Fluorescence In Situ Hybridization Assay Research Articles

[Genetic analysis for a female carrying idic(Y)(p11.32) with Disorders of sex development].

To explore the genetic basis for a patient with Disorders of sex development (DSD). A female patient who had presented at the Linyi People's Hospital due to primary amenorrhea on April 6, 2022 was selected as the study subject. Conventional chromosomal karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), fluorescence quantitative PCR and Sanger sequencing were carried out for the patient. The patient, a 14-year-old female, had featured short statue, multiple nevi, and primary amenorrhea. She was found to have a karyotype of 46,X,idic(Y)(p11.3)[59]/45,X[39]/47,X,idic(Y)(p11.3)×2[2]. The result of FISH assay was 46,X,der(Y).ish idic(Y)(p11.3)(SRY+)[59]/45,X[39]/47,X,der(Y)×2.ish idic(Y)(p11.3)(SRY+)[2]. That of CMA was arr[GRCh37](X)×1,(Y)×0-1,arr[GRCh37]Yp11.32(118552_472090)×1. The patient had no deletion in the AZF region of Y chromosome, and was negative for variant of SRY gene. Combining the above results, her molecular karyotype was determined as mos 46,X,idic(Y)(p11.32)[59]/45,X[39]/47,X,idic(Y)(p11.32)×2[2].ish 46,X,idic(Y)(p11.32)(DXZ1+,DYZ1++,DYZ3++,SRY+)[59]/45,X(DXZ1+,DYZ1-,DYZ3-,SRY-)[39]/47,X,der(Y)×2.ish idic(Y)(p11.32)(DXZ1+,DYZ1++,DYZ3++,SRY+)[2].arr[GRCh37](X)×1, (Y)×0-1，arr[GRCh37]Yp11.32(118552_472090)×1. The patient was diagnosed with mosaicism DSD with idic(Y)(p11.32). The abnormal mosaicism karyotype probably underlay the DSD in this patient.

Hsa_circ_0079875 functions as a competitive endogenous RNA to promote hepatocellular carcinoma progression

ABSTRACT Circular RNA (circRNA) can influence the development of hepatocellular carcinoma (HCC) as a competitive endogenous RNA (ceRNA). However, there are still many circRNAs whose functions are unknown. Our research explores the role of a novel circRNA, hsa_circ_0079875, in HCC. The expression of hsa_circ_0079875 in HCC was verified by next-generation sequencing, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and fluorescence in situ hybridization (FISH). The distribution of hsa_circ_0079875 in HCC cells was investigated by RNA subcellular isolation and FISH assays. The functional effects on HCC proliferation, invasion, migration, cell cycle, and apoptosis were verified by overexpression and knockdown of hsa_circ_0079875. Moreover, xenograft mouse models and immunohistochemistry experiments were used to assess the function of hsa_circ_0079875 in vivo. Hsa_circ_0079875 was up-regulated in HCC tissues and mainly distributed in the cytoplasm. Higher hsa_circ_0079875 leads to larger tumor tissue, more microvascular invasion(MVI) and higher AFP levels, which in turn leads to a poor prognosis. Overexpression of hsa_circ_0079875 can promote the proliferation, migration, and invasion of HCC cells and inhibit apoptosis in vitro and in vivo. Knocking down hsa_circ_0079875 has the opposite effect. Sequencing and biological information predicted the target miRNA and mRNA of hsa_circ_0079875. Further bioinformatics and clinical correlation analysis revealed that hsa_circ_0079875 promote the malignant biological behaviors of HCC through hsa_circ_0079875/miR-519d-59/NRAS ceRNA net. Therefore, hsa_circ_0079875 can be a potential prognostic marker and therapeutic target for HCC.

RET Fusion Testing in Patients With NSCLC: The RETING Study

IntroductionRET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. MethodsThis situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. ResultsThe most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). ConclusionsIn-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.

Genomic and cytogenetic analyses reveal satellite repeat signature in allotetraploid okra (Abelmoschus esculentus)

BackgroundSatellite repeats are one of the most rapidly evolving components in eukaryotic genomes and play vital roles in genome regulation, genome evolution, and speciation. As a consequence, the composition, abundance and chromosome distribution of satellite repeats often exhibit variability across various species, genome, and even individual chromosomes. However, we know little about the satellite repeat evolution in allopolyploid genomes.ResultsIn this study, we investigated the satellite repeat signature in five okra (Abelmoschus esculentus) accessions using genomic and cytogenetic methods. In each of the five accessions, we identified eight satellite repeats, which exhibited a significant level of intraspecific conservation. Through fluorescence in situ hybridization (FISH) experiments, we observed that the satellite repeats generated multiple signals and exhibited variations in copy number across chromosomes. Intriguingly, we found that five satellite repeats were interspersed with centromeric retrotransposons, signifying their involvement in centromeric satellite repeat identity. We confirmed subgenome-biased amplification patterns of these satellite repeats through existing genome assemblies or dual-color FISH, indicating their distinct dynamic evolution in the allotetraploid okra subgenome. Moreover, we observed the presence of multiple chromosomes harboring the 35 S rDNA loci, alongside another chromosomal pair carrying the 5 S rDNA loci in okra using FISH assay. Remarkably, the intensity of 35 S rDNA hybridization signals varied among chromosomes, with the signals predominantly localized within regions of relatively weak DAPI staining, associated with GC-rich heterochromatin regions. Finally, we observed a similar localization pattern between 35 S rDNA and three satellite repeats with high GC content and confirmed their origin in the intergenic spacer region of the 35 S rDNA.ConclusionsOur findings uncover a unique satellite repeat signature in the allotetraploid okra, contributing to our understanding of the composition, abundance, and chromosomal distribution of satellite repeats in allopolyploid genomes, further enriching our understanding of their evolutionary dynamics in complex allopolyploid genomes.

Prioritization of Fluorescence In Situ Hybridization (FISH) Probes for Differentiating Primary Sites of Neuroendocrine Tumors with Machine Learning.

Determining neuroendocrine tumor (NET) primary sites is pivotal for patient care as pancreatic NETs (pNETs) and small bowel NETs (sbNETs) have distinct treatment approaches. The diagnostic power and prioritization of fluorescence in situ hybridization (FISH) assay biomarkers for establishing primary sites has not been thoroughly investigated using machine learning (ML) techniques. We trained ML models on FISH assay metrics from 85 sbNET and 59 pNET samples for primary site prediction. Exploring multiple methods for imputing missing data, the impute-by-median dataset coupled with a support vector machine model achieved the highest classification accuracy of 93.1% on a held-out test set, with the top importance variables originating from the ERBB2 FISH probe. Due to the greater interpretability of decision tree (DT) models, we fit DT models to ten dataset splits, achieving optimal performance with k-nearest neighbor (KNN) imputed data and a transformation to single categorical biomarker probe variables, with a mean accuracy of 81.4%, on held-out test sets. ERBB2 and MET variables ranked as top-performing features in 9 of 10 DT models and the full dataset model. These findings offer probabilistic guidance for FISH testing, emphasizing the prioritization of the ERBB2, SMAD4, and CDKN2A FISH probes in diagnosing NET primary sites.

[Clinicopathological and molecular features of metaplastic thymoma].

Objective: To investigate the clinicopathological features, and molecular genetic alterations of metaplastic thymoma (MT). Methods: A total of ten MT cases, diagnosed from 2011 to 2021, were selected from the Department of Pathology of Jinling Hospital, Nanjing University Medical School, Nanjing, China for clinicopathological and immunohistochemical (IHC) examination and clinical follow-up. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and YAP1 C-terminus (YAP1-CT) IHC were performed to detect YAP1::MAML2 fusions. Results: There were four males and six females, ranging in age from 29 to 60 years (mean 50 years, median 54 years). Microscopically, all tumors showed a typical biphasic morphology consisting of epithelial components and gradually or abruptly transitioning spindle cell components. The two components were present in varying proportions in different cases. Immunophenotypically, the epithelial cells were diffusely positive for CKpan, CK5/6 and p63. The spindle cells were diffusely positive for vimentin and focally positive for EMA. TdT was negative in the background lymphocytes. Ki-67 proliferation index was less than 5%. YAP1 and MAML2 break-apart FISH analyses showed that all ten cases had narrow split signals with a distance of nearly 2 signal diameters and may be considered false-negative. Using YAP1::MAML2 fusion FISH assays, abnormal fusion signals were observed in all the ten cases. NGS demonstrated YAP1::MAML2 fusions in all eight cases with adequate nucleic acids; in two cases the fusions were detected by DNA sequencing and in eight cases by RNA sequencing. All ten cases of MT demonstrated loss of YAP1 C-terminal expression in epithelioid cells. Conclusions: MT is a rare and low-grade thymic tumor characterized by a biphasic pattern and YAP1::MAML2 fusions. Break-apart FISH assays may sometimes show false-negative results due to the proximity of YAP1 and MAML2, while YAP1 C-terminal IHC is a highly sensitive and specific marker for MT. Loss of YAP1 C-terminal expression can also be used to screen YAP1::MAML2 fusions for possible MT cases.

Deep and Durable Cytogenetic and Molecular Responses with Pemigatinib in Myeloid/Lymphoid Neoplasms with Fibroblast Growth Factor Receptor 1 Rearrangement: The Fight-203 Study

Introduction:Myeloid/lymphoid neoplasmswith fibroblast growth factor receptor 1 rearrangement (MLN FGFR1) are aggressive hematologic neoplasms caused by various reciprocal translocations involving chromosome band 8p11, resulting in tyrosine kinase fusion genes (eg, ZMYM2::FGFR1 from t[8;13][p11;q12] or BCR::FGFR1 from t[8;22][p11;q11]) and constitutive FGFR1 activationand downstream signaling. Pemigatinib is a potent and selective oral inhibitor of FGFR1-3 approved for the treatment of adults with relapsed or refractory MLN FGFR1. Efficacy and safety data for 41 patients with MLN FGFR1 in the ongoing FIGHT-203 study of pemigatinib were previously reported. Median daily pemigatinib dose was 9.5 mg; treatment-emergent adverse events led to dose interruptions in 66% of patients. Rates of complete response (CR) among the 38 patients evaluable for clinical response and rates of complete cytogenetic response (CCyR) for the 40 patients evaluable for cytogenetic response were 74% and 70%, respectively. CR and CCyR occurred in >80% of patients with chronic phase disease and in >50% of patients with blast phase disease. Here we present an analysis of centralized fluorescence in situ hybridization (FISH) evaluation of bone marrow (BM) cells and serial characterization of levels of FGFR1 fusion transcripts in this same cohort. Methods: FIGHT-203 (NCT03011372) is an ongoing open-label, multicenter, phase 2 study evaluating pemigatinib in patients ≥18 years old with MLN FGFR1 and ≥1 prior therapy. The starting dose was pemigatinib 13.5 mg daily (2 weeks on, 1 week off). Following protocol amendments, patients without prior therapy were eligible to enroll, and the dosing regimen was changed to a continuous schedule. Patients were censored for cytogenetic and molecular follow-up at the time of transplantation. CCyR was defined as 0 metaphases positive for the respective reciprocal translocation in ≥20 evaluated BM metaphases on local and/or central karyotyping or 0 cells (or not exceeding the lower level of detection of the probe) with the FGFR1 rearrangement in ≥200 BM cells on local and/or central break-apart FISH assays (central FISH prioritized). As part of the translational research plan, fusion partner genes were identified with next-generation sequencing (NGS; FusionPlex, Archer, Boulder, CO, USA) and targeted PCR profiling of RNA extracted from whole blood; droplet digital PCR (ddPCR) assays were developed to monitor the frequencies of specific FGFR1 fusion transcripts in RNA extracted from whole blood samples (estimated limit of detection, 0.05 copies/μL). Results: As of June 30, 2021, 39 of 41 enrolled and treated patients were evaluable for translational analyses. Among the 39 patients with whole blood samples, FGFR1 fusion partner genes identified on translational testing were ZMYM2 (n=17), BCR (n=11), FGFR1OP (n=4), TPR (n=3), TRIM24 (n=2), and C14orf93, CCDC6, CNTRL and ETV6 (n=1 each). Two of 39 patients had 2 fusions detected ( ZMYM2 and BCR, n=1; ZMYM2 and TPR, n=1). C14orf93, CCDC6 and ETV6 have not been previously identified as FGFR1 fusion partner genes. Central FISH assessment of BM cells confirmed FGFR1-aberrantcells were reduced to undetectable levels in responding patients but rebounded in some patients during periods of treatment interruption (Figure A). Serial ddPCR of whole blood samples demonstrated marked reductions in FGFR1 fusion transcripts for the 34 patients with ≥3 measures while on treatment (Figure B). Among patients with CR, 81.0% had a >2-log reduction and 47.6% had a >3-log reduction in detectable FGFR1 fusion transcripts. Smaller reductions in FGFR1 fusion transcripts were observed in patients with stable disease. Changes in FGFR1 fusion transcripts correlated with FGFR1 FISH results (Pearson coefficient = 0.81) and reflected changes in treatment intensity. Conclusion:Several new FGFR1 fusion partner genes, including C14orf93, CCDC6, and ETV6 were identified with NGS and PCR profiling. Pemigatinib treatment resulted in marked decreases in the percentages of cells with the FGFR1 rearrangement on FISH, as well as 2-3 log reductions in FGFR1 fusion transcripts in patients with MLN FGFR1. Further investigations are ongoing to determine the relationship between pemigatinib dose intensity and the depth and durability of molecular response.

High Deletion Burden Identified By Whole Genome Sequencing Is Associated with Enhanced Risk in del17p CLL Patients

Presence of deletion17p (del17p) in frontline (1L) and relapsed/refractory (R/R) CLL has been associated with shorter time to treatment initiation, early relapse after therapy and inferior overall survival (OS) (Mato et.al., Haemotologica 2022). However, there exists notable clinical heterogeneity in terms of outcome within this group (Tam et.al., Blood 2009, Stephens et.al; AJH 2015). Previous reports in 1L and R/R CLL have identified percentage of del17p nuclei by fluorescence in situ hybridization (FISH), having TP53 mutations on the second allele, copy number alterations and complex karyotype (CK) as additional risk factors within the del17p population (Yu et al., CCR 2017; Baliakas et al., Blood 2019). Here we profile a cohort of wait and watch (W&W, n=14), 1L treated (n=36) and R/R CLL (n=3) patients by whole genome sequencing (WGS) and RNA sequencing (RNAseq) to gain insights into genomic factors and gene expression pathways contributing to the high-risk nature of del17p across the spectrum of CLL patients. Using the Mayo Clinic CLL Database, we identified 53 patients with del17p (identified by FISH testing in routine clinical practice at diagnosis) who had stored peripheral blood mononuclear cells (PBMCs) available. RNA and DNA from matched peripheral blood (tumor) and DNA from CD19- (germline) samples at diagnosis (n=50) and relapse (n=3) was extracted using Qiagen RNeasy (RNA) and Purene (DNA) kits and subjected to RNAseq and WGS respectively. Additional FISH assays (del11q, trisomy 12, del13q) and CK by chromosomal banding analysis (CBA) were performed. Baseline clinical characteristics, time to first treatment, types of treatment, and survival were abstracted from the CLL Database. The Mayo Clinic IRB approved this study. Copy number analysis identified deletions in 3p, 4p, 8p, 9p, 9q and gains in 2p, 8q, 15q and 17q chromosomal lesions occurring in patients. We explored the association of clinical and genetic factors, and genomic instability signatures with survival to identify high-risk features in del17p patients. Both total number of deletions by WGS and number of chromosomal aberrations by CBA as a continuous variable were significantly associated with unfavorable OS in the entire cohort (n=53) in a univariate analysis (Hazard ratio, HR= 2.29, p= 0.00054 and HR= 2.33, p=0.0054 respectively). However, there was no significant association of number of gained genes by WGS or aneuploidy scores with survival (HR=0.74, p=0.37). In patients with CK data (n=41), deletion burden, but not gains, were highly correlated to chromosomal aberrations by CBA. Using cutpointr package for identifying deletion threshold corresponding to CK≥ 3, we defined patients with deletion burden ≥ 514 as enhanced-high risk (EHR; median OS = 4.83 years) compared to the group with high-risk (HR; median OS=9.94 years) (p=0.025). There was no significant difference in number of deleted genes between WW and treated patients. The EHR group is enriched in patients with high clonal cell fraction of del17p by FISH, unmutated IGHV and CK≥ 3. The EHR group remains significant in a multi-variable analysis with IGHV-mutation status, ZAP70, and age (HR: 5.16, 95% CI: 1.04 - 25.6, p=0.04). Mutational analysis identified 35 known CLL driver mutations with TP53 (86%), ATM (15%), NOTCH1(13%), MGA (13%), CHD2 and MED12(11%) as the top mutated genes. Total number of driver genes mutated was not significantly different between EHR and HR subgroups, but the EHR subset was significantly enriched with mutations in NOTCH1 (21%), and MGA (21%) as compared to no mutations in the HR group in these genes. Deleted genes (≥ 514) was prognostic in bi-allelic TP53 aberration patients as well (p=0.021) Differential expression gene-set enrichment analysis indicated a significant enrichment of DNA repair, cellular response to DNA damage and downregulation of inflammatory responses in the EHR group. CK is a known prognostic marker which includes both chromosomal losses and gains. In this small cohort of del17p we were able to show the prognostic value of deletions only, but not gains, as captured by WGS. Using genome wide sequencing, we identify increasing genomic deletions as a feature of enhanced-high risk del17p. While deletion burden cut-offs identified here are specific to our research method and require further validation in additional independent cohorts, EHR subgroup remained significant after adjusting for other known prognostic variables .

Wash-free FISH of bacterial ribosomal RNAs by benzo[a]pyrene-modified oligonucleotides

Rapid and reliable bacterial detection methods are essential for appropriate antibiotic treatment of infectious diseases. Fluorescence in situ hybridization (FISH) is one of the most promising detection methods for the culture-independent detection of bacteria. However, standard FISH assays involve stringent washing procedures that result in poor reproducibility of assay results. Therefore, reliable FISH assays require fluorescent oligonucleotides that produce strong fluorescence in the presence of complementary RNAs. Here, we report the synthesis and photophysical properties of OMCbpy fluorescent oligonucleotides, which contain a 2′-O-benzo[a]pyrenylmethylcytidine (Cbpy) unit in the middle of each 2′-O-methyl RNA sequence. Fluorescence studies showed that the fluorescence emission of OMCbpy was enhanced up to 39-fold in the presence of complementary RNAs under slightly acidic conditions. Fluorescence microscopy analysis revealed that wash-free FISH with OMCbpy allows for detection of 16S ribosomal RNAs in fixed Escherichia coli (E. coli) cells with high reproducibility.

UroVysion™ fluorescence in situ hybridization (FISH) possibly has a high positive rate in carcinoma of non-urothelial lineages.

Background: Positive UroVysion™ fluorescence in situ hybridization (FISH) is generally considered as urothelial carcinoma (UC). We clarify if UroVysion™ FISH can be positive in carcinoma of non-urothelial lineages (CNUL), and verify the consistency of urine FISH and histological FISH in CNUL. Methods: All CNUL subjects detected by urine FISH assay due to haematuria from Tongji Hospital were screened. Meanwhile, 2 glandular cystitis and 2 urothelial carcinoma were served as negative or positive control. Paraffin-embedded tissue sections of all subjects were sent to the pathology department for histological FISH detection. Results: A total of 27 patients were included in this study, including 9 with adenocarcinomas, 11 with squamous cell carcinomas, and 7 with other tumour types. The overall positive rate in urine FISH was 64.00% (16/25) in patients with CNUL, 77.78% (7/9) in those with adenocarcinoma and 54.55% (6/11) in those with squamous carcinoma. There was a significant difference in the GLP p16 gene deletion rate between UC and CNUL (100% vs. 8.00%, p = 0.017). Histological FISH results showed that the histological results of 19 patients were consistent with their urine FISH results, and only one patient with stage Ⅲa urachal carcinoma had inconsistent histological FISH results (positive) and urine FISH (negative) results. Conclusion: We demonstrated for the first time the application value of FISH in CNUL on urine samples. Positive urine FISH tests indicate not only UC, but also CNUL. UroVysion™ FISH possibly has a high positive rate in CNUL. CNUL and UC have different genetic changes shown by FISH.

Preoperative fluorescence in situ hybridization analysis as a predictor of tumor recurrence in patients with non-muscle invasive bladder cancer: a bi-institutional study

BackgroundNon-muscle invasive bladder cancer (NMIBC) is known for its elevated recurrence rate, necessitating an enhancement in the current risk stratification for recurrence. The urine-based fluorescence in situ hybridization (FISH) assay has emerged as a noninvasive auxiliary tool for detecting bladder cancer. The aim of this study was to explore the potential relationship between the preoperative FISH assay and recurrence, and to develop a FISH-clinical nomogram for predicting the recurrence-free survival (RFS) in NMIBC patients.MethodsIn total, 332 eligible patients were enrolled from two hospitals. The SYSMH cohort was randomly assigned to the training set (n = 168) and the validation set I (n = 72) at a ratio of 7:3, while the SYSUTH cohort was allocated to the validation set II (n = 92). The correlation between the preoperative FISH assay and recurrence was determined through the Cox regression analysis. The least absolute shrinkage and selection operator (LASSO) Cox regression algorithm was used for model construction. The performance of the model was assessed by its discrimination, calibration, and clinical usefulness.ResultsWe uncovered that chromosome 7 aneuploidy, p16 locus loss, number of the positive FISH sites, and the FISH test result were significantly associated with tumor recurrence. Then, a FISH-clinical nomogram incorporating the FISH test result, T stage, associated CIS, tumor grade, and tumor status was developed. It showed favorable calibration and discrimination with a C-index of 0.683 (95%CI, 0.611–0.756) in the training set, which was confirmed in the validation set I and validation set II with C-indexes of 0.665 (95%CI, 0.565–0.765) and 0.778 (95%CI, 0.665–0.891), respectively. Decision curve analysis revealed the clinical usefulness of the nomogram. Moreover, our proposed nomogram significantly outperformed the guideline-recommended EORTC and CUETO scoring models.ConclusionOur study confirmed the prognostic value of the preoperative FISH assay and proposed a FISH-clinical nomogram to predict RFS in NMIBC patients. Our nomogram can serve as a more precise tool for recurrence risk stratification, which may optimize disease management in bladder cancer and improve patient prognosis.

Detection of clinically actionable gene fusions by next-generation sequencing-based RNA sequencing of non-small cell lung cancer cytology specimens: A single-center experience with comparison to fluorescence insitu hybridization.

Genomic profiling is needed to identify actionable alterations in non-small cell lung cancer (NSCLC). Panel-based testing such as next-generation sequencing (NGS) is often preferred to interrogate multiple alterations simultaneously. In this study, we evaluate the utility of an RNA-based NGS assay to detect genomic alterations in NSCLC cytology specimens and compare these results to fluorescence in situ hybridization (FISH) testing. A retrospective review was performed of 264 NSCLC cytology specimens that were concurrently tested for gene fusions by RNA-based NGS and ALK, RET, and/or ROS1 by FISH. Genomic alterations were detected in 29 cases by NGS, including ALK, RET, ROS1, NTRK, NUTM1, and FGFR3 fusions and MET exon 14 skipping alterations. Of the 20 cases with ALK, RET, and ROS1 fusions detected by NGS, 16 (80%) were concordant with the corresponding FISH results. Three cases showed discordance, where EML4::ALK (n=2) and SLC34A2::ROS1 (n=1) fusions were not detected by the corresponding FISH assay; one case with EZR::ROS1 was inadequate for FISH. No gene fusions were detected in 181 cases by NGS and 54 cases failed testing. The concordance rates for detecting ALK, RET, and ROS1 fusions using NGS and FISH were 97%, 100%, and 99.5%, respectively. RNA-based NGS can be used to detect gene fusions in NSCLC cytology cases with high concordance with FISH results. However, RNA-based NGS may have high failure rates and therefore a low threshold for reflexing inadequate cases to an orthogonal testing method is essential for comprehensive genomic profiling.

Loss of YAP1 C-terminus expression as an ancillary marker for metaplastic thymoma: a potential pitfall in detecting YAP1::MAML2 gene rearrangement.

Metaplastic thymoma is a rare thymic tumour characterized by Yes Associated Protein 1 (YAP1) and Mastermind Like Transcriptional Coactivator 2 (MAML2) gene fusions resulting from an intrachromosomal inversion of chromosome 11. Immunohistochemistry with an antibody directed against the C-terminus of YAP1 has shown loss of expression inYAP1-rearranged vascular neoplasms, poromas, and porocarcinomas. This study aimed to validate ananti-YAP1 C-terminal antibody as an ancillary immunohistochemical marker for the diagnosis of metaplastic thymoma. Ten metaplastic thymomas were selected for the current study. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed to detect YAP1::MAML2 fusions. We then performed immunohistochemistry to detect YAP1 C-terminus expression in 10 metaplastic thymomas, 50 conventional thymomas (10 each of type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, and type B3 thymoma) and seven thymic carcinomas. All 10 cases showed narrow split signals with a distance of nearly two signal diameters and sometimes had false-negative results in YAP1 and MAML2 break-apart FISH (BA-FISH). Abnormal colocalized signals of the YAP1::MAML2 fusion were observed in all 10 cases using fusion FISH (F-FISH) assays. Eight of 10 cases with adequate nucleic acids were successfully sequenced and all showed YAP1::MAML2 fusions; in two cases the fusions were detected by both DNA and RNA sequencing and in six cases by RNA sequencing only. YAP1::MAML2 fusion transcripts were identified in four cases by RT-PCR. Metaplastic thymoma showed loss of YAP1 C-terminus expression in all 10 (100%) cases. All other thymic neoplasms showed retained YAP1 C-terminus expression. YAP1 C-terminus immunohistochemistry is a highly sensitive and specific ancillary marker that distinguishes metaplastic thymoma from its mimics. BA-FISH assays could not effectively detect YAP1::MAML2 fusions due to the proximity of the two genes. Loss of YAP1 C-terminus expression is a reliable surrogate for the detection of YAP1::MAML2 fusions in metaplastic thymoma.

Validation of Dual-Color Dual In Situ Hybridization for HER2/neu Gene in Breast Cancer.

Human epidermal growth factor (HER2/neu) gene amplification, a poor prognostic factor in invasive breast cancer, has shown substantial utility as a predictive marker, with significantly improved survival following anti-HER2 therapies like trastuzumab. Dual-color dual in situ hybridization (D-DISH), a recently introduced fully automated assay for HER2/neu evaluation on light microscopy, has several advantages over fluorescence in situ hybridization (FISH). To standardize and validate the D-DISH assay using FISH as the gold standard and assess interobserver reproducibility in interpreting the D-DISH assay. D-DISH was performed using the latest HER2 Dual ISH DNA Probe Cocktail assay (Ventana Medical Systems Inc, Tucson, Arizona) in 148 cases of invasive breast cancer. The same block was used for performing immunohistochemistry by Ventana PATHWAY anti-HER2/neu (4B5) antibody and FISH assay by ZytoLight SPEC ERBB2/CEN17 Dual Color Probe. D-DISH was separately interpreted by 4 pathologists blinded to FISH results. Concordance of 98.65% and a Cohen κ value of 0.97 were observed between FISH and D-DISH. Intraclass correlation coefficient (0.93-0.97) and κ values (0.98-1.0) for interobserver reproducibility showed almost perfect agreement by D-DISH. Interobserver reproducibility was also evaluated for genomic heterogeneity, HER2 group categorization, and polysomy (κ values 0.42-0.74, 0.89-0.93, and 0.98-1.0, respectively). We successfully validated the latest version of D-DISH assay as a substitute for FISH in predicting HER2 gene status with significant interobserver reproducibility, concluding that this D-DISH assay may be introduced in routine diagnostic services as a reflex test to ascertain HER2 gene status.

A Robust FISH Assay to Detect FGFR2 Translocations in Intrahepatic Cholangiocarcinoma Patients.

FGFR fusions retaining the FGFR kinase domain are active kinases that are either overexpressed or constitutively activated throughout diverse cancer types. The presence of FGFR translocations enhances tumor cell proliferation and contributes to significant sensitivity to FGFR kinase inhibitors. FGFR2 as an actionable target in intrahepatic cholangiocarcinoma (iCCA) has been tested in many clinical trials. FISH (fluorescence in situ hybridization) and NGS (next-generation sequence) are well-known tools to investigate the translocations of FGFR with multiple or unknown translocation partners. A rapid and robust FISH assay was developed and validated to detect FGFR2 translocations from FFPE specimens in iCCA. The analytical performance of the FISH assay was evaluated for probe localization, probe sensitivity and specificity, and assay precision. Twenty-five archival FFPE specimens from local iCCA patients were tested for FGFR2 translocations. FISH results were correlated with that of NGS on some samples. Biallelic translocations and a novel FGFR2 translocation involving the partner gene, SHROOM3, t(4;10) (q21;q26), were identified in a local iCCA patient.

Downregulated Phosphoglycerate Kinase 1 Attenuates Cerebral Ischemia–Reperfusion Injury by Reversing Neuroinflammation and Oxidative Stress through the Nuclear Factor Erythroid 2 Related Factor 2/ARE Pathway

Understanding the role and mechanism of astrocytes in inflammation and oxidative response is crucial for developing therapeutic strategies to reduce inflammation and oxidative injury in cerebral ischemia–reperfusion injury (CIRI). In this study, we investigated the regulatory effects of phosphoglycerate kinase 1 (PGK1) on inflammation and oxidative response after CIRI in male adult Sprague-Dawley (SD) rats and using primary astrocytes obtained from neonatal SD rats, and explored its related mechanisms. We established a rat model of middle cerebral artery occlusion-reperfusion (MCAO/R) by suture occlusion, and an oxygen-glucose deprivation/reoxygenation model of astrocytes using oxygen-free, glucose-free, and serum-free cultures. AAV8-PGK1-GFP was injected into the left ventricle 24 h before modeling. Real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, co-immunoprecipitation (CoIP) assay, fluorescence in situ hybridization (FISH), and western blotting were used to elucidate the in-depth mechanisms of PGK1 in CIRI. PGK1 overexpression significantly exacerbated neurological deficits, increased cerebral infarct volume, and aggravated nerve cell injury in rats after MCAO/R. Using FISH and CoIP assays, we verified the localization of PGK1 and Nrf2 in primary astrocytes. Further rescue experiments showed that Nrf2 knockdown eliminated the protective effect of CBR-470-1 (a PGK1 inhibitor) on CIRI. Lastly, we confirmed that PGK1 aggravates CIRI by inhibiting the Nrf2/ARE pathway. In conclusion, our findings suggest that inhibiting PGK1 attenuates CIRI by reducing the release of inflammatory and oxidative factors from astrocytes by activating the Nrf2/ARE signaling pathway.

Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells.

Fluorescence in situ hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. We present an improved method for FISH probe production that yields high-purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The degree of labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO, and Alexa dyes) was mostly >90%, comparable with commercial probes. The ease and low cost of production allowed the generation of probe sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for Polr2a (RNA polymerase II subunit 2a) and Gapdh, and of the long noncoding RNAs Malat1 and Neat1 Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the Gabbr1 and Noc2l transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

LncRNA OIP5-AS1/miR-410-3p/Wnt7b axis promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via regulating the Wnt/β-catenin pathway

LncRNA OIP5-AS1 has a common gene imbalance in various cancers and tumours, which plays an important role in regulating its biological function. However, there are few studies on lncRNA OIP5-AS1 in rheumatoid arthritis (RA). The purpose of the present study was to investigate the role of lncRNA OIP5-AS1 in the pathogenesis of RA. In the present study, we established an adjuvant arthritis (AA) rat model to obtain primary fibroblast-like synoviocytes (FLSs);The subcellular localisation of lncRNA OIP5-AS1 was detected by fluorescence in situ hybridisation (FISH) assay; Cell proliferation of FLSs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay;IL-1β, IL-6 and TNF-α concentrations were measured by enzyme-linked immunosorbent assay (ELISA);Quantitative real-time PCR (qRT-PCR), Western blots(WB) and immunofluorescence were used to detect the expression of lncRNA OIP5-AS1/miR-410-3p/wnt7b signal axis and Wnt/β-catenin signal pathway related indicators in FLSs. FISH assay confirmed the presence of lncRNA OIP5-AS1 in the cytoplasm, suggesting that it acts as a competing endogenous RNA (ceRNA). qRT-PCR showed that the expression of lncRNA OIP5-AS1 was upregulated in FLSs, while the expression of miR-410-3p was downregulated in FLSs. We also found that lncRNA OIP5-AS1 knockdown inhibited the proliferation and inflammation of FLSs. Moreover, the expression of Wnt7b, the downstream target gene of miR-410-3p, and the activation of the Wnt/β-catenin signalling pathway were also inhibited by lncRNA OIP5-AS1 knockdown. These results suggested that lncRNA OIP5-AS1 promotes the activation of the Wnt/β-catenin signalling pathway by regulating the miR-410-3p/Wnt7b signalling axis, thereby participating in the occurrence and development of RA.

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.