Fluorescence In Situ Hybridization Assay Research Articles

Application of fluorescence in situ hybridization technique to verify the clonalities of non-clonal cytogenetic abnormalities identified in Myelodysplastic syndrome

To assess the value of fluorescence in situ hybridization (FISH) technique for the verification of the clonalities of non-clonal cytogenetic abnormalities (n-CCA) identified by conventional chromosome banding analysis (CBA) in patients with Myelodysplastic syndrome (MDS). Clinical data and results of karyotyping and FISH assays for 91 patients of MDS with n-CCA identified by CBA were retrospectively analyzed. In total 94 non-clonal +8, 5q-, -7/7q- or 20q- were detected by CBA, among which 43 (45.7%) were verified to be clonal abnormalities by FISH. The detection rates for +8, 5q-, -7/7q- and 20q- by FISH were 47.6% (30/63), 25% (2/8), 41.7% (5/12), 40% (2/5) and 66.7% (4/6), respectively, with the positive cells accounting for 4% to 90% of all counted cells, with a median value of 7%. The 91 patients were divided into three groups including ≥ 20, 10 ~< 20 and < 10 based on the numbers of metaphase cells in CBA, and the detection rates by FISH for the three groups were 43.7% (31/71), 33.3% (3/9) and 63.6% (7/11), respectively, which showed no statistically difference (P > 0.05). Continuous CBA and FISH surveys were conducted for 26 patients who received supportive treatment, and the results revealed that 91.7% (11/12) of FISH-verified positive abnormalities had persisted, whereas 92.9% (13/14) of the n-CCA verified as negative by FISH was transient. Nearly half of the CBA identified n-CCA have been verified as clonal aberrations by FISH, and the FISH detection rate showed no correlation with the number of metaphase cells. FISH test is strongly recommended for verifying the clonalities of n-CCA detected by CBA, and continuous cytogenetic survey of the patients with MDS is necessary.

Abstract HER2-07: HER2-07 Genomic Characterization of Primary and Metastatic HER2-low Breast Cancers

Abstract Background: Varying levels of HER2 expression without ERBB2 gene amplification can be detected by immunohistochemistry (IHC) in approximately 60% of all invasive breast cancers (BCs). HER2-low-expressing BCs have recently been shown to respond to novel anti-HER2 antibody drug conjugates. Several studies have demonstrated that HER2-low BCs do not seem to constitute a distinct clinical and transcriptomic subtype as compared to HER2-0 BCs. Here we sought to define the clinicopathologic features and repertoire of somatic genetic alterations in HER2-low BCs. Methods: We retrieved clinical, pathological, and genomic data of BCs that were subjected to targeted sequencing using the FDA-cleared MSK-IMPACT assay from April 2014 to December 2021. After removing cases where any available biopsy had HER2 3+ and/or positive ERBB2 fluorescence in situ hybridization (FISH), 3608 samples (primary=1347; post-treatment/metastatic=2261) were included. Tumors were classified as HER2-low if they had an HER2 IHC score of 1+ or 2+ with a non-amplified FISH assay and HER2-0 if they had an HER2 IHC score of 0. Somatic mutations and DNA copy number alterations from MSK-IMPACT were analyzed. Multiple testing correction using the Benjamini-Hochberg method was applied to control for the false discovery rate (q). Q values &amp;lt; 0.1 were considered significant. Results: Among 3608 HER2- BCs, 1460 (40%) and 2148 (60%) were HER2-0 and HER2-low, respectively. Hormone receptor (HR) expression was significantly higher in HER2-low than HER2-0 tumors in both primary (781 [68.3%] vs 362 [31.7%]; p&amp;lt; 0.001) and metastatic (1031 [60.5%] vs 673 [39.5%]; p&amp;lt; 0.001) samples. A higher proportion of HER2-low tumors was found in metastatic than primary samples (59% vs 41%; p&amp;lt; 0.001) in this cohort. No difference in histology subtype, tumor grade, disease stage (among primary tumors), mutational signatures, and tumor mutational burden was found overall and when cases were stratified by HR expression. In HR-positive BCs, HER2-0 BCs harbored higher frequency of TP53 (33% vs 25%; odds ratio [OR] 1.49, 95% confidence interval [CI] 1.25-1.78, q&amp;lt; 0.001) and CDKN1A (1% vs 0%; OR 17.47, 95% CI 2.48-756.37, q=0.02) alterations than HER2-low BCs. Similar findings were observed in metastatic but not in primary HR-positive BCs. No differences were detected in HR-negative BCs stratified into HER2-low and HER2-0. Given the potential misclassification that exists between IHC HER2-0 and HER2-1+, we then conducted an exploratory analysis splitting the HER2-low group into HER2 1+ and 2+. TP53 alterations remained significantly enriched in HER2-0 compared to HER2-1+ HR-positive tumors (33% vs 24%; OR 1.55, 95% CI 1.28-1.87, q&amp;lt; 0.001). In HR-positive BCs, HER2-2+ displayed a higher frequency of genetic alterations in genes encoding for transcription factors, such as MYC (14.2% vs 7.3%; OR 2.09, 95% CI 1.44-3.04, q=0.02) and YAP1 (2% vs 0.3%; OR 6.86, 95% CI 1.7-39.57, q=0.1), and DNA damage response, such as FAM175A (1.6% vs 0%; OR 18.23, 95% CI 2.43-807.73, q=0.03) and BRCA2 (4% vs 1%; OR 3.09, 95% CI 1.49-6.55, q=0.1), than HER2-0 tumors. In HR-negative HER2-2+ tumors, a higher frequency of PIK3CA mutations was observed in comparison to HER2-0 (36.9% vs 19.5%; OR 2.41, 1.4-4.1, q=0.1), overall and in the metastatic setting. Conclusions: HER2-low BCs seem not to represent a distinct pathologic subtype. At the genomic level, however, some differences were identified and these became more conspicuous upon subclassification of HER2 IHC expression into 1+ and 2+. Further investigation into methods that more accurately detect and quantify low levels of HER2 expression in BC samples as well as better characterize the biology behind the HER2-low/ultralow expression is warranted. Citation Format: Antonio Marra, Anton Safonov, Joshua Drago, Emanuela Ferraro, Pier Selenica, Andrea Gazzo, Giuseppe Curigliano, Shanu Modi, Pedram Razavi, Jorge Reis-Filho, Sarat Chandarlapaty. HER2-07 Genomic Characterization of Primary and Metastatic HER2-low Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-07.

Validation of Fluorescence in Situ hybridization (FISH) Assay Using An Analyte-Specific Reagent in Detecting Aneuploidies of Chromosomes 13, 18, 21, X, and Y in Prenatal Diagnosis

Fluorescence &lt;em&gt;In-Situ&lt;/em&gt; hybridization (FISH) is a sensitive and highly efficient technique commonly used in routine diagnostics. Most of these tests that use analyte-specific reagents are not approved by US Food and Drug Administration (FDA) but are developed by individual test laboratories. There is an emerging demand for prenatal diagnosis of aneuploidies by FISH. Since most of these assays are laboratory-developed tests, it is essential to validate them prior to their use in diagnosis. However, validation procedures of these assays are oversight despite the presence of several validation guidelines. To validate FISH assay using analyte-specific reagents in detecting aneuploidies of chromosomes 13, 18, 21, X, and Y as per American College of Medical Genetics (ACMG) guidelines in 2016. Analyte-specific reagents supplied by Oxford Gene Technologies were used in the validation process using blood and amniotic fluid samples obtained from healthy male and female adults and fetuses respectively. The validation process includes probe localization, evaluation of assay specificity, and establishment of lower cut-off and reportable reference ranges. Probe localization indicated a 100% specificity for all probes tested. Interphase FISH on uncultured amniotic fluid demonstrated significantly high (≥95%) overall disomic signal patterns for all autosomes and sex chromosomes tested. The reportable 95% confidence interval was 94.84, 94.84, 95.24, 94.54, and 94.54 for chromosomes 13, 18, 21, X, and Y respectively. The present study illustrates an experimental design in validating laboratory-developed FISH assay using analyte-specific reagents in detecting aneuploidies of chromosomes 13, 18, 21, X, and Y as per ACMG guidelines. Test probes used in the present study are consistent with probe localization characteristics, assay specificity, and reportable reference ranges recommended by ACMG. Therefore, the FISH assay used in the present study could be recommended as a supplementary prenatal diagnostic test that can be carried out along with standard chromosomal analysis.

Prenatal diagnosis for a fetus with 5p deletion syndrome

To explore the genetic basis for a fetus with club foot detected upon mid-pregnancy ultrasonography. Amniotic fluid of the fetus and peripheral blood samples of its parents were collected and subjected to G-banding karyotype analysis and copy number variation sequencing (CNV-seq). The result was verified by fluorescence in situ hybridization (FISH). The fetus and its parents all had a normal karyotype. CNV-seq analysis revealed that the fetus has harbored a 23.12 Mb on chromosome 5 and a 21.46 Mb duplication on chromosome 7. FISH assay has verified that its mother has carried a cryptic t(5;7)(p14.3;q33) translocation. CNV-seq combined with FISH can effectively detect cryptic chromosome aberrations, and can help to reduce severe birth defects and provide a basis for prenatal genetic counseling.

Reckoning the Status of HER2/neu Protein and Oncogene in Breast Cancer Specimens: Comparison of IHC Assay with FISH Assay at a Tertiary Care Centre, West India

Introduction: Breast carcinoma is the most prevalent malignant neoplasm among women with more than one million new cases per year. The benefit of humanised anti-Human Epidermal growth factor Receptor 2 (HER2)/neu monoclonal antibody trastuzumab (Herceptin) in HER2/neu-positive breast cancers has been well documented. Immunohistochemistry (IHC) for protein overexpression and Fluorescence In Situ Hybridisation (FISH) for detecting HER2/neu gene amplification is widely used. Some laboratories use IHC as primary test with FISH for subset of cases while using FISH as primary investigation followed by IHC if needed is done by others. Aim: To evaluate IHC assay in breast carcinoma cases for HER2/neu as screening test before FISH. Materials and Methods: A cross-sectional descriptive type of observational study was conducted in the Department of Pathology and Multi-Disciplinary Research Unit, SMS Medical College and attached hospitals Jaipur, India, between April 2020 and December 2021. A total 122 samples of invasive breast carcinoma were included in study for IHC and FISH analysis. On paraffin embedded breast tumour tissue sections, IHC was performed using mouse monoclonal antibody targeting the intracellular domain of HER2/neu protein and FISH was implemented by dual colour probes targeting the HER2/neu gene on chromosome 17. Cases were classified according to HER2/neu status on IHC interpretation and FISH interpretation as per American Society of Clinical Oncology/ the College of American Pathologists (ASCO/CAP) HER2/neu-2018 testing guidelines. Chi-square test, kappa coefficient and Z-test were applied for statistical analysis. The p-value&lt;0.05 was considered significant. Results: Of the 122 patients of Invasive Duct Carcinoma (IDC), the mean age was 50.3±12.7 years with a age range of 25-70 years. HER2/neu IHC score 3+ (over expression) was seen in 19 (15.6%) cases while 2+ (equivocal) result was obtained in 84 (68.8%) cases and 19 (15.6%) cases showed negative (1+/0) HER2/neu expression. Out of 19 IHC positive cases, all cases were amplified by FISH and all 19 IHC negative cases, were non amplified by FISH. There were 84 IHC equivocal cases of which 37 (44%) cases were amplified, 45 (53.6%) non amplified and 2 (2.4%) cases were equivocal when analysed by FISH. Also, 11 (9%) cases were found Centromere Enumeration Probe (CEP) amplified in the study, they were negative or equivocal on IHC and none was IHC positive. Conclusion: The study concluded that combined FISH and IHC methodologies could optimise information on HER2/neu status in breast cancer patients. Also, testing algorithm is emphasised where laboratories may use IHC as a screening method and FISH can be used as accurate and specific method in IHC (2+) equivocal cases. Thus, patients with HER2/neu status positive of IHC (3+) or FISH (gene amplified) can be proposed to be treated with herceptin (trastuzumab).

CircHIPK3 promotes bone microvascular endothelial cell proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling in steroid-induced osteonecrosis of the femoral head.

Circular RNA homeodomain interacting protein kinase 3 (circHIPK3) has been implicated in facilitating angiogenesis in various conditions. However, its role in steroid-induced osteonecrosis of the femoral head (ONFH) remains unclear. To investigate whether circHIPK3 promotes bone microvascular activity and angiogenesis by targeting miR-7 and Krüppel-like factor 4 (KLF4)/vascular endothelial growth factor (VEGF) signaling in ONFH. Fifty patients with steroid-induced ONFH undergoing hip-preserving surgery or total hip arthroplasty were included in this study. The expression of circHIPK3, miR-7 and KLF4 was evaluated using reverse transcription polymerase chain reaction (RT-PCR) in necrotic and healthy samples of the femoral head. Bone microvascular endothelial cells (BMECs) were extracted and cultured with 0.1 mg/mL hydrocortisone to create a hormonally deficient cell model. These BMECs were then transfected with either circHIPK3 overexpressing or silencing plasmids with or without miR-7 mimics. The MTT assays were used to detect cell proliferation. Scratch assays were used to assess the migration ability of the BMECs. The tube formation was carried out using an in vitro Matrigel angiogenesis assay. Annexin V-FITC/PI and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to assess the degree of apoptosis. Western blot assays were carried out to discern KLF4 and VEGF expression. The interactions of circHIPK3, miR-7 and KLF4 were confirmed using luciferase, RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and fluorescence in situ hybridization (FISH) assays. The circHIPK3 and KLF4 expression was decreased, whereas miR-7 expression was increased in necrotic tissues compared to non-necrotic samples. Both circHIPK3 and KLF4 expression correlated negatively with miR-7. The overexpression of circHIPK3 promoted the proliferative, migratory and angiogenic capabilities of the BMECs, while adding an miR-7 mimic reversed these effects. At the same time, the overexpression of circHIPK3 reduced the apoptosis rate of the BMECs and increased KLF4 and VEGF protein expression, but adding an miR-7 mimic reversed these effects. The FISH, RNA pull-down, RIP, and luciferase assays revealed an interaction between circHIPK3, miR-7 and KLF4. The circHIPK3 promotes BMEC proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling.

The genetic landscape of SMARCB1 alterations in SMARCB1-deficient spectrum of mesenchymal neoplasms

SMARCB1 biallelic inactivation resulting in SMARCB1/INI1 deficiency drives a wide range of malignancies, including many mesenchymal tumors. However, the specific types of SMARCB1 alterations and spectrum of cooperating mutations among various types of sarcomas has not been well investigated. We profiled SMARCB1 genetic alterations by targeted DNA sequencing and fluorescence in situ hybridization (FISH) in a large cohort of 118 soft tissue and bone tumors, including SMARCB1-deficient sarcomas (78, 66%): epithelioid sarcomas, epithelioid peripheral nerve sheath tumors, poorly differentiated chordomas, malignant rhabdoid tumors, and soft tissue myoepithelial tumors, as well as non-SMARCB1-deficient sarcomas (40, 34%) with various SMARCB1 genetic alterations (mutations, copy number alterations). SMARCB1 loss by immunohistochemistry was present in 94% SMARCB1 pathogenic cases. By combined sequencing and FISH assays, 80% of SMARCB1-deficient tumors harbored homozygous (biallelic) SMARCB1 loss, while 14% demonstrated heterozygous SMARCB1 loss-of-function (LOF) alterations, and 6% showed no demonstrable SMARCB1 alterations. FISH and sequencing were concordant in the ability to detect SMARCB1 loss in 48% of cases. Epithelioid sarcomas most commonly (75%) harbored homozygous deletions, while a subset showed focal intragenic deletions or LOF mutations (nonsense, frameshift). In contrast, most soft tissue myoepithelial tumors (83%) harbored SMARCB1 nonsense point mutations without copy number losses. Additionally, clinically significant, recurrent co-occurring genetic events were rare regardless of histotype. By sequencing, extended 22q copy number loss in genes flanking the SMARCB1 locus (22q11.23) occurred in one-third of epithelioid sarcomas and the majority of poorly differentiated chordomas. Poorly differentiated chordomas and soft tissue myoepithelial tumors showed significantly worse overall and disease-free survival compared to epithelioid sarcomas. Overall, SMARCB1 LOF alterations predominate and account for SMARCB1 protein loss in most cases: majority being biallelic but a subset were heterozygous. In contrast, SMARCB1 alterations of uncertain significance can be seen in diverse sarcomas types and does not indicate a SMARCB1-deficient entity.

Role of CDKN2A deletion in grade 2/3 IDH-mutant astrocytomas: need for selective approach in resource-constrained settings.

The authors aimed to assess the frequency of homozygous CDKN2A deletion in isocitrate dehydrogenase (IDH)-mutant diffuse astrocytomas (grade 2/3) and to narrow down the clinicopathological indications in which the CDKN2A fluorescence in situ hybridization (FISH) assay is cost-effective in resource-constrained settings. IDH-mutant astrocytomas were analyzed for ATRX, p53, MIB1-LI, and p16 expression using immunohistochemistry. The FISH assay was used to evaluate CDKN2A deletion and 1p/19q codeletion. Survival outcomes were assessed according to the different molecular markers. A total of 150 adult patients with IDH-mutant grade 2 (n = 95) and grade 3 (n = 55) astrocytomas (145 primary and 5 recurrent) were analyzed. Using a cutoff value of 30% for defining significant homozygous CDKN2A deletion, none of the grade 2 and 10.9% (6/55) of grade 3 astrocytomas showed this deletion (4 primary and 2 recurrent grade 3 tumors) and were reclassified as grade 4. This mutation was more frequent in recurrent (40%, 2/5) than primary (2.76%, 4/145) gliomas. Half (3/6, 50%) of the CDKN2A-deleted cases demonstrated poor outcomes; 2 of these cases experienced recurrence at 12 and 36 months after surgery, and 1 died at 5 months. The majority of CDKN2A-deleted cases showed marked cellularity (100%), pleomorphism (100%), brisk mitosis (83.3%), and tumor giant cell formation (83.4%). None of the cases with retained p16 expression harbored this deletion. Both overall survival (p = 0.039) and progression-free survival (p = 0.0045) were found to be worse in cases with p16 loss. Selectively performing CDKN2A FISH only in high-risk cases with histomorphological features of anaplasia, p16 loss, or recurrent tumors achieved a sensitivity and negative predictive value of 100%. This approach would have resulted in saving 41.1% of the original expenditure ($6900 US per 150 samples) and 27.6 person-minutes per sample without compromising the identification of deleted cases. Homozygous CDKN2A deletion is conspicuously absent in grade 2 and rare in primary grade 3 IDH-mutant astrocytomas. The authors propose that restricting use of the FISH assay to cases showing histomorphological features of anaplasia, p16 loss, or recurrent tumors will help this platform to be utilized in the most cost-effective manner in resource-constrained settings.

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

Long noncoding RNA SNHG8 promotes chemoresistance in gastric cancer via binding with hnRNPA1 and stabilizing TROY expression.

To determine SNHG8's function and potential mechanisms in gastric cancer (GC) chemoresistance. We assessed SNHG8 expression in GC cell lines, GC/CDDP cell lines (cell lines treated with cisplatin), and 42 GC tissues and SNHG8 levels in the lncRNA microarray analysis of AGS/CDDP and AGS cell lines. We also examined GC cell viability in vivo and in vitro and its apoptosis level with Flow cytometry assays. SNHG8 was localized in subcells using fluorescence in situ hybridization (FISH) and cell fraction assays, hnRNPA1's link to SNHG8 was determined utilizing RNA immunoprecipitation (RIP) and FISH assays, gene expression profiles were assessed employing RNA transcriptome sequencing, and hnRNPA1's relationship with TROY was ascertained with the RIP assay. SNHG8 increased significantly in GC cell lines and GC tissues. However, a decrease in its expression promoted sensitivity to chemotherapy and inhibited DNA damage repair in vitro and in vivo. SNHG8 appeared to regulate TROY expression via linking with hnRNPA1. Reducing TROY levels considerably stimulated GC cell chemosensitivity, whereas heightening them partially rescued the rate of chemoresistance caused by downregulating SNHG8. In summary, the "SNHG8/hnRNPA1-TROY" axis is crucial to GC chemoresistance.

Circ_0002099 is a novel molecular therapeutic target for bladder cancer.

Bladder cancer (BLCA) acts as one of the most common malignant tumors in the urinary system without ideal therapy. We performed the present study to explore the role and mechanism of Circ_0002099 in BLCA progression. RNase R treatment assay and actinomycin D treatment assay were used to confirm the circular structure of Circ_0002099. Nuclear-cytoplasmic fractionation assay and fluorescence in situ hybridization (FISH) were used to indicatethe subcellular localization of Circ_0002099. The CCK-8 assay, colony formation assay, wound-healing assay, Transwell assay, and animal experiment were used to reveal the facilitative effect of Circ_0002099 on BLCA both in vitro and in vivo. Furthermore, bioinformatic analysis, western blot analysis, FISH,and dual-luciferase reporter assay were conducted to demonstrate the role of Circ_0002099 in BLCA progression. The results indicated that Circ_0002099 was significantly upregulated in BLCA and could enhance the progression of BLCA in vivo and in vitro. Furthermore, dual-luciferase reporter assay and FISHassay revealed that Circ_0002099 could regulate miR-217-5p/miR-103a-3p/Kirsten RAS (KRAS) axis in BLCA. In addition, rescue experiments confirmed that miR-217-5p/miR-103a-3p could rescue the facilitative effect of Circ_0002099 on BLCA progression. Moreover, FUS(FUSed in sarcoma)was identified to regulate the Circ_0002099-miR-217-5p/miR-103a-3p/KRAS axis in BLCA progression. The present study suggested that FUS-medicated Circ_0002099 could promote the epithelial-mesenchymal transitionprocess in BLCA progression via miR-217-5p/miR-103a-3p/KRAS axis-WNT/β-catenin axis. It could be a promising prognostic biomarker and therapeutic target for BLCA.

Circular RNA circRASSF5 Functions as an Anti-Oncogenic Factor in Hepatocellular Carcinoma by Acting as a Competitive Endogenous RNA Through Sponging miR-331-3p

ObjectiveRecently, emerging studies have validated that circular RNAs participate in multiple biological progresses in various human malignant tumors, including hepatocellular carcinoma (HCC). However, until now, the elucidated mechanism of circular RNAs is only the tip of the iceberg. In this study, we firstly identify a novel circular RNA circRASSF5 (the only circular RNA derived from the RASSF5 gene), and attempt to investigate its biological function and underlying mechanism in HCC.MethodsqRT-PCR, Western blotting and IHC were applied to detect the expression of related genes. CCK-8 assay, EdU staining, wound healing and transwell assays were used to investigate HCC proliferation, migration and invasion abilities. Animal model studies were included to investigate the function of circRASSF5 in HCC tumorigenesis and metastasis. RNA pull-down assay, luciferase reporter assay and FISH (fluorescence in situ hybridization) assay were performed to explore the potential biological mechanism underlying circRASSF5 function in HCC.ResultsCircRASSF5 is obviously downregulated in both HCC tissues and cell lines. Low level of circRASSF5 is negatively associated with larger tumor size, severe vascular invasion, more portal vein tumor embolus and unfavorable prognosis. Loss-of-function assay reveals that circRASSF5 remarkably impedes the growth and metastasis of HCC cells in vitro and in vivo. Mechanistically, circRASSF5 directly interacts with miR-331-3p as a sponge, and then enhances the expression of PH domain and leucine-rich repeat protein phosphatase (PHLPP), thus restraining the progression of HCC cells.ConclusionAltogether, we validate that circRASSF5 is a tumor suppressor in HCC, which competitively sponges with miR-331-3p and then enhances the tumor inhibitory effect of PHLPP, indicating the potential application value of circRASSF5 for HCC diagnosis and clinical treatment.

Development and application of universal ND-FISH probes for detecting P-genome chromosomes based on Agropyron cristatum transposable elements.

Fluorescence in situ hybridization (FISH) is a basic tool that is widely used in cytogenetic research. The detection efficiency of conventional FISH is limited due to its time-consuming nature. Oligonucleotide (oligo) probes with fluorescent labels have been applied in non-denaturing FISH (ND-FISH) assays, which greatly streamline experimental processes and save costs and time. Agropyron cristatum, which contains one basic genome, "P," is a vital wild relative for wheat improvement. However, oligo probes for detecting P-genome chromosomes based on ND-FISH assays have not been reported. In this study, according to the distribution of transposable elements (TEs) in Triticeae genomes, 94 oligo probes were designed based on three types of A. cristatum sequences. ND-FISH validation showed that 12 single oligo probes generated a stable and obvious hybridization signal on whole P chromosomes in the wheat background. To improve signal intensity, mixed probes (Oligo-pAc) were prepared by using the 12 successful probes and validated in the diploid accession A. cristatum Z1842, a small segmental translocation line and six allopolyploid wild relatives containing the P genome. The signals of Oligo-pAc covered the entire chromosomes of A. cristatum and were more intense than those of single probes. The results indicate that Oligo-pAc can replace conventional genomic in situ hybridization (GISH) probes to identify P chromosomes or segments in non-P-genome backgrounds. Finally, we provide a rapid and efficient method specifically for detecting P chromosomes in wheat backgrounds by combining the Oligo-pAc probe with the Oligo-pSc119.2-1 and Oligo-pTa535-1 probes, which can replace conventional sequential GISH/FISH assays. Altogether, we developed a set of oligo probes based on the ND-FISH assays to identify P-genome chromosomes, which can promote utilization of A. cristatum in wheat improvement programs.

A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia.

Simple SummaryPatients with Fanconi Anemia (FA) are at high risk of developing oral squamous cell carcinoma with poor prognosis. Oral brush biopsy–based cytology represents an attractive non-invasive advantage in the characterization of (pre) malignant oral lesions, but a sufficient number of abnormal cells is mandatory to make the diagnosis. The aim of this 2-phases retrospective study is to introduce a new multi-color FISH assay including a 9p21 FISH assay to determine the oral lesions that require treatment, with just a few cells. We demonstrate a good accuracy of the multicolor FISH assay if applied on oral brush biopsy-based specimens from FA patients, using different algorithms to determine chromosomal aneuploidy. Nevertheless, some false positive results were observed and the detection of a genetically altered field in the oral cavity as a possible reason for what may hamper the application of multi-color FISH is discussed.Background: Fanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of oral squamous cell carcinoma. These cancers can be detected with oral brush biopsy-based cytology even at early stages. This study aims to determine the diagnostic accuracy of a new multi-color fluorescent in situ hybridization (FISH) assay consisting of probes for CCND1, TERC, MYC and centromere of chromosome 6, as well as a 9p21 FISH assay consisting of probes for CDKN2A and centromere of chromosome 9 for the detection of oral (pre) malignant lesions in FA. Methods: (I) Cutoffs for the dichotomization of positive or negative multi-color FISH results are determined and (II) retrospectively validated by using archived oral brush biopsy specimens from individuals with Fanconi anemia. In addition, the specimens for cutoff determination were re-hybridized with the 9p21 FISH assay. Results: A cutoff of six or more chromosomal aneuploid cells for a positive FISH result was determined in the cutoff study on 160 biopsy specimens. The validating of this cutoff on 152 specimens showed at best a sensitivity of 87% and a specificity of 82.9%. Conclusion: Multi-color FISH is a sufficient tool to detect chromosomal aneuploidy in oral (pre) malignant lesions of individuals with Fanconi anemia. However, some false positive results may hamper the application as an adjuvant method to oral brush biopsy-based cytology in an oral cancer surveillance program.

Mosaic trisomy 20: discrepancy between cyto-and molecular genetic technologies in prenatal diagnosis

To provide genetic counseling and prenatal diagnosis for a fetus with mosaic trisomy 20. Chromosomal karyotyping, chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) were carried out for a pregnant woman with advanced maternal age. The karyotype of amniotic fluid sample was 47,XN,+20, whilst the result of CMA was normal. To verify this discrepancy, CMA was performed again with the cultured amniotic fluid, which yielded a result of 47,XN,+20. FISH assay of the amniotic fluid sample was nuc ish(D20Z1)×3[11]/(D20Z1)×2[89], which indicated that about 11% of fetal cells were trisomy 20. After the fetus was born, the karyotype of peripheral blood sample was normal. The amniotic fluid sample might be mosaic trisomy 20, and a dominant growth of 47,XN,+20 cells had occurred during the culture process, resulting in alteration of amniotic fluid cell composition. Mosaic trisomy 20 indicated by FISH may be attributed to confined placental mosaicism or somatic mosaicism of trisomy 20.

Karyotyping of Lymphocytes and Epithelial Cells of Distinct Embryonic Origin Does Not Help to Predict the Turner Syndrome Features

Background: In Turner syndrome (TS), fluorescent in situ hybridization (FISH) karyotyping offers an alternative to classical karyotyping. Objective: We tested the added value of FISH karyotyping from lymphocytes (mesodermal origin), buccal cells (ectodermal origin), and a rear-tongue smear (endodermal origin) to determine the 45,X cell line fraction and its impact on patient phenotype. Design and Patients: Classical karyotyping and three FISH assays were done in 153 girls and women previously diagnosed with TS in four university hospitals. The 45,X cell line fraction was determined for each method and correlated with the major phenotypic signs. Results: Classical karyotyping identified 45,X/46,XX mosaicism in 77/153 subjects (50%), 45,X monosomy in 52/153 (34%), and other karyotypes in 24/153 (16%). FISH from lymphocytes verified 45,X in 47/52 original cases, whereas 4/52 had 45,X/46,XX and 1/52 45,X/47,XYY mosaicism. The 45,X cell line fraction was higher in FISH from lymphocytes compared to classical karyotyping (median 86.4% vs. 70.0%; p < 0.001), while there was no difference for FISH from buccal or rear-tongue smear cells. The mean 45,X cell line fraction was more abundant in patients with several of the characteristic phenotypic signs compared to patients without them (p < 0.01), but the predictive power was insufficient. Conclusion: FISH analysis confirmed the findings of classical karyotyping; only a few 45,X monosomy cases were reclassified as mosaics. The 45,X cell line fraction did not show clinically meaningful prediction of the phenotype. FISH analysis of buccal or rear-tongue epithelial cells may be a non-inferior, less invasive alternative to classical karyotyping.

Challenges and limitation of MTAP immunohistochemistry in diagnosing desmoplastic mesothelioma/sarcomatoid pleural mesothelioma with desmoplastic features

AimGenomic-based ancillary assays including immunohistochemistry (IHC) for BRCA-1 associated protein-1 (BAP1) and methylthioadenosine phosphorylase (MTAP), and fluorescence in situ hybridization (FISH) for CDKN2A are effective for differentiating pleural mesothelioma (PM) from reactive mesothelial proliferations. We previously reported a combination of MTAP and BAP1 IHC effectively distinguishes sarcomatoid PM from fibrous pleuritis (FP). Nevertheless, cases of sarcomatoid PM with desmoplastic features (desmoPM) are encountered where the IHC assessment is unclear. Methods and resultsWe evaluated assessment of MTAP IHC, BAP1 IHC, and CDKN2A FISH in 20 desmoPM compared to 24 FP. MTAP and BAP1 IHC could not be assessed in 11 (55 %) and 10 (50 %) cases, respectively, due to loss or faint immunoreactivity of internal positive control cells, while CDKN2A FISH could be evaluated in all cases. The sensitivities for MTAP loss, BAP1 loss, and CDKN2A homozygous deletion in desmoPM were 40 %, 10 %, and 100 %. A combination of MTAP loss and BAP1 loss yielded 45 % of sensitivity. ConclusionsMTAP IHC is a useful surrogate diagnostic marker in differentiating ordinary sarcomatoid PM from FP, but its effectiveness is limited in desmoPM. CDKN2A FISH is the most effective diagnostic assays with 100 % sensitivity and specificity in discriminating desmoPM from FP in the facilities where the FISH assay is available.

Correlation of ROS1 (D4D6) Immunohistochemistry with ROS1 Fluorescence In Situ Hybridization Assay in a Contemporary Cohort of Pulmonary Adenocarcinomas.

Sambit K. MohantyObjective Repressor of Silencing ( ROS1 ) gene rearrangement in the lung adenocarcinomas is one of the targetable mutually exclusive genomic alteration. Fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), next-generation sequencing, and reverse transcriptase polymerase chain reaction assays are generally used to detect ROS1 gene alterations. We evaluated the correlation between ROS1 IHC and FISH analysis considering FISH as the gold standard method to determine the utility of IHC as a screening method for lung adenocarcinoma. Materials and Methods A total of 374 advanced pulmonary adenocarcinoma patients were analyzed for ROS1 IHC on Ventana Benchmark XT platform using D4D6 rabbit monoclonal antibody. FISH assay was performed in parallel in all these cases using the Vysis ROS1 Break Apart FISH probe. Statistical Analysis The sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values, and accuracy were evaluated. Results A total of 17 tumors were positive either by IHC or FISH analysis or both (true positive). Four tumors were positive by IHC (H-score range: 120-270), while negative on FISH analysis (false positive by IHC). One tumor was IHC negative, but positive by FISH analysis (false negative). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, negative predictive value, and accuracy were 94.4% (confidence interval [CI]: 72.71-99.86%), 63.6% (CI: 30.79-89.07%), 2.6 (CI: 1.18-5.72), 0.09 (CI: 0.01-0.62), 80.95% (CI: 65.86-90.35%), 87.5% (CI: 49.74-98.02%), and 82.76%, respectively. Conclusion ROS1 IHC has high sensitivity at a cost of lower specificity for the detection of ROS1 gene rearrangement. All IHC positive cases should undergo a confirmatory FISH test as this testing algorithm stands as a reliable and economic tool to screen ROS1 rearrangement in lung adenocarcinomas.

A Combination of MTAP and p16 Immunohistochemistry Can Substitute for CDKN2A Fluorescence In Situ Hybridization in Diagnosis and Prognosis of Pleural Mesotheliomas.

Homozygous deletion (HD) of CDKN2A is one of the most frequent genetic abnormalities in pleural mesotheliomas. HD of CDKN2A by fluorescence in situ hybridization (FISH) is a reliable marker of malignancy in mesothelial proliferations; however, evaluation of CDKN2A deletion requires FISH. The 9p21 locus includes both CDKN2A and MTAP (methylthioadenosine phosphorylase); the latter is frequently codeleted with CDKN2A. To examine the question of whether immunohistochemistry for MTAP and p16, the protein product of CDKN2A, can serve as a surrogate for CDKN2A HD by FISH. A random selection of 125 pleural mesothelioma cases was divided into 3 groups for evaluation of p16 and MTAP expression compared with FISH for CDKN2A deletion: 53 with HD, 39 with heterozygous deletion, and 33 without deletion. By itself, loss of p16 nuclear expression (<1% staining) showed a high sensitivity (96%) but low specificity (43%) for CDKN2A HD by FISH. MTAP cytoplasmic expression loss (≤30% staining) showed a 97% specificity and 69% sensitivity. The combination of p16 nuclear (<1% staining) and MTAP cytoplasmic (≤30% staining) loss demonstrated both high specificity (96%) and high sensitivity (86%). Patients with retained p16 expression (≥1%) had the best prognosis, whereas a p16 (<1%)/MTAP loss combination was associated with a dismal prognosis. MTAP immunohistochemical staining is a valid surrogate marker for CDKN2A HD by FISH; however, to obtain the same accuracy as the FISH assay, a combination of nuclear p16 and cytoplasmic MTAP staining is recommended. These findings correlate with prognosis.

Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

ObjectiveTo investigate the mechanisms of super-enhancer-associated LINC01485/miR-619-5p/RUNX2 signaling axis involvement in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsOsteogenic differentiation of hBMSCs was induced in vitro. The expression levels of LINC01485 and miR-619-5p during osteogenesis were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Osteogenic differentiation was examined by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, ALP activity measurement, and Alizarin Red S (ARS) staining assays. Thereafter, the effects of LINC01485 and miR-619-5p on osteogenic differentiation of hBMSCs were evaluated by performing loss- and gain-of-function experiments. Subsequently, a fluorescence in situ hybridization (FISH) assay was employed to determine the cellular localization of LINC01485. Bioinformatics analysis, RNA antisense purification (RAP) assay, and dual-luciferase reporter assays were conducted to analyze the interactions of LINC01485, miR-619-5p, and RUNX2. Rescue experiments were performed to further delineate the role of the competitive endogenous RNA (ceRNA) signaling axis consisting of LINC01485/miR-619-5p/RUNX2 in osteogenic differentiation of hBMSCs.ResultsThe expression of LINC01485 was up-regulated during osteogenic differentiation of hBMSCs. The overexpression of LINC01485 promoted osteogenic differentiation of hBMSCs by up-regulating the expression of osteogenesis-related genes [e.g., runt-related transcription factor 2 (RUNX2), osterix (OSX), collagen type 1 alpha 1 (COL1A1), osteocalcin (OCN), and osteopontin (OPN)], and increasing the activity of ALP. ALP staining and ARS staining were also found to be increased upon overexpression of LINC01485. The opposing results were obtained upon LINC01485 interference in hBMSCs. miR-619-5p was found to inhibit osteogenic differentiation. FISH assay displayed that LINC01485 was mainly localized in the cytoplasm. RAP assay results showed that LINC01485 bound to miR-619-5p, and dual-luciferase reporter assay verified that LINC01485 bound to miR-619-5p, while miR-619-5p and RUNX2 bound to each other. Rescue experiments illustrated that LINC01485 could promote osteogenesis by increasing RUNX2 expression by sponging miR-619-5p.ConclusionLINC01485 could influence RUNX2 expression by acting as a ceRNA of miR-619-5p, thereby promoting osteogenic differentiation of hBMSCs. The LINC01485/miR-619-5p/RUNX2 axis might comprise a novel target in the bone tissue engineering field.