPSPS gene in two pairs of homologous chromosomes, at the telomeric region. The glyphosate-resistant H. glaucum (Hg-YP1) also showed amplification of EPSPS gene at telomeric regions in two pairs of homologous chromosomes, but the signals were brighter and appeared as cluster of EPSPS genes. Similarly, the glyphosate-susceptible B. diandrus (Bd-S) plants showed faint signals of EPSPS gene on two homologous chromosomes, at the telomeric position. However, samples of two glyphosate-resistant, B. diandrus, Bd-SA988 and Bd-Vic showed much brighter hybridization signals of EPSPS gene, located at the telomere on two homologous chromosomes, suggesting an increase in EPSPS gene copies at this position. Overall, unequal crossover during meiosis may have triggered the initial EPSPS gene duplication sparking the evolution of glyphosate resistance.

False-positive XXY results by interphase FISH in cytogenetically normal XX individuals: two cases highlighting the necessity of additional laboratory follow-up

BackgroundInterphase fluorescence in situ hybridization (FISH) is commonly used for rapid aneuploidy detection in clinical settings. While FISH-based aneuploidy detection provides rapid results desirable for patient management, it usually only utilizes one probe per chromosome, which may lead to rare false-positive findings.Case presentationHere we report two interphase FISH results, which were false-positive for XXY in cytogenetically normal XX individuals. Both false-positive cases were due to hybridization of the Y chromosome centromeric probe DYZ3 to the pericentromeric region of chromosome 15. In both cases, chromosomal microarray revealed no detectable Y chromosome material, suggesting the hybridizations of the DYZ3 probe to chromosome 15 likely represent benign heterochromatic variants of no clinical significance. In one case, the DYZ3 hybridization was also identified in the phenotypically unaffected mother, further suggesting this is likely a rare variant of no clinical significance.ConclusionsThis report marks the first documentation of hybridization of the DYZ3 probe to another chromosome in cytogenetically normal individuals. Our report has important clinical implications, because DYZ3 is widely used by clinical laboratories for Y chromosome detection. Our findings underscore the necessity of confirming abnormal aneuploidy detection FISH results with additional laboratory methods such as chromosomal microarray analysis.

BIOM-69. THE ONCOGENIC FLIP IN PATIENTS WITH LEPTOMENINGEAL METASTATIC DISEASE (LMD): LONGITUDINAL DETECTION IN CEREBROSPINAL FLUID TUMOR CELLS (CSF-TCS) REVEALS IMPLICATIONS FOR DIFFERENTIAL TREATMENT OF THE LMD TUMOR

Abstract INTRODUCTION Patients with LMD have poor prognosis and limited treatment options. Oncogene amplification of primary, metastatic, and CNS metastatic tumors can be heterogeneous. Therefore, patients with LMD may benefit from assessment of clinically relevant biomarkers in CSF, which may guide the choice of a targeted therapy specifically for the LMD tumor. CNSide is a CLIA-validated laboratory-developed test ordered commercially at the discretion of physicians for CSF-TC enumeration, immunocytochemical (ICC) and fluorescence in situ hybridization (FISH) analysis of oncogene amplification. We longitudinally analyze oncogenes in CSF-TCs in patients with LMD of various primary cancers. METHODS CSF was collected from patients with suspected or confirmed LMD; 613 tests were ordered on 218 individual patients with breast (N=105 patients), lung (N=65), gastrointestinal (N=10), and other cancers. Using CNSide, CSF-TCs were isolated and tested via ICC (ER, PD-L1, and PR), and FISH (ALK, cMET, cMyc, EGFR, FGFR1, HER2, NTRK1, NTRK3, PTEN, RET, and ROS1). RESULTS In patients with lung cancer, ALK was detected in 14% (17/118) of samples, CMET in 61% (78/128), HER2 in 73% (16/22), and RET in 4% (4/90). In patients with breast cancer, HER2 was detected in 39% (65/168) of samples, FGFR1 in 32% (19/60), ER in 26% (44/168) and PR in 4% (5/120). 66 patients underwent 2+ CSF draws; 24 underwent 5+. Among these, there were 13 ICC flips (7 acquired mutations) and 58 FISH probe detection flips (26 acquired mutations). 20/66 patients (30.3%) had at least one flip in their ordered biomarkers. CONCLUSION CNSide can be used to detect oncogene amplification on CSF-TCs of patients with LMD, and mutational status of the LMD tumor may differ from the original tumor biopsy. CSF-TC analysis may provide therapeutic insights that vary from the original tumor and could open the door to additional treatment targets for patients struck with LMD.

Deep-Learning-Based Approach in Cancer-Region Assessment from HER2-SISH Breast Histopathology Whole Slide Images.

Fluorescence in situ hybridization (FISH) is widely regarded as the gold standard for evaluating human epidermal growth factor receptor 2 (HER2) status in breast cancer; however, it poses challenges such as the need for specialized training and issues related to signal degradation from dye quenching. Silver-enhanced in situ hybridization (SISH) serves as an automated alternative, employing permanent staining suitable for bright-field microscopy. Determining HER2 status involves distinguishing between "Amplified" and "Non-Amplified" regions by assessing HER2 and centromere 17 (CEN17) signals in SISH-stained slides. This study is the first to leverage deep learning for classifying Normal, Amplified, and Non-Amplified regions within HER2-SISH whole slide images (WSIs), which are notably more complex to analyze compared to hematoxylin and eosin (H&E)-stained slides. Our proposed approach consists of a two-stage process: first, we evaluate deep-learning models on annotated image regions, and then we apply the most effective model to WSIs for regional identification and localization. Subsequently, pseudo-color maps representing each class are overlaid, and the WSIs are reconstructed with these mapped regions. Using a private dataset of HER2-SISH breast cancer slides digitized at 40× magnification, we achieved a patch-level classification accuracy of 99.9% and a generalization accuracy of 78.8% by applying transfer learning with a Vision Transformer (ViT) model. The robustness of the model was further evaluated through k-fold cross-validation, yielding an average performance accuracy of 98%, with metrics reported alongside 95% confidence intervals to ensure statistical reliability. This method shows significant promise for clinical applications, particularly in assessing HER2 expression status in HER2-SISH histopathology images. It provides an automated solution that can aid pathologists in efficiently identifying HER2-amplified regions, thus enhancing diagnostic outcomes for breast cancer treatment.

TMOD-26. GENERATION OF A CHROMOSOME 8 GAIN;NF1-/- HIPSC-DERIVED SCHWANN CELL PRECURSOR MODEL

Abstract Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome caused by a germline mutation in the NF1 gene. The loss of the second copy of NF1 within Schwann cell precursors (SCPs) contributes to the formation of plexiform neurofibromas (PNs) found in 30-50% of NF1 patients. PNs can progress into malignant peripheral sheath tumors (MPNSTs), an aggressive form of cancer and a leading cause of mortality in NF1 patients. Our lab previously demonstrated frequent chromosome 8 (Chr8) gain in NF1-MPNST patient-derived xenografts (PDX) and patient tumors. To investigate Chr8 gain’s role, we created isogenic NF1-/- iPSC cell lines with and without Chr8 gain. Trisomy 8 fibroblasts were obtained and single-cell cloned to establish wildtype (WT) and Chr8 gain populations. Washington University’s Genome Engineering & Stem Cell Center (GESC@MGI) reprogrammed these cells into human induced pluripotent stem cells (hiPSCs). Using synthetic gRNA and CRISPR/Cas9, NF1 knockout lines were generated. Four iPSC cell lines were differentiated into SCPs, and cell survival and proliferation were assessed using Incucyte cell survival and CellTiter-Glo® Assay. Fluorescence in situ hybridization (FISH) and karyotyping validated the human iPSCs. qPCR revealed high expression of iPSC markers OCT3/4 and SOX2 in all iPSC lines. After differentiation, SCP markers (GAP43 and ITGA4) were upregulated in WT, NF1-/-, and Chr8 gain;NF1-/- SCP lines, demonstrating successful differentiation. Furthermore, Chr8 gain; NF1-/- SCPs displayed enhanced proliferation and increased cell survival compared to WT and NF1-/- SCPs, suggesting improved characteristics in the presence of Chr8 gain. In summary, our research provides insights into Chr8 gain and NF1 loss effects on SCP differentiation, proliferation, and survival. Understanding these molecular mechanisms may contribute to developing targeted therapies for NF1.

BIOM-70. CSF TUMOR CELL (CSF-TC) DETECTION, QUANTIFICATION AND BIOMARKER ASSESSMENT HELPS IN CLINICAL MANAGEMENT OF BREAST CANCER AND NON-SMALL CELL LUNG CANCER PATIENTS HAVING LEPTOMENINGEAL DISEASE (FORESEE STUDY, NCT05414123)

Abstract Leptomeningeal metastases (LM) or the presence of cancer cells in the cerebrospinal fluid (CSF)/pia remains a challenging disease to diagnosis and treat. Current standard of care methods to diagnose or assess treatment response of LM (Clinical Evaluation, MRI and Cytology) have limited sensitivity and specificity and are poorly quantifiable. This creates challenges for physicians to manage LM or determine the best course of treatment. The FORESEE Study was a multi-center, prospective clinical trial evaluating a novel diagnostic platform, CNSide, that aimed to overcome these challenges. The FORESEE study enrolled patients with breast or non-Small Cell Lung Cancer (NSCLC) with suspicion of or confirmed LM. CNSide can detect and quantify tumor cells in the CSF from patients with breast cancer or NSCLC having a suspicious or confirmed LM. This platform also is able to identify actionable mutations in the CSF via Fluorescent In Situ Hybridization (FISH) and next-generation sequencing. The primary end point of the FORESEE study was clinical utility and the impact of CNSide on physician treatment decisions. Secondary endpoints included evaluating the clinical performance of CNSide vs. cytology in tumor cell detection (sensitivity, specificity, PPV and NPV). The study enrolled 40 patients (22 breast cancer and 18 NSCLC). Each patient underwent standard of care diagnostic evaluations at baseline and at three follow up timepoints through their treatment. CNSide aided clinical decision making in 93% (50/54) of the clinical decisions. At baseline 39 patients were assessed and confirmed a positive LM diagnosis in 15% (6/39), a negative LM diagnosis in 8% (3/39), progression in 15% (6/39), resolution in 15% (6/39) and no progression, or resolution in 36% (14/39) of the patients. CNSide informed the specific drug selected for treatment in 26% (10/39) patients at baseline and demonstrated clinical high utility. The FORESEE data is undergoing ongoing analysis; updated analysis will be presented at the meeting.

Prenatal diagnosis analysis of three cases of Turner syndrome fetuses with complex mosaic small supernumerary marker chromosomes

To explore the value of applying multiple genetic testing techniques for the prenatal diagnosis of Turner syndrome fetuses with complex mosaic small supernumerary marker chromosomes (sSMC). Chromosomal karyotypes of amniotic fluid samples from 5 030 pregnant women who had undergone amniocentesis at the Prenatal Diagnosis Center of the Third Affiliated Hospital of Zhengzhou University from January to December 2022 were retrospectively reviewed. Three fetuses with complex mosaicism fetuses (carrying 2 types of sSMC) were selected as the study subjects. Genetic tests including G-banded chromosomal karyotyping analysis, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and copy number variation sequencing (CNV-seq) were used to clarify the origin and mosaic status of the sSMC. This study has been approved by the Medical Ethics Committee of the Third Affiliated Hospital of Zhengzhou University (No. 2023-159-01). G-banded chromosomal analysis of fetus 1 showed a karyotype of 45,X[64]/46,X,+mar1[13]/46,X,+mar2[3]. FISH results showed that 52% of of its cells had contained one X chromosome signal, whilst 48% contained two X chromosome signals. CMA results revealed the fetus had harbored a 32.32 Mb and a 50.93 Mb deletion in Xp22.33p21.1 and Xq22.2q28 regions, respectively, in addition with mosaic deletions of approximately 1.43 copies, 1.78 copies and 1.43 copies in the Xp21.1p11.1, Xq11.1q21.1 and Xq21.2q22.2 regions, respectively. The fetus 2 had a karyotype of 45,X[27]/46,X,+mar1[14]/46,X,+mar2[12]. FISH results indicated that 88% of its cells contained one X chromosomes signal and two Y chromosome signals, and 12% contained signals for one X chromosomes signal and one Y chromosome signal. CNV-seq results revealed a deletion of 7.74 Mb in the Yq11.222q11.23 region and a mosaic duplication of approximately 1.738 copies in the Yp11.31q11.221 region. The fetus 3 had a karyotype of 45,X[60]/46,X,+mar1[11]/46,X,+mar2[6]. FISH results showed that 28% of its cells contained one X chromosome signal, and 72% contained tow X chromosome signals. CNV-seq results revealed deletions of 55.60 Mb and 53.50 Mb in the Xp22.33p11.1 and Xq22.1q28 regions, respectively, along with a mosaic deletion of approximately 1.85 copies in the Xp11.1q13.2 region and a mosaic repeats of approximately 2.66 copies in the Xq13.2q22.1 region. The sSMCs in the 3 fetuses had all originated from sex chromosomes and were of complex mosaic type. After genetic counseling, the three couples had all opted to terminate the pregnancy. The combined use of multiple genetic testing techniques has determined the origin and structure of complex mosaic sSMCs and provided a basis for prenantal diagnosis and genetic counseling.

A clinicopathological and molecular series of five TFEB-altered renal cell carcinoma (RCC) cases: highlighting an aggressive subset of TFEB-rearranged RCC concomitant with TFEB amplification/gene copy number gains.

The classification of TFEB-altered renal cell carcinoma (RCC) has been revised to include TFEB-rearranged RCC and TFEB-amplified RCC in the 2022 World Health Organization (WHO) Classification of Tumors of the Urinary System. Given the wide spectrum of TFEB-altered RCC in terms of morphology and clinical behavior, an accurate diagnosis is challenging yet crucial, particularly in aggressive cases. Moreover, the concurrence of TFEB gene rearrangement and amplification/gene copy number (GCN) gains was also observed, but there was limited knowledge of these cases. We presented three TFEB-rearranged RCC cases, one TFEB-amplified RCC case, and one case of concomitant TFEB-rearranged and -amplified RCC, comparing the similarities and differences among these three subgroups. Furthermore, we summarized the clinicopathological and molecular features of TFEB-rearranged RCC concomitant with TFEB amplification/GCN gains from the literature and the present study. TFEB-altered RCCs exhibit significant heterogeneity in morphology and clinical behavior while displaying similar immunohistochemical profiles, including positive staining for Melan-A, PAX8, and CD117, and negative staining for CK7. A typical biphasic "rosette-like" morphology has been observed in a proportion of TFEB-rearranged RCC concomitant with TFEB amplification/GCN gains, which has been noted in TFEB-rearranged RCC, but not in cases with only TFEB amplification. Notably, TFEB-rearranged RCCs concomitant with TFEB amplification/GCN gains tend to be aggressive, in contrast to the often indolent nature of TFEB-rearranged cases, irrespective of the extent of TFEB gene copy increase. Therefore, a TFEB FISH assay is essential for unclassified RCC cases that exhibit melanocytic marker expression, and fluorescent signals should be counted and interpreted acurrately.

FISH combined with RT-PCR facilitates classification of Chinese adult patients with B-other ALL through improved identification of ZNF384 rearrangement

ZNF384 gene rearrangements are a distinct subtype of adult B cell acute lymphoblastic leukemia (B-ALL). We screened 46 B-other ALL patients for ZNF384 fusions using fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR). Clinical data, treatment response, and minimal residual disease (MRD) status were analyzed. Ten (21.7%) patients were ZNF384-r positive (nine by FISH, nine by RT-PCR, eight by both). FISH showed atypical signals, including 3’ signal gain and 5’ signal deletion. EP300 was the main fusion partner (n = 5). TAF15::ZNF384, SYNRG::ZNF384, CREBBP::ZNF384, and TCF3::ZNF384 fusions were found in one patient each; one case’s partner gene is unknown. One patient was MRD-negative at the end of the first induction, lower than in patients without ZNF384-r. ZNF384-r incidence matched B-other ALL incidence in Chinese patients. Combined FISH and RT-PCR improved detection. ALL with ZNF384-r has unique features, and lower MRD-negative response may indicate a negative impact on traditional treatments.

Deep learning method for detecting fluorescence spots in cancer diagnostics via fluorescence in situ hybridization

Fluorescence in Situ Hybridization (FISH) is a technique for macromolecule identification that utilizes the complementarity of DNA or DNA/RNA double strands. Probes, crafted from selected DNA strands tagged with fluorophore-coupled nucleotides, hybridize to complementary sequences within the cells and tissues under examination. These are subsequently visualized through fluorescence microscopy or imaging systems. However, the vast number of cells and disorganized nucleic acid sequences in FISH images present significant challenges. The manual processing and analysis of these images are not only time-consuming but also prone to human error due to visual fatigue. To overcome these challenges, we propose the integration of medical imaging with deep learning to develop an automated detection system for FISH images. This system features an algorithm capable of quickly detecting fluorescent spots and capturing their coordinates, which is crucial for evaluating cellular characteristics in cancer diagnosis. Traditional models struggle with the small size, low resolution, and noise prevalent in fluorescent points, leading to significant performance declines. This paper offers a detailed examination of these issues, providing insights into why traditional models falter. Comparative tests between the YOLO series models and our proposed method affirm the superior accuracy of our approach in identifying fluorescent dots in FISH images.

EFFECT BRIGHTNESS COLOR GUPPY FISH (Poecilia reticulata)

Guppy fish (Poecilia reticulata) is a popular ornamental fish because of their unique color patterns, the color of ornamental fish will fade if their feed does not contain enough pigmentation to keep them bright. This study aimed to determine the effect of adding Spirulina sp. powder with different dosage to the feed on the color brightness of guppy fish. This study used a completely randomized design (CRD) with five treatments (feed added with Spirulina sp. powder as much as 0%, 0.3%, 0.6%, 0.9%, and 1.2%) and three replications. The test fish were male HB Red guppy fish with a size of 3 cm and a density of 3 fish/aquarium. Water changes (siphoning) was done every morning, and feeding was done twice a day. The parameters observed in this study were the chroma value, survival rate, and water quality. The result showed that the effect of adding Spirulina sp. powder to the feed at a dose of 0.9% and 1.2% had a significant effect on the color brightness. The survival rate indicates that the outcomes were not significantly different between treatments from a 100% success rate. The adding of Spirulina sp. powder increases the brightness of Guppy fish. The brightness color is very important in the Guppy fish farming, because color is the main indicator in ornamental fish. The water has a good quality, with an average temperature of 25.8-26°C and a pH of 7.9-8.0.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Detection of Alternative Lengthening of Telomeres via Chromogenic In Situ Hybridization for the Prognostication of PanNETs and Other Neoplasms

Molecular studies have shown alternative lengthening to telomeres (ALT) to be an important prognostic biomarker of shorter relapse-free survival (RFS) for patients with pancreatic neuroendocrine tumors (PanNETs) and other neoplasms. However, the preferred method of detecting ALT in tissue is by fluorescence in situ hybridization (FISH), which has several clinical limitations. These issues necessitate the creation of a chromogenic ALT assay that can be easily implemented into routine practice. A chromogenic in situ hybridization (CISH) assay was developed using genetically modified osteosarcoma cell lines, 20 normal pancreata, 20 ALT-positive PanNETs, and 20 ALT-negative PanNETs. Thereafter, it was validated on a multiinstitutional cohort of 360 surgically resected PanNETs and correlated with multiple clinicopathologic features, RFS, and FISH results. Separately, 109 leiomyosarcomas (LMS) were evaluated by both CISH and FISH, and, similarly, the prognostic significance of ALT status was assessed. Upon optimization, ALT–CISH was identified in 112 of 360 (31%) primary PanNETs and was 100% concordant with FISH testing. ALT correlated with several adverse prognostic findings and distant metastasis (all P < .004). The 5-year RFS for patients with ALT-positive PanNETs was 35% as compared with 94% for ALT-negative PanNETs. By multivariate analysis, ALT was an independent prognostic factor for shorter RFS. Similarly, ALT was associated with shorter RFS in patients with LMS and, analogous to PanNETs, a negative, independent prognostic factor. ALT–CISH was developed and validated in not only PanNETs but also sarcomas, specifically LMS. CISH testing has multiple advantages over FISH that facilitate its widespread clinical use in the detection of ALT and prognostication of patients with diverse neoplasms.

Prospective Validation of a One-Step RT-qPCR-based Test for Quantifying HER2 Gene Expression in Breast Cancer.

To Validate a one-step RT-qPCR as a reliable diagnostic tool in HER-2 positive breast cancer. Further, establishing this tool as a standard procedure to quantify HER-2 expression in Breast cancer patient. Here we report a prospective validation study that shows the concordance of one-step RT-qPCR in assessing the HER2 levels in formalin-fixed paraffin-embedded (FFPE) tissue samples with the current paradigm of diagnosis such as Immunohistochemistry (IHC) and Fluorescence-in-situ Hybridization (FISH). We collected 275 FFPE samples from the Department of Pathology, Ibn Rochd University Hospital Center. IHC was carried out in all the samples and those with a score of 2+ were also analyzed using FISH. We extracted mRNA from FFPE samples and performed RT-qPCR, and the results were compared with those obtained using IHC/FISH. HER2 mRNA levels were quantified and normalized using the reference genes RPL30 and RPL37, based on our earlier reports. HER2 cut-off value was fixed at 11.954 corresponding to the combination of the best sensitivity and specificity (93.4% and 100%) respectively, and with positive predictive values PPV that reached 100% and a negative predictive value NPV that reached 89.4%. The results showed 100% concordance with FISH. The Kappa coefficient was 0.863 which indicated concordance with IHC. The area under the curve (AUC) which is an important parameter that determines the diagnostic accuracy of the test was calculated as AUC=0.955. The results were comparable to some of the recent studies published in a similar direction. Our study shows that one-step RT-qPCR-based quantitation is an accurate and reproducible test to record HER2 gene expression for a better treatment orientation focusing on maximal therapeutic and survival outcome.

Quercetin inhibits endothelial & hepatocellular carcinoma cell crosstalk via reducing extracellular vesicle-mediated VEGFR2 mRNA transfer.

This study aims to investigate the regulatory effects of quercetin extracellular vesicles (EVs)-mediated expression of vascular endothelial growth factor receptor 2 (VEGFR2) in hepatocellular carcinoma (HCC)-derived circulating tumor cells (CTCs) and the underlying mechanisms. CTCs were isolated from patients with pathologically diagnosed HCC, with VEGFR2 expression visualized by fluorescence in situ hybridization (FISH). The human HCC cell line Huh-7 and SK-HEP-1 were used for in vitro studies to assess EVs uptake, VEGFR2 mRNA transfer, invasion, migration, cancer stem cell (CSC) properties, and VEGF secretion. Results showed that VEGFR2 mRNA was commonly expressed in HCC-CTCs, with a higher incidence in biphenotypic CTCs. Its expression was limited in HCC cell lines, but present in certain liver cells. In vitro experiments confirmed that VEGFR2 mRNA could be transferred to HCC cells via EVs from primary tumor endothelial cells (PTECs), which was impaired by quercetin treatment. Quercetin significantly reduced VEGFR2 mRNA and protein expression in HCC cells, weakened their invasive and metastatic capacities, and diminished VEGFR2-mediated CSC properties. In vivo, quercetin reduced VEGF secretion, impaired angiogenesis, slowed tumor growth, and decreased the number and proportion of VEGFR2-positive CTCs. In summary, VEGFR2 mRNA is present in HCC-CTCs, potentially sourced from PTECs-derived EVs. Quercetin effectively inhibits VEGFR2 expression, impacting HCC cell invasion, metastasis, and CSC characteristics. Besides, it reduces VEGFR2-positive CTCs in vivo. These effects support its therapeutic potential in HCC treatment by targeting the angiogenesis and tumor dissemination pathway.

Unfavorable Prognostic Impact of HER2 2+/FISH-Negativity in Older Patients with HER2-Negative and High-Risk Breast Cancer.

Human epidermal growth factor receptor 2 (HER2)-low breast cancer, consisted of carcinomas with HER2 protein 1+ or 2+ without gene amplification, has been considered a biologically heterogeneous disease. Limited research separately investigated the prognostic significance of HER2 2+ without gene amplification, and no evidence can be identified in older patients. In this dedicated cohort of older patients with HER2-negative and high-risk breast cancer, we analyzed the real-world prognosis after standard adjuvant chemotherapy, and investigated the associations of survival with HER2 2+ without gene amplification. From January 2016 to December 2021, older patients (≥65 years) with breast cancer were reviewed, and HER2-negative/high-risk disease receiving standard adjuvant chemotherapy was included. HER2-negativity was defined as immunohistochemistry (IHC) score 0, 1+ or 2+ without gene amplification by fluorescent in situ hybridization (FISH). Cox proportional hazards regression analyses were performed to assess the associations of HER2 2+/FISH-negativity with disease-free survival (DFS), which was estimated by the Kaplan-Meier method and compared by the Log rank test. This cohort consisted of 121 consecutive older patients. With a median follow-up of 46 months, 12 patients had a DFS event. By univariate and multivariate analyses, HER2 2+/FISH-negativity was the only independent predictor for worse DFS (hazard ratio 5.56; P=0.046). Patients with HER2 2+/FISH-negativity had significantly poorer DFS compared with those with HER2 0 or 1+ (Log rank test, P=0.029). In both hormone receptor (HR)-positive (Log rank test, P=0.052) and HR-negative (Log rank test, P=0.125) subgroups, HER2 2+/FISH-negativity showed a marginally significant adverse influence on DFS. In older patients with HER2-negative/high-risk breast cancer undergoing standard adjuvant chemotherapy, our findings suggest that HER2 2+/FISH-negativity has an independent negative impact on prognosis.