Validation of terminal Schwann cell gene marker expression by fluorescent in situ hybridization using RNAscope.

Recent RNA-seq studies have generated a new crop of putative gene markers for terminal Schwann cells (tSCs), non-myelinating glia that cap axon terminals at the vertebrate neuromuscular junction (NMJ). While compelling, these studies did not validate the expression of the novel markers using in situ hybridization techniques. Here, we use RNAscope technology to study the expression of top candidates from recent tSC and non-myelinating Schwann cell marker RNA-seq studies. Our results validate the expression of these markers at tSCs but also demonstrate that they are present at other sites in the muscle tissue, specifically, at muscle spindles and along intramuscular nerves.

CIC rearranged sarcomas: A single institution experience of the potential pitfalls in interpreting CIC FISH results.

AIM: The aim of this study was to establish how reliable FISH CIC analysis using an IVD (in vitro diagnostic) commercial probe is. METHODS AND RESULTS: A series of 19 CIC-DUX4 sarcomas were evaluated. The samples presenting CIC-DUX4 fusion transcript detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Sanger sequencing and/or Next Generation Sequencing were selected for Fluorescent in Situ Hybridization (FISH) CIC analysis with CIC break-apart IVD probe and compared to molecular analysis. CIC FISH analysis showed 26% of false negatives. CONCLUSION: Our results indicate that, in the setting of CIC-DUX4 fusion positive small round cell sarcomas, CIC FISH using IVD commercial probe may lead to false-negative results. This novel study evaluates the diagnostic use of a commercial IVD CIC probe for FISH.

The success rate of interphase fluorescence in situ hybridization in plasma cell disorders can be improved using unconventional sources of plasma cells.

BACKGROUND: Immunomagnetic cell sorting (IMCS) is a preferred technique for the enrichment of plasma cells (PC) before fluorescence in situ hybridization (FISH). Here, we share our real-world experience regarding the success rate of IMCS, its limitations, and the utility of alternate sources to obtain a successful FISH in various PC disorders. MATERIALS AND METHODS: A retrospective analysis was performed in patients with a PC neoplasm, who underwent bone marrow (BM) examination, and FISH testing over 30 months. In all cases with an unsuccessful IMCS, an attempt was made to identify the cause of failure. RESULTS: Immunomagnetic cell sorting of PCs was successful in 395/450 cases (87.8%; 77/98 cases (78.6%) with <10% PCs and 318/352 (90.3%) with ≥10% PCs in BM aspirate; P = .003). Among cases with unsuccessful IMCS (<10% PCs; n = 21 and ≥10% PCs; n = 34), an alternate source could be used successfully in 34 (62%) patients and includes air-dried trephine biopsy imprint smears (n = 28) with aggregates or sheets of PCs, fine-needle aspiration smears/biopsy from plasmacytoma (n = 5), and ascitic fluid (n = 1). 284/395 (71.9%) patients with successful IMCS and all 34 cases with an alternate source of PCs showed at least one cytogenetic abnormality on four-probe FISH. CONCLUSION: Variations in the sample quality together with significant variation in the number of PCs between BM aspirate and the trephine biopsy imprint smears/biopsy reduce the success rate of IMCS in a real-world scenario and necessitate utilization of patient-specific alternate sources of PCs like a trephine biopsy imprint or cytology smears from extramedullary sources for successful FISH testing in PC neoplasms.

Comparison of bias and resolvability in single-cell and single-transcript methods.

Single-cell and single-transcript measurement methods have elevated our ability to understand and engineer biological systems. However, defining and comparing performance between methods remains a challenge, in part due to the confounding effects of experimental variability. Here, we propose a generalizable framework for performing multiple methods in parallel using split samples, so that experimental variability is shared between methods. We demonstrate the utility of this framework by performing 12 different methods in parallel to measure the same underlying reference system for cellular response. We compare method performance using quantitative evaluations of bias and resolvability. We attribute differences in method performance to steps along the measurement process such as sample preparation, signal detection, and choice of measurand. Finally, we demonstrate how this framework can be used to benchmark different methods for single-transcript detection. The framework we present here provides a practical way to compare performance of any methods.

Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization.

BACKGROUND: The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. METHODOLOGY: Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. RESULTS: FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. CONCLUSIONS: The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host-parasite interactions.

Pitfalls in RET Fusion Detection Using Break-Apart FISH Probes in Papillary Thyroid Carcinoma.

OBJECTIVE: A standardized procedure of fused REarranged during Transfection (RET) gene detection using fluorescence in situ hybridization (FISH) remains to be established in papillary thyroid carcinoma (PTC). Our purpose was to investigate false-negative and false-positive events and their FISH signal characteristics. METHODS: A total of 111 PTC cases were analyzed using break-apart FISH probes for RET status evaluation. All FISH results were validated using fusion-induced asymmetric transcription assay (FIATA)-based reverse transcription droplet digital PCR (RT-ddPCR). Then, suspected RET-positive cases were tested using quantitative reverse transcription-PCR (RT-qPCR), followed by next-generation sequencing (NGS) for recognizing fusion variants. RESULTS: Thirty RET+ cases were revealed, including 20 CCDC6 (exon 1)-RET (exon 12), 6 NCOA4 (exon 8)-RET (exon 12), 1 NCOA4 (exon 7)-RET (exon 12), 1 CCDC186 (exon 7)-RET (exon 12), 1 ERC1 (exon 12)-RET (exon 12) and 1 SPECC1L (exon 9)-RET (exon 12) tumors. All RET fusion cases occurred in the BRAF- population, with a prevalence of 41.7% (30/72). Four cases of 8% to 13% (cutoff was 7.6%) dominant isolated 3' green (IG) FISH signals were RET-. One FISH- case with isolated 5' red (IR) signals with 94% abnormal tumor cells was demonstrated to be positive, harboring the NCOA4 (exon 7)-RET (exon 12) variant. Compared with RET fusions characterized by dominant break-apart signals with 29% to 100% aberrant cells, RET + with dominant IG-signal patterns all showed more frequent FISH+ cells (84%-92%). RET+ PTC with a break-apart signal pattern was more frequently found in unifocal lesions than in multifocal/bilateral tumors (P = 0.049). CONCLUSIONS: A false-positive or false-negative event may exist for RET status detection in PTCs using the traditional FISH scoring method with break-apart probes.

Analysis of Cellular EMT States Using Molecular Biology and High Resolution FISH Labeling.

Metastasis results from the ability of cancer cells to grow and to spread beyond the primary tumor to distant organs. Epithelial-to-Mesenchymal Transition (EMT), a fundamental developmental process, is reactivated in cancer cells, and causes epithelial properties to evolve into mesenchymal and invasive ones. EMT changes cellular characteristics between two distinct states, yet, the process is not binary but rather reflects a broad spectrum of partial EMT states in which cells exhibit various degrees of intermediate epithelial and mesenchymal phenotypes. EMT is a complex multistep process that involves cellular reprogramming through numerous signaling pathways, alterations in gene expression, and changes in chromatin morphology. Therefore, expression of key proteins, including cadherins, occludin, or vimentin must be precisely regulated. A comprehensive understanding of how changes in nuclear organization, at the level of single genes clusters, correlates with these processes during formation of metastatic cells is still missing and yet may help personalized prognosis and treatment in the clinic. Here, we describe methods to correlate physiological and molecular states of cells undergoing an EMT process with chromatin rearrangements observed via FISH labeling of specific domains.

Fluorescence In Situ Hybridization (FISH) Detection of Chromosomal 12p Anomalies in Testicular Germ Cell Tumors.

Gains of genetic material or internal rearrangements of chromosome 12p, including 12p overrepresentation or isochromosome 12p [i(12p)], are observed in virtually all germ cell tumors (GCT), in all histologic subtypes, and from various body locations. The chromosomal region involved in these alterations contains the growth and survival promoting oncogene KRAS (12p12.1). Gains or rearrangements of 12p characterize GCT from in situ to chemoresistant stages. Fluorescence in situ hybridization (FISH) detection of chromosome 12p anomalies is a sensitive and specific test for the diagnosis of germ cell tumors. Here we provide a detailed protocol for FISH detection of isochromosome 12p and chromosome 12p overrepresentation. The method is helpful for diagnosis of germ cell origin, and for selection of patients who may benefit from cisplatin-based chemotherapy.

Causes behind error rates for predictive biomarker testing: the utility of sending post-EQA surveys.

External quality assessment (EQA) schemes assess the performance of predictive biomarker testing in lung and colorectal cancer and have previously demonstrated variable error rates. No information is currently available on the underlying causes of incorrect EQA results in the laboratories. Participants in EQA schemes by the European Society of Pathology between 2014 and 2018 for lung and colorectal cancer were contacted to complete a survey if they had at least one analysis error or test failure in the provided cases. Of the 791 surveys that were sent, 325 were completed including data from 185 unique laboratories on 514 incorrectly analyzed or failed cases. For the digital cases and immunohistochemistry, the majority of errors were interpretation-related. For fluorescence in situ hybridization, problems with the EQA materials were reported frequently. For variant analysis, the causes were mainly methodological for lung cancer but variable for colorectal cancer. Post-analytical (clerical and interpretation) errors were more likely detected after release of the EQA results compared to pre-analytical and analytical issues. Accredited laboratories encountered fewer reagent problems and more often responded to the survey. A recent change in test methodology resulted in method-related problems. Testing more samples annually introduced personnel errors and lead to a lower performance in future schemes. Participation to quality improvement projects is important to reduce deviating test results in laboratories, as the different error causes differently affect the test performance. EQA providers could benefit from requesting root cause analyses behind errors to offer even more tailored feedback, subschemes, and cases.

An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics.

Since the revolutionary discovery of RNA interference (RNAi) more than 20 years ago, synthetic small interfering RNAs (siRNAs) have held great promise as therapeutic agents for treating human diseases by the specific knockdown of disease-causing gene products. To facilitate the development of siRNA therapeutics, a robust, high-throughput in vitro assay for measuring gene silencing is imperative during the initial siRNA lead sequence identification and, later, during the lead optimization with chemically modified siRNAs. There are several potential assays for measuring gene expression. Quantitative reverse transcription PCR (qRT-PCR) has been widely used to quantitate messenger RNA (mRNA). This method has a few disadvantages, however, such as the requirement for RNA isolation, complementary DNA (cDNA) generation, and PCR reaction, which are labor-intensive, limit the assay throughput, and introduce variability. We chose a high-content imaging assay, bDNA FISH, that combines the branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by siRNAs because it is sensitive and robust with a short reagent procurement and assay development time. We also built a fully automated liquid-handling platform for executing bDNA FISH assays to increase throughput, and the system has a capacity of generating 192 concentration-response curves in a single run. We have successfully developed and executed the bDNA FISH assays for multiple targets using this automated platform to identify and optimize siRNA candidate molecules. Examples of the bDNA FISH assay for selected targets are presented.

Quantitative Impact of the 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) Practice Guideline Update on Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: A Systematic Analysis.

CONTEXT.­: The global impact of the new 2018 American Society of Clinical Oncology/College of American Pathologists human epidermal growth factor receptor 2 (HER2) practice guideline update on the overall HER2 status designation, compared with the prior 2013 iteration, is unknown. OBJECTIVES.­: To report the quantitative impact of the new guideline on HER2 status distribution. DESIGN.­: The analysis comprised a retrospective cohort of patients from the authors' institution, combined with other peer-reviewed publications that assessed the impact of the 2018 guideline in relation to the 2013 guideline. RESULTS.­: Our study revealed that the new guideline led to an average 9% reclassification rate for the overall HER2 status, with a net gain in overall HER2 negative designation. This is largely due to reclassification of the equivocal (Group 4) groups. Unexpectedly, infrequent but consistent discordance between Group 1/5 and fluorescence in situ hybridization results are observed across studies (1.8%; 73 of 3965 cases where fluorescence in situ hybridization and immunohistochemistry are both reported). CONCLUSIONS.­: Early clinical recognition of these resultant changes, including emerging issues of tumor heterogeneity, and potential discordance between immunohistochemistry to fluorescence in situ hybridization, is important for accurate clinical assessment of individual HER2 test results.

HER2 Testing for Breast Cancer in the Genomics Laboratory: A Sea Change for Fluorescence In Situ Hybridization.

CONTEXT.­: Guidelines for HER2 testing in breast cancer have changed over time, from the US Food and Drug Administration guideline to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines published in 2007, 2013, and 2018. OBJECTIVE.­: To investigate the change in assignment of HER2 status in breast cancers with equivocal (2+) immunohistochemistry (IHC) results by fluorescence in situ hybridization (FISH) following implementation of the ASCO/CAP 2018 guideline. DESIGN.­: The study included 3556 invasive breast cancers that were HER2 equivocal (2+) by IHC and were submitted to our FISH laboratory after July 2018. Reflex testing (with repeat IHC staining) was performed on certain categories of FISH results known as groups 2, 3, and 4. Concomitant review of IHC and FISH was performed on these reflex cases per 2018 guideline recommendations. The FISH data were analyzed to compare US Food and Drug Administration and ASCO/CAP 2007, 2013, and 2018 interpretations. RESULTS.­: Of 3548 invasive breast cancers with complete data available, the percentage agreement for FISH according to different guidelines was highest for ASCO/CAP 2018 versus US Food and Drug Administration (96.5%), followed by ASCO/CAP 2018 versus 2007 (93.8%), and lowest with ASCO/CAP 2018 versus 2013 (83.7%). Per the 2018 guideline, reflex IHC testing was performed on 633 breast cancers (17.8%); the majority of reflex testing results were negative (541 of 633; 85.5%). The overall distribution of HER2 FISH results (per the 2018 guideline) was 88.5% negative and 11.5% positive. CONCLUSIONS.­: By eliminating the equivocal FISH category, the 2018 ASCO/CAP guideline significantly reduced the HER2 FISH-positive rate in tumors with equivocal (2+) IHC results.

[Expert consensus on prenatal fluorescence in situ hybridization].

Although non-invasive prenatal testing has been widely used, it has certain limitations. As the gold standard of prenatal diagnosis, G-banding karyotype analysis is time-consuming and laborious. Fluorescence in situ hybridization (FISH), as a method for detecting samples with non-radioactive signals, does not require cell culture and has a short turnover time, and can diagnose aneuploidies of chromosomes 13, 18, 21, X, Y with efficiency, which can solve the problems such as insufficient testing ability and long diagnosis period for karyotype analysis. To standardize the procedures of prenatal FISH assay and enhance laboratory quality management, the Expert Committee of the Prenatal Screening and Diagnosis Laboratory of the Clinical Test Center of the National Health Commission and the Inter-laboratory Quality Assessment Committee of the Neonatal Genetic and Metabolic Disease Screening Laboratory have formulated this consensus.

Prenatal diagnosis of BACs-on-Beads assay in 1520 cases from Fujian Province, China.

BACKGROUND: The aim of this study was to evaluate the application of BACs-on-Beads (BoBs™) assay for rapid detection of chromosomal abnormalities for prenatal diagnosis (PND). METHODS: A total of 1520 samples, including seven chorionic villi biopsy samples, 1328 amniotic fluid samples, and 185 umbilical cord samples from pregnant women were collected to detect the chromosomal abnormalities using BoBs™ assay and karyotyping. Furthermore, abnormal specimens were verified by chromosome microarray analysis (CMA) and fluorescence in situ hybridization (FISH). RESULTS: The results demonstrated that the success rate of karyotyping and BoBs™ assay in PND was 98.09% and 100%, respectively. BoBs™ assay was concordant with karyotyping for Trisomy 21, Trisomy 18, and Trisomy 13, sex chromosomal aneuploidy, Wolf-Hirschhorn syndrome, and mosaicism. BoBs™ assay also detected Smith-Magenis syndrome, Williams-Beuren syndrome, DiGeorge syndrome, Miller-Dieker syndrome, Prader-Willi syndrome, Xp22.31 microdeletions, 22q11.2, and 17p11.2 microduplications. However, karyotyping failed to show these chromosomal abnormalities. A case of 8q21.2q23.3 duplication which was found by karyotyping was not detected by BoBs™ assay. Furthermore, all these chromosomal abnormalities were consistent with CMA and FISH verifications. According to the reports, we estimated that the detection rates of karyotyping, BoBs™, and CMA in the present study were 4.28%, 4.93%, and 5%, respectively, which is consistent with the results of a previous study. The respective costs for the three methods were about $135-145, $270-290, and $540-580. CONCLUSION: BoBs™ assay is considered a reliable, rapid test for use in PND. A variety of comprehensive technological applications can complement each other in PND, in order to maximize the diagnosis rate and reduce the occurrence of birth defects.

Simultaneous Detection of RNAs and Proteins with Subcellular Resolution.

We describe the detailed methods of "immunoFISH" to analyze the expression level and the spatial localization of RNA transcripts and proteins on cultured cells and formal-fixed, paraffin-embedded (FFPE) tissue sections. On cultured cells, we detect specific transcripts using the Stellaris fluorescence in situ hybridization (FISH) probes labeled with fluorophores that target multiple regions along the desired transcripts and proteins combining the immunofluorescent staining. On FFPE tissue sections, we use the RNAscope FISH probes, modified branched DNA (bDNA) probes to amplify the RNA signals, followed by immunofluorescent staining for protein detection. The abundance, composition, and spatial distribution are determined by signals from fluorescently labeled proteins and individual transcripts of images acquired using high-resolution fluorescence microscopy.

High-risk cytogenetics in multiple myeloma: Further scrutiny of deletions within the IGH gene region enhances risk stratification.

Multiple myeloma is a clonal malignancy of plasma cells in the bone marrow. Risk stratification is partly based on cytogenetic findings that include abnormalities of the IGH locus as determined by fluorescence in situ hybridization (FISH), such as rearrangements that result in either standard-risk or high-risk gene fusions. IGH deletions have been evaluated as a group in multiple myeloma patients with respect to cumulative outcomes but have provided limited guidance. Whether these deletions have the potential to result in gene fusions and thus further stratify patients is unknown. We identified 229 IGH deletions in patients referred for plasma cell dyscrasia genetic testing over 5.5 years. Follow-up was conducted on 208 of the deletions with dual fusion FISH probes for standard-risk (IGH-CCND1) and high-risk IGH gene fusions (IGH-FGFR3, IGH-MAF, IGH-MAFB). Of all deletions identified with follow-up, 44 (21%) resulted in a gene fusion as detected by FISH, 15 (7%) of which were fusion partners associated with high-risk multiple myeloma. All fusion-positive 3'-IGH deletions (6 fusions) resulted in high-risk IGH-FGFR3 fusions. Of the 15 high-risk fusion-positive cases, eight were without other high-risk cytogenetic findings. This study is the first to evaluate the presence of IGH gene fusions upon identification of IGH deletions and to characterize the deletion locus. Importantly, these findings indicate that follow-up FISH studies with dual fusion probes should be standard of care when IGH deletions are identified in multiple myeloma.

A rare case of NIPT discrepancy caused by the placental mosaicism of three different karyotypes, 47,XXX, 47,XX,+21, and 48,XXX,+21.

BACKGROUND: Placental mosaicism is one of the major reasons for noninvasive prenatal testing (NIPT) discrepancy. Herein, we discovered a rare case of placenta with complex karyotypes that caused false-positive and false-negative results in noninvasive prenatal testing. METHODS: Next-generation sequencing (NGS) and Quantitative fluorescent polymerase chain reaction (QF-PCR) were performed on the cord blood sample, fetal tissues, and eight placental biopsies. Fluorescent In Situ Hybridization (FISH) and karyotyping were also carried to confirm the fetal genome status. RESULTS: The results suggested that the fetal chromosome was 47,XXX and the placenta had three karyotypes of 48,XXX,+21, 47,XX,+21, and 47,XXX. QF-PCR indicated that the extra chromosome 21 and chromosome X were all from the father. It is speculated that the zygote may have 48,XXX,+21 karyotype and trisomy rescue could be the main mechanism for the development of the homogeneous fetus and complex mosaic placenta. CONCLUSION: Overall, the complicated nature of our case underlines the importance of discussing with parents the possibility of both atypical and discordant results during preconfirmatory amniocentesis counseling and consent.

Design of a Comprehensive Fluorescence in Situ Hybridization Assay for Genetic Classification of T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) results from deregulation of a number of genes via multiple genomic mechanisms. We designed a comprehensive fluorescence in situ hybridization (CI-FISH) assay that consists of genomic probes to simultaneously investigate oncogenes and oncosuppressors recurrently involved in chromosome rearrangements in T-ALL, which was applied to 338 T-ALL cases. CI-FISH provided genetic classification into one of the well-defined genetic subgroups (ie, TAL/LMO, HOXA, TLX3, TLX1, NKX2-1/2-2, or MEF2C) in 80% of cases. Two patients with translocations of the LMO3 transcription factor were identified, suggesting that LMO3 activation may serve as an alternative to LMO1/LMO2 activation in the pathogenesis of this disease. Moreover, intrachromosomal rearrangements that involved the 10q24 locus were found as a new mechanism of TLX1 activation. An unequal distribution of cooperating genetic defects was found among the six genetic subgroups. Interestingly, deletions that targeted TCF7 or TP53 were exclusively found in HOXA T-ALL, LEF1 defects were prevalent in NKX2-1 rearranged patients, CASP8AP2 and PTEN alterations were significantly enriched in TAL/LMO leukemias, and PTPN2 and NUP214-ABL1 abnormalities occurred in TLX1/TLX3. This work convincingly shows that CI-FISH is a powerful tool to define genetic heterogeneity of T-ALL, which may be applied as a rapid and accurate diagnostic test.