Fluorescence In Situ Hybridization Technique Research Articles

Comparison between Immunocytochemistry, FISH and NGS for ALK and ROS1 Rearrangement Detection in Cytological Samples.

The detection of ROS1 and ALK rearrangements is performed for advanced-stage non-small cell lung cancer. Several techniques can be used on cytological samples, such as immunocytochemistry (ICC), fluorescence in situ hybridization (FISH) and, more recently, next-generation sequencing (NGS), which is gradually becoming the gold standard. We performed a retrospective study to compare ALK and ROS1 rearrangement results from immunocytochemistry, FISH and NGS methods from 131 cytological samples. Compared to NGS, the sensitivity and specificity of ICC were 0.79 and 0.91, respectively, for ALK, and 1 and 0.87 for ROS1. Regarding FISH, the sensitivity and specificity were both at 1 for ALK and ROS1 probes. False-positive cases obtained by ICC were systematically corrected by FISH. When using ICC and FISH techniques, results are very close to NGS. The false-positive cases obtained by ICC are corrected by FISH, and the true-positive cases are confirmed. NGS has the potential to improve the detection of ALK and ROS1 rearrangements in cytological samples; however, the cost of this technique is still much higher than the sequential use of ICC and FISH.

Molecular classification of soft tissue sarcomas for adequate diagnosis: A study on the northeast population of Morocco

BackgroundSoft tissue sarcomas (STS) are a heterogeneous group of tumors. For adequate therapeutic management, an accurate diagnosis is necessary. In Morocco, the diagnosis is essentially based on the morphological and immunohistochemical study. Compared to other techniques, fluorescence in situ hybridization (FISH) is easier to develop and less expensive. This study aims to assess the feasibility and utility of implementing FISH technique to improve diagnostic accuracy and establish a good classification. Material and methodsThis is a retrospective cohort study. 211 cases of mesenchymal tumors were included. Hematoxylin Eosin Safran (HES) staining was performed in all cases followed by immunohistochemistry (IHC). FISH was performed in all cases with suspected STS. The probes used were EWSR1, MDM2 and SS18. The performance of FISH and histopathological test were evaluated by the ROC curve method (receiver operating characteristic). We evaluated the concordance between FISH and real time PCR by Cohen test. ResultsThe real-time PCR technique showed good agreement with the FISH test by a Kappa coefficient of 60% (p = 0.035). FISH was able to confirm that it is more accurate (Youden’s Index = 91%) than histological/immunohistochemical analysis (Youden’s Index = 51%), as well as the positive predictive value was higher (100%) with an ROC curve finding a larger area under the curve of 0.953 (95% CI: 0.918–0.988), p = 0.000 which supports that FISH shows high performance to present an accurate final diagnosis. ConclusionThis is the first and the largest Moroccan series for the molecular diagnosis of STS by FISH. Our study shows that paraffin FISH is a sensitive and specific ancillary tool in the diagnosis of STS when used in the appropriate clinicopathological context.

Ré-utilisation de lames d’immunohistochimie pour la réalisation de FISH : une solution pertinente d’épargne tissulaire

Diagnostic updates, an increased precision of tumor sub-type classification and the development of new diagnostic biomarkers (immunohistochemistry (IHC), Fluorescence in situ hybridization (FISH) and other molecular pathology techniques), have a significant impact on pathologists' management of tissue samples. The objective of this work was to test and validate the FISH technique on detached IHC slides. An IHC technique was first performed on 30 tissue samples. After detachment of the lamella, a FISH technique was then performed according to the usual protocol with a centromeric probe. A validation cohort (n=10) with duplicate testing using a traditional FISH technique and an IHC slide with a detached lamella was then carried out. Finally, a cohort of 20 "old" cases (IHC carried out over 2years ago) was also tested. Different types of probes (specific locus, break apart) have been used. All the slides were interpreted by a technician and a pathologist. Evaluation criteria were: the general interpretability of the slide; the percentage of labeled nuclei; intensity of the signal and the presence or absence of autofluorescence. FISH was interpretable in 100% of recently treated cases and 90% of "old" cases with a satisfactory intensity and a high percentage of labeled nuclei, without autofluorescence. The results of our study show that the reuse of IHC slides for performing FISH is a powerful means of economizing tissue samples, especially for small samples and in the absence of archived representative material.

Telocytes and Their Structural Relationships With the Sperm Storage Tube and Surrounding Cell Types in the Utero-Vaginal Junction of the Chicken.

Telocytes (TCs) are a new type of mesenchymal cells that have been discovered recently in many organs and tissues. However, studies of TCs in the avian reproductive system are still at the beginning. Chickens are one of the world's most popular domesticated animals, providing inexpensive but valuable proteins and nutrients from chickens and eggs to nourish the human bodies. Chickens have important scientific value; thus, understanding the reproductive system regulations seems to be important. The utero-vaginal junction is involved in the regulation of sperm storage. The sperm storage tube (SST) in the utero-vaginal junction stores sperm. The purpose of this study was to investigate the existence of TCs in the utero-vaginal junction of the chicken, and their structural relationships with the sperm storage tube and surrounding cell types. We studied the morphology, ultrastructure, and immune characterization of TCs.MethodsThe utero-vaginal junction of 4-month-old healthy adult chickens (n = 10) were used for Masson's staining, fluorescent in situ hybridization technique (FISH), and transmission electron microscopy (TEM) analysis. The results showed that TCs were present in the utero-vaginal junction. TCs appear as CD34 immunopositive and C-kit immunopositive. They were identified especially via small-body and long-protrusion telopodes (Tps) containing Podomers (Pm) and Podoms (Pd). The Tps were bent, folded, and intertwined with each other, sometimes in the shape of a labyrinth. The Tps were embedded between collagen fiber bundles, smooth muscle bundles, and around blood vessels and releasing vesicles. TCs surround these glands, forming heteromorphic cell connections with surrounding lymphocytes and plasma cells, smooth muscle cells, blood vessels, collagen fibers, and fibroblast-formed homotypic or allotypic connections in a complex three-dimensional network structure. This study provides a morphological basis for the possible role of TCs in regulating the utero-vaginal junction physiological role and in intercellular communication.

Discerning the Putative U and V Chromosomes of Saccharina japonica (Phaeophyta) by Cytogenetic Mapping of Sex-Linked Molecular Markers

Saccharina japonica, an importantly industrial species in brown seaweeds, has a typical alternate life history of heteromorphic generations and UV sex determination system. But up to now, the sex chromosomes, U and V, in this kelp remain to be evidenced cytologically. In the present study, the female-linked (SJ-f_000170 and MSj68-58-2) and male-linked (SJ-13_001840 and MSj68-16-2) molecular markers developed on the basis of Ectocarpus sex-determining regions (SDRs) were selected after PCR amplification and Southern blotting analysis. Mono-color fluorescence in situ hybridization (FISH) to the kelp metaphase chromosomes with each of these confirmed sex-linked markers as a probe indicated that the hybridization signals were confined to either female or male gametophytes exclusively. The shape and size of hybridized chromosomes and the relative location of these sex-linked markers on them suggested that the marked chromosomes in a sex were the same, which was further confirmed by dual-color FISH observations. SJ-f_000170 and SJ-13_001840, corresponding to each sex, were therefore used to map the sporophyte metaphase chromosomes by dual-color FISH technique, and they were hybridized on different chromosomes as expected. The sporophyte metaphase chromosomes as hybridized by SJ-f_000170 and SJ-13_001840 resembled those in either female or male gametophytes, suggesting that they were the putative U and V, respectively, chromosomes in S. japonica. To further demonstrate the reliability of this inference, genomic information of the screened bacterial artificial chromosome (BAC) clones carrying two female-linked markers, for example, from the constructed BAC libraries of the kelp female gametophytes was provided. The insert sequencing of two selected BAC clones revealed that repetitive elements were rich whereas gene density was poor, which was characterized as non-recombining SDR sequences in brown seaweed Ectocarpus sp. and liverwort Marchantia polymorpha. The present study laid a solid foundation for unveiling the mystery of S. japonica sex chromosomes.

Concordance of HER2 status tested by IHC and FISH in biopsy and surgical resection specimens and comparison with clinicopathological features in gastric carcinoma.

HER2-targeted therapy has been shown to benefit HER2-positive gastric cancer. It is very important to determine the HER2 expression level correctly to select the appropriate test and sampling method. In this study, we investigated the frequency of overexpression of HER2 and intratumoral heterogeneity of HER2-positive cases, comparison of HER2 used immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) performance in biopsy and resection specimens, the correlation of HER2 status between biopsy and resection specimens, and its relationship with clinicopathological findings. Formalin-fixed, paraffin-embedded specimens of a total of 40 surgically resected and biopsy specimens of gastric cancer were analyzed. HER2 status was examined using both IHC and FISH techniques, and the findings and their association with different clinicopathological parameters were evaluated. The concordance rate between the results of IHC and FISH in biopsy and resection specimens was 96.6% and 86.6%, respectively. In paired 20 cases, the overall concordance rate of HER2-IHC and HER2-FISH status between biopsy and resection specimens was 90% and 100%, respectively. HER2-IHC analysis revealed that 5/40 cases were IHC 2+ and only 1 of 5 IHC 2+ cases demonstrated HER2-FISH amplification. Our results showed that HER2-IHC was well concordant with FISH in cases with a score of 0/1+ or 3+ and demonstrates strong concordance between biopsy and resection specimens. FISH should be performed when the IHC result is equivocal. In our study, no statistically significant correlation was observed between HER2 positivity and clinicopathological parameters. Overall, both biopsy and resection specimens are appropriate for HER2 testing.

Genetic diversity of ribosomal loci (5S and 45S rDNA) and pSc119.2 repetitive DNA sequence among four species of Aegilops (Poaceae) from Algeria

In continuation of our previous research we carried out the karyological investigation of 53 populations of four Aegilops species (A. geniculata, A. triuncialis, A. ventricosa, and A. neglecta) sampled in different eco-geographical habitats in Algeria. The genetic variability of the chromosomal DNA loci of the same collection of Aegilops is highlighted by the Fluorescence In Situ Hybridization technique (FISH) using three probes: 5S rDNA, 45S rDNA, and repetitive DNA (pSc119.2). We found that the two rDNA loci (5S and 45S) hybridized with some chromosomes and showed a large genetic polymorphism within and between the four Aegilops species, while the repetitive DNA sequences (pSc119.2) hybridized with all chromosomes and differentiated the populations of the mountains with a humid bioclimate from the populations of the steppe regions with an arid bioclimate. However, the transposition of the physical maps of the studied loci (5S rDNA, 45S rDNA, and pSc119.2) with those of other collections revealed the existence of new loci in Aegilops from Algeria.

Amplified Fluorescence in Situ Hybridization by Small and Bright Dye-Loaded Polymeric Nanoparticles.

Detection and imaging of RNA at the single-cell level is of utmost importance for fundamental research and clinical diagnostics. Current techniques of RNA analysis, including fluorescence in situ hybridization (FISH), are long, complex, and expensive. Here, we report a methodology of amplified FISH (AmpliFISH) that enables simpler and faster RNA imaging using small and ultrabright dye-loaded polymeric nanoparticles (NPs) functionalized with DNA. We found that the small size of NPs (below 20 nm) was essential for their access to the intracellular mRNA targets in fixed permeabilized cells. Moreover, proper selection of the polymer matrix of DNA-NPs minimized nonspecific intracellular interactions. Optimized DNA-NPs enabled sequence-specific imaging of different mRNA targets (survivin, actin, and polyA tails), using a simple 1 h staining protocol. Encapsulation of cyanine and rhodamine dyes with bulky counterions yielded green-, red-, and far-red-emitting NPs that were 2-100-fold brighter than corresponding quantum dots. These NPs enabled multiplexed detection of three mRNA targets simultaneously, showing distinctive mRNA expression profiles in three cancer cell lines. Image analysis confirmed the single-particle nature of the intracellular signal, suggesting single-molecule sensitivity of the method. AmpliFISH was found to be semiquantitative, correlating with RT-qPCR. In comparison with the commercial locked nucleic acid (LNA)-based FISH technique, AmpliFISH provides 8-200-fold stronger signal (dependent on the NP color) and requires only three steps vs ∼20 steps together with a much shorter time. Thus, combination of bright fluorescent polymeric NPs with FISH yields a fast and sensitive single-cell transcriptomic analysis method for RNA research and clinical diagnostics.

Localization of ITS and 5S rDNA on the chromosomes of Ulva prolifera using fluorescence in situ hybridization

ABSTRACT Ulva prolifera has been identified as the dominant species of the world’s largest green tides occurring in the Yellow Sea, China, using the molecular markers of internal transcribed spacer (ITS) and 5S ribosomal DNA (5S rDNA). In this study, these two markers were successfully located on the chromosomes of U. prolifera using the fluorescence in situ hybridization (FISH) technique. The ITS rDNA probe was localized on chromosome number 1 of gametophytes and the 5S rDNA probe appeared on chromosome number 6. Dual-colour FISH was also performed, and the locations of the two markers were consistent with single-colour localization, indicating a precise gene location. The localization of these rDNAs using the FISH technique facilitated the identification of individual chromosomes and a karyotype analysis of Ulva, which is beneficial for understanding the blooming mechanism of green tides in the Yellow Sea.

BCR/ABL Fusion Gene by FISH Technique: What is the Appropriate Cut Off for Diagnosis and Monitoring Response to Treatment?

Objective: Fluorescence in situ Hybridisation (FISH) is a widely used and useful cytogenetic technique for diagnosis and monitoring treatment responses in chronic myeloid leukaemia (CML). The positive or negative FISH result is interpreted based on individual standard normal cut off percentage, but universal cut off is unaddressed. We aimed to determine the performance of the FISH technique based on our routine normal cut off. Materials and Methods: A retrospective descriptive and analytical study was conducted on CML patients followed-up at Srinagarind Hospital. Data has been collected from laboratory records over the past ten years. We excluded patients who were not completely tested FISH and chromosome or RQ-PCR. Results: 675 FISH tests from 255 CML patients were analyzed. Specimens were mainly from bone marrow (98.2%) Chromosome analysis (G-banding) showed no metaphase in 31.9%. FISH test was positive in 99 samples with normal cut off at 9.7%. The falsenegative rate of FISH was 2.6% (0.65% by using the standard cut off at 1%) and the false positive rate was 3%. Conclusion: The FISH results should be interpreted carefully. Undue high level of normal cut off causes a high false-negative rate of FISH. The standard normal cut off for double signal FISH (D-FISH) was 1%. However, the cutoff FISH interpretation should be individually set and regularly validated. The FISH result should also be construed with G-banding, and RQ-PCR, which will improve the accuracy and will prevent misdiagnosed of CML. Keywords: Fluorescence in situ Hybridisation (FISH); Chronic Myeloid Leukemia (CML); Cut off; False positive; False negative

Frequency of ALK gene rearrangement and its association with survival rate among Upper Egypt Non-Small cell lung cancer patients

Background: Anaplastic Lymphoma Kinase(ALK)gene rearrangement shows ethnic variations worldwide. There isn’t a population-based study measuring its prevalence among the Egyptian population till now. Aim: To measure ALK gene rearrangement prevalence correlated with survival rates among a sample of Upper Egypt Non-Small Cell Lung Cancer (NSCLC) patients. Patients and Methods: The study included twenty-six NSCLC patients. 21 of them were males and the rest were females, the enrolled patients had a median age of 60 years. 8 presented with pleural effusion, while 4 showed brain metastases, and 10 had liver metastases. Most of the cases were given a diagnosis with adenocarcinoma, squamous cell carcinoma (14, 6 cases, respectively), and the rest were given a diagnosis with undifferentiated carcinoma. Fluorescence Insitu Hybridization (FISH) technique was used for detection of rearranged ALK gene from FFEP Tissue Sections. Vysis ALK Break Apart FISH Probe Kit was used, it’s composed of two probes green-labeled one to bind to the 5′ end of the ALK gene, and another orange labeled one to bind to the 3′ end. Tumor cells with 15 % or more separate green 5′ and red 3′ signals or isolated red 3′ signals were considered positive for the rearranged ALK gene, while cells with yellowish signals (representing overlapped signals between green and red probes) were considered negative for ALK gene rearrangement. A fluorescence microscope supplied with a Leica CCD camera was used to detect clinicopathological characteristics of rearranged ALK gene, image processing was done using an imaging system and software. Results: Patients were followed up for 63 months, 15 of them were positive for the rearranged ALK gene. All patients were treated with chemotherapy and they had different objective response rates (ORR), ALK+ve cohort 73.3% (PR:3, SD:8), ALK–ve cohort 100% (PR:6, SD:5). The overall survival rate (OSR) was 51,6%, and two- year progression-free survival rate (PFSR) was 68,8%. Univariate analysis was done between ALK+ve / ALK–ve for different risk groups (age, gender, histological subtype, the site of metastasis, and grade). Patients with adenocarcinoma showed better OSR than those with squamous cell carcinoma, PConclusions: Patients with ALK gene rearrangement are resistant to chemotherapy, so they should be directed for individualized treatment, the prevalence of ALK+ve patients could be more than expected among the Egyptian population. further confirmation is required through multiple center population-based studies. Policymakers are invited for validating central laboratory, they are also recommended to enhance accessibility for diagnostic facilities and services, and adopting inhibitors of tyrosine kinase (TKI) as a new effective treatment strategy for NSCLC cases.

Effect of Vitamin A-Fortified Rice on the Gut Microbiota of Thai Lactating Women and Their Exclusively Breastfed Infants

Abstract The optimal vitamin A (VA) status of lactating women is important for mothers and their breastfed infants, especially in protecting against infectious diseases. Vitamin A fortified rice is one of the food-base intervention strategy which has the potential to improve VA status. Vitamin A and gut microbiota are interrelated in their effect on human health and immunity however no specific relationship has been proved in these groups of population. This study aimed to determine the effect of VA fortified rice on the gut microbiota changes of lactating woman-exclusively breastfed infant pairs. A double-blind, randomized controlled trial (RCT) of VA fortified rice was conducted in 70 lactating women-infants pairs for 14 weeks. Gut microbiota was measured using the fluorescent in situ hybridization (FISH) and next generation sequencing (NGS) technique. Based on the FISH technique, the numbers of Clostridium spp. /Enterobacter spp. were significantly lower (P < 0.05) in mothers fed VA-fortified rice at the end of the study. In contrast, the abundance of Bifidobacterium spp. and Lactobacillus spp. of infants whose mothers fed with VA-fortified rice was significantly higher (P < 0.05) than the control group. The NGS technique confirmed that results with the increasing of Lactobacillus, B. longum and B. Choerinum in the infant of intervention group. In conclusion, VA-fortified rice was efficacious in decreasing Clostridium spp. /Enterobacter spp. in lactating women and raising the number of Bifidobacterium spp. and Lactobacillus spp. in their breastfed infants. Keywords: Breastfeeding, Gut microbiota, Lactating woman-infant pairs, Randomized controlled trial, Vitamin A

A Universal Labeling Strategy for Nucleic Acids in Expansion Microscopy.

Expansion microscopy (ExM) enables the nanoscale imaging of ribonucleic acids (RNAs) on a conventional fluorescence microscope, providing information on the intricate patterns of gene expression at (sub)cellular resolution and within spatial context. To extend the use of such strategies, we examined a series of multivalent reagents that allow the labeling and grafting of deoxyribonucleic acid (DNA) oligonucleotide probes in a unified approach. We show that the reagents are directly compatible with third-generation in situ hybridization chain reaction RNA FISH (fluorescence in situ hybridization) techniques while displaying complete retention of the targeted transcripts. Furthermore, we validate and demonstrate that our labeling method is compatible with multicolor staining. Through oligonucleotide-conjugated antibodies, we demonstrate excellent performance in ×4 ExM and ×10 ExM, achieving a resolution of ∼50 nm in ×10 ExM for both pre- and postexpansion labeling strategies. Our results indicate that our multivalent molecules enable the rapid functionalization of DNA oligonucleotides for ExM.

Friends with Benefits: An Inside Look of Periodontal Microbes’ Interactions Using Fluorescence In Situ Hybridization—Scoping Review

Fluorescence in situ hybridization (FISH) has proven to be particularly useful to describe the microbial composition and spatial organization of mixed microbial infections, as it happens in periodontitis. This scoping review aims to identify and map all the documented interactions between microbes in periodontal pockets by the FISH technique. Three electronic sources of evidence were consulted in search of suitable articles up to 7 November 2020: MEDLINE (via PubMed), Scopus (Elsevier: Amsterdam, The Netherlands), and Web of Science (Clarivate Analytics: Philadelphia, PA, USA) online databases. Studies that showed ex vivo and in situ interactions between, at least, two microorganisms were found eligible. Ten papers were included. Layered or radially ordered multiple-taxon structures are the most common form of consortium. Strict or facultative anaerobic microorganisms are mostly found in the interior and the deepest portions of the structures, while aerobic microorganisms are mostly found on the periphery. We present a model of the microbial spatial organization in sub- and supragingival biofilms, as well as how the documented interactions can shape the biofilm formation. Despite the already acquired knowledge, available evidence regarding the structural composition and interactions of microorganisms within dental biofilms is incomplete and large-scale studies are needed.

Identification of Brachyspira spp. in the cecum of broiler chickens using histology and in situ diagnostic assays

The genus Brachyspira corresponds to the group of bacteria formerly classified into the genus Serpulina and includes several commensal and pathogenic intestinal spirochetes that affect pigs, poultry, and other animal species, including humans. In birds, some pathogenic species of this genus causes a condition known as avian intestinal spirochetosis, which remains underdiagnosed, thereby causing serious economic losses. Brachyspira is a fastidious organism that necessitates the employment of fast and efficient identification techniques. The aim of this study was to identify Brachyspira spp. using histology, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in formalin-fixed paraffin embedded (FFPE) tissue samples from the cecum of commercial poultry. Samples were collected from 129 birds aged between 35 and 45 days from commercial broiler farms. For evaluation, routine histology processing (H&E) and the histochemical technique, periodic acid–Schiff (PAS) were done. Additionally, FFPE tissue samples were evaluated for FISH and IHC. The histological lesions were analyzed and graded after H&E staining, and the goblet cells were counted and compared using PAS staining with the positive and negative samples obtained through FISH and IHC. For FISH, probes labeled with Brachyspira spp., B. pilosicoli, B. hyodysenteriae, and B. intermedia were used, whereas rabbit polyclonal antibody specific for Brachyspira spp. was used for IHC. Of 129 samples, 82 were positive with IHC and 86 were positive with FISH. The samples positive for the genus Brachyspira in the FISH technique were tested for B. pilosicoli, B. hyodysenteriae, and B. intermedia in which 56 were positive for B. pilosicoli, 75 for B. hyodysenteriae and 80 for B. intermedia. There was an increase in goblet cells in the samples positive for FISH and IHC. The techniques used were effective and gave corresponding results, thus serving as a fast and efficient tool for diagnosis.

Abstract LB190: DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations

Abstract Genomic DNA anomalies such as copy number variations (gene duplication, amplification, deletion) and gene rearrangements are important biomarkers and drug targets in many cancer types. DNA in-situ hybridization (ISH) is the gold standard method to directly visualize these molecular alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissues at single-cell resolution within a histological section. However, currently available fluorescent ISH (FISH) assays provide limited morphological detail due to the use of fluorescent nuclear staining compared to chromogenic staining. Furthermore, FISH techniques rely on expensive fluorescence microscopes, risk loss of fluorescent signal over time and involve tedious imaging at high magnifications (100X). There is thus an unmet need for a sensitive and robust chromogenic DNA-ISH assay that can enable high-resolution detection of genomic DNA targets with the ease of bright-field microscopy. We present here DNAscope - a novel chromogenic DNA-ISH assay - for detecting and visualizing genomic DNA targets under a standard light microscope. DNAscope is based on the widely used RNAscope® double-Z probe design and signal amplification technology and provides unparalleled sensitivity and specificity with large signal dots readily visualized at 40X magnification and with full morphological context. Furthermore, DNAscope ensures specific DNA detection without interference from RNA due to the use of a novel RNA removal method. Using a duplex chromogenic detection assay in red and blue, we demonstrate highly specific and efficient detection of gene rearrangements (ALK, ROS1, RET and NTRK1), gene amplification (ERBB2, EGFR, MET) and deletion (TP53 and CDKN2A). The DNAscope assay has been carefully optimized for probe signal size and color contrast to enable easy interpretation of signal patterns under conventional light microscopy or digital pathology. Compared to conventional FISH assays, DNAscope probes are standard oligos that are designed in silico to be free of any repetitive sequences and can be rapidly synthesized for any DNA target. In conclusion, the DNAscope assay provides a powerful and convenient alternative to commonly used FISH assays in many cancer research applications. Citation Format: Li-Chong Wang, Farzaneh Tondnevis, Courtney Todorov, Jayson Gaspar, Aparna Sahajan, Vasudha Murlidhar, Bingqing Zhang, Xiao-Jun Ma. DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB190.

Evaluation of MLPA as a comprehensive molecular cytogenetic tool to detect cytogenetic markers of chronic lymphocytic leukemia in Egyptian patients

BackgroundChronic lymphocytic leukemia (CLL) is the most common form of adult leukemia. This disease is genetically heterogeneous, and approximately 85% of patients with CLL harbor chromosomal aberrations that are considered effective prognostic biomarkers. The most frequent aberrations include deletions in 13q14, followed by trisomy 12, and deletions in 11q22.3 and 17p13 (TP53). Currently, fluorescence in situ hybridization (FISH) is the most widely used molecular cytogenetic technique to detect these aberrations. However, FISH is laborious, time-consuming, expensive, and has a low throughput. In contrast, multiplex ligation-dependent probe amplification (MLPA) is a reliable, cost-effective, and relatively rapid technique that can be used as a first-line screening tool and complement with FISH analysis. This study aimed to evaluate the contributions of MLPA as a routine standalone screening platform for recurrent chromosomal aberrations in CLL in comparison to other procedures. Thirty patients with CLL were screened for the most common genomic aberrations using MLPA with SALSA MLPA probemix P038-B1 CLL and FISH. ResultsIn 24 of the 30 cases (80%), the MLPA and FISH results were concordant. Discordant results were attributed to a low percentage of mosaicism. Moreover, the MLPA probemix contains probes that target other genomic areas known to be linked to CLL in addition to those targeting common recurrent CLL aberrations. ConclusionsThe usage of MLPA as the first screening platform followed by FISH technique for only the negative cases is the most appropriate approach for CLL diagnosis and prognosis.

Molecular Cytogenetic Study in Patients with Acute Lymphoblastic Leukemia (ALL) in Erbil Province

Background: Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and is also important in older adults. Chromosome number or structure abnormalities are seen in approximately 90% of children and 70% of adult patients with ALL. The aim of this study was to determine the prevalence of these chromosomal abnormalities in ALL patients using Fluorescence in situ Hybridization (FISH) technique and to define the frequency of chromosomal abnormalities of ALL patients in adults and children in Erbil Province. Methods: In this cross-sectional study, we evaluated karyotype results in blood samples, that collected from 55 patients with ALL (in both sexes) in Nanakaly Hospital in Erbil Province. Thirty healthy individuals were selected as the control group. Patients ages ranged between 1 to 63 years old. The samples were centrifuged to extract nucleated cells. The cells were then subjected to hypotonic shock, fixed with methanol and acetic acid. A cell suspension was then prepared for FISH technique. After examining the samples with fluorescent microscope, the obtained data along with demographic and baseline characteristics of patients were entered in SPSS software, then statistically analyzed. Results: The prevalence of chromosomal abnormalities among ALL group was 52.7% (n = 29). Of these, 31¬% (9 people) had abnormalities in chromosome number and 69¬% (20 people) had abnormalities in chromosome structure. The most common chromosomal abnormality was translocation t (9; 22), which accounted for 31¬% of all abnormalities and its prevalence among ALL patients was 16.4¬%. Clonal trisomy and t (12; 21) also accounted for 13.8% and 10.3% of abnormalities, respectively. Clonal trisomy was the most common abnormality in chromosome number, accounting for 44.4% (n = 4) of abnormalities. Only one patient with single chromosome X (X0) pattern was observed in patients. There was no significant (P> 0.05) relationship between the incidence of chromosomal abnormalities with gender, family history, history of surgery and bacterial infection, occupation, place of residence, smoking and blood type as stated from the questionnaire form. Conclusion In the current study, concluded that at least one chromosomal abnormality was found in more than half of all patients with ALL. Structural abnormalities were more common than chromosome number abnormalities. Normal karyotype was more frequent in this current study. The frequencies of some cytogenetic abnormalities such as hyperdiploidy and t (9;22) in the current study findings were comparable to those reported in the literature. The results of this study in Erbil Province can be used as baseline information for treatment decision and research purposes in ALL patients. We recommend the use of advanced molecular techniques in the future to better elucidate cryptic cytogenetic abnormalities.

Chorus2: design of genome-scale oligonucleotide-based probes for fluorescence insitu hybridization.

SummaryOligonucleotide (oligo)‐fluorescence in situ hybridization (FISH) has rapidly becoming the new generation of FISH technique in plant molecular cytogenetics research. Genome‐scale identification of single‐copy oligos is the foundation of successful oligo‐FISH experiments. Here, we introduce Chorus2, a software that is developed specifically for oligo selection. We demonstrate that Chorus2 is highly effective to remove all repetitive elements in selection of single‐copy oligos, which is critical for the development of successful FISH probes. Chorus2 is more effective than Chorus, the original version of the pipeline, and OligoMiner for repeat removal. Chorus2 allows to select oligos that are conserved among related species, which extends the usage of oligo‐FISH probes among phylogenetically related plant species. We also implemented a new function in Chorus2 that allows development of FISH probes from plant species without an assembled genome. We anticipate that Chorus2 can be used in plants as well as in mammalian and other non‐plant species. Chorus2 will broadly facilitate the design of FISH probes for various types of application in molecular cytogenetics research.

Clinical Cytogenetics of the Dog: A Review.

Simple SummaryThe cytogenetic analysis of dogs is mainly focused on the diagnosis of disorders of sex development (DSD) and cancers. Unfortunately, the study of canine chromosomes is a challenging task due a high chromosome number (2n = 78) and the one-arm morphology of all autosomes. For years, the application of conventional cytogenetic techniques, Giemsa staining and G and DAPI (4′,6-diamidino-2-phenylindole) bandings, allowed the identification of sex chromosome aneuploidies and centric fusions. An advanced clinical cytogenetic diagnosis is also needed due to the fact that the dog is a valuable animal model in biomedical research. The application of hybridization methods, such as fluorescence in situ hybridization (FISH) and array comparative genome hybridization (aCGH), facilitated the detection of other chromosomal rearrangements. It can be foreseen that a wide use of modern molecular techniques (e.g., SNP microarray and next generation sequencing) will substantially extend the knowledge on canine chromosome mutations.The dog is an important companion animal and has been recognized as a model in biomedical research. Its karyotype is characterized by a high chromosome number (2n = 78) and by the presence of one-arm autosomes, which are mostly small in size. This makes the dog a difficult subject for cytogenetic studies. However, there are some chromosome abnormalities that can be easily identified, such as sex chromosome aneuploidies, XX/XY leukocyte chimerism, and centric fusions (Robertsonian translocations). Fluorescence in situ hybridization (FISH) with the use of whole-chromosome painting or locus-specific probes has improved our ability to identify and characterize chromosomal abnormalities, including reciprocal translocations. The evaluation of sex chromosome complement is an important diagnostic step in dogs with disorders of sex development (DSD). In such cases, FISH can detect the copy number variants (CNVs) associated with the DSD phenotype. Since cancers are frequently diagnosed in dogs, cytogenetic evaluation of tumors has also been undertaken and specific chromosome mutations for some cancers have been reported. However, the study of meiotic, gamete, and embryo chromosomes is not very advanced. Knowledge of canine genome organization and new molecular tools, such as aCGH (array comparative genome hybridization), SNP (single nucleotide polymorphism) microarray, and ddPCR (droplet digital PCR) allow the identification of chromosomal rearrangements. It is anticipated that the comprehensive use of chromosome banding, FISH, and molecular techniques will substantially improve the diagnosis of chromosome abnormalities in dogs.