Fluorescence In Situ Hybridization Probes Research Articles

Rapid assessment of hyperdiploidy in plasma cell disorders using a novel multi‐parametric flow cytometry method

Trisomies of odd numbered chromosomes are seen in nearly half of patients with multiple myeloma (MM) and typically correlate with a hyperdiploid state and better overall survival (OS). We compared DNA ploidy of monoclonal plasma cells (as a surrogate for the presence of trisomies) assessed simultaneously by PCPRO (plasma cell proliferative index), a novel method that estimates DNA index by multi-parametric flow cytometry to fluorescence in situ hybridization (FISH) in 1703 patients with plasma cell disorders. The distribution of ploidy was hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), tetraploid/near-tetraploid: 108 (6%). FISH identified trisomies in 82% (621/756) of patients with hyperdiploidy by PCPRO and no trisomy by FISH was observed in 88% (730/834) of patients without hyperdiploidy. 95% (795/834) of patients without hyperdiploidy on PCPRO had one or less trisomy by FISH. Sensitivity and specificity of PCPRO for detecting hyperdiploidy was 86% (621/725) and 84% (730/865), respectively. Sensitivity increased to 94% (579/618) for patients with more than one trisomy. Newly diagnosed MM patients with hyperdiploidy on PCPRO (147/275) had better OS compared to nonhyperdiploid patients (median not reached vs 59 months, P = 0.008) and better progression free survival (median: 33 vs 23 months, P = 0.03). Within the hyperdiploidy group, patients with high-hyperdiploidy (DNA index: 1.19-1.50) versus those with low-hyperdiploidy (DNA index: 1.05-1.18) had superior OS (3 year OS of 88% vs 68% P = 0.03). Ploidy assessment by flow cytometry can provide rapid, valuable prognostic information and also reduces the number of copy number FISH probes required and hence the cost of FISH.

Fluorescence In Situ Hybridization and Rehybridization Using Bacterial Artificial Chromosome Probes.

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.

Poorly differentiated pulmonary synovial sarcoma with SYT gene amplification: A case report.

Fluorescence in situ hybridization (FISH) and reverse-transcription polymerase chain reaction (RT-PCR) analysis may be used for the diagnosis of synovial sarcoma (SS), particularly of the poorly differentiated type. While the majority of the studies report that the SYT FISH probe is considered to be break-apart in SS, with two orange and two green signals, the SYT probe in the present case of a 52-year-old male patient with pulmonary SS displayed orange and green signal separation, along with SYT orange signal amplification. RT-PCR was used to verify that the SYT gene amplification was another form of expression of SYT-SSX gene fusion t(X; 18)(p11; q11). In this case, the tumour sample obtained by biopsy was small; therefore, the definitive diagnosis of poorly differentiated SS originating from the lung with SYT gene amplification was confirmed by FISH and RT-PCR. Therefore, these mature biomarkers, which are available as immunohistochemical stains in the molecular pathology laboratory, may help pathologists to diagnose intractable soft tissue tumours based only on small cytological specimens.

Evaluation of FISH for Blood Cultures under Diagnostic Real-Life Conditions.

BackgroundThe study assessed a spectrum of previously published in-house fluorescence in-situ hybridization (FISH) probes in a combined approach regarding their diagnostic performance with incubated blood culture materials.MethodsWithin a two-year interval, positive blood culture materials were assessed with Gram and FISH staining. Previously described and new FISH probes were combined to panels for Gram-positive cocci in grape-like clusters and in chains, as well as for Gram-negative rod-shaped bacteria. Covered pathogens comprised Staphylococcus spp., such as S. aureus, Micrococcus spp., Enterococcus spp., including E. faecium, E. faecalis, and E. gallinarum, Streptococcus spp., like S. pyogenes, S. agalactiae, and S. pneumoniae, Enterobacteriaceae, such as Escherichia coli, Klebsiella pneumoniae and Salmonella spp., Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Bacteroides spp.ResultsA total of 955 blood culture materials were assessed with FISH. In 21 (2.2%) instances, FISH reaction led to non-interpretable results. With few exemptions, the tested FISH probes showed acceptable test characteristics even in the routine setting, with a sensitivity ranging from 28.6% (Bacteroides spp.) to 100% (6 probes) and a specificity of >95% in all instances.ConclusionIf sophisticated rapid diagnostic methods like mass spectrometry from blood culture materials are not available, FISH provides an option for rapid differentiation for laboratories in resource-limited settings.

Cell-Free DNA Analysis Demonstrates Comprehensive Genomic Profiling and a Facile Method to Sensitively in Multiple Myeloma

Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by complex cytogenetic and molecular abnormalities including translocations involving the immunoglobin heavy chain locus and mutations involving numerous oncogenic signaling pathways. Fluorescence in situ hybridization (FISH) has emerged as the most useful current cytogenetic assessment and provide a new level of insight into the correlation of myeloma prognosis risk model. However, the identification or sorting of malignant cells is required before FISH probes and only involved expansion of the types of probe and number of detectable targets is reached. Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with cancer precision medicine and patient prognosis.Methods: In this retrospective cohort study, we identified 37 patients from 9 relapsed/refractory (RR) and 33 newly diagnosed (ND) patients were analyzed for chromosomal copy number imbalance using the ultrasensitive chromosomal aneuploidy detector (UCAD) platform.Results: Chromosome copy number aberration (CNA) were frequently (82.6%, N=46) detected in MM plasma cell free DNA. Applying UCAD to cfDNA, FISH in CD138 purified bone marrow aspirates, and some matched bone marrow biopsies, we find concordance in copy number alterations (~81%) between liquid and tumor biopsies. Significant copy number changes, including 1q gains, 13q deletion and 17p deletion could be found in 57.89%, 54.05%, and 16.67% in plasma of MM, which is higher percentage than FISH assay (46.81%, 28.26%, and 8.89%), respectively. Besides, chromosome 6p and 6q were determined the higher frequency aberration from UCAD. Moreover, a higher frequency of copy number aberrations and variations was detected in RR patients than ND (100% vs 78.4%, respectively), obviously CNAs heterogeneity displayed in advanced disease. In the inconsistent some samples, UCAD from the plasma and bone marrow showed the similar results, which indicated the FISH is underdetermined and insensitivity in some patients' routine inspection.Conclusion: We conclude that cfDNA analysis as an adjunct to BM biopsy represents a noninvasive and broaden the applicability strategy for comprehensive genomic profiling and therapeutic monitoring of MM. DisclosuresNo relevant conflicts of interest to declare.

Phylogeography and Historical Biogeography of the Astyanax bimaculatus Species Complex (Teleostei: Characidae) in Coastal Southeastern South America.

Astyanax bimaculatus, a ubiquitous species in many Neotropical basins, is characterized by a complex taxonomy and are currently considered a species complex. The goal of this study was to analyze 31 populations (N = 136) of this species from southeastern Brazil using cytogenetic techniques: conventional staining, nucleolar organizer region (Ag-NOR), C-banding, and 18S and 5S recombinant DNA fluorescent in situ hybridization (FISH) probes; and molecular techniques: S72, RAG2, and COI. All populations were 2n = 50 (6m + 20sm +18st +6a); Ag-NORs were predominantly simple, C-banding revealed high variation levels within and among basins, and the FISH probes 18S and 5S were restricted to chromosome pairs 14 and 7, respectively. The S72 was uninformative for phylogenetic analyses, and RAG2 showed no variation among populations. The COI gene revealed three haplogroups. The most basal was composed of Pandeiros population (São Francisco Basin) that diverged in the Middle Miocene. The second was composed of A. altiparanae from the Upper Paraná Basin and Espírito Santo Stream (Paraíba do Sul Basin), whereas the third was composed of Astyanax lacustris from São Francisco and coastal basins. The second and third haplogroups diverged in the Pleistocene, indicating that diversification of the bimaculatus complex was driven by tectonic activity and sea-level fluctuations.

In situ visualisation of the abundant Chloroflexi populations in full-scale anaerobic digesters and the fate of immigrating species.

Anaerobic digestion is a key process for the conversion of waste organics to biogas for energy and is reliant on the synergistic activities of complex microbial communities. Members of the phylum Chloroflexi are often found to be abundant in these systems, yet little is known of their role, with most members yet to be cultured or identified. The aim of this study was to characterize the Chloroflexi communities present in full-scale anaerobic digesters receiving excess sludge from wastewater treatment plants. The core genus-level-phylotypes were identified from extensive 16S rRNA gene amplicon sequencing surveys of 19 full-scale systems over a 6 year period. The T78 and Leptolinea, and the RB349 and SJA-170, were found to be the most abundant genera of mesophilic and thermophilic digesters, respectively. With the exception of Leptolinea, these phylotypes are known only by their 16S rRNA gene sequence, and their morphology and metabolic potentials are not known. Fluorescence in situ hybridisation (FISH) probes were designed for these phylotypes, with their application revealing a similar thin filamentous morphology, indicating a possible role for these organisms in maintaining floc structure. The new FISH probes provide a useful tool for future efforts to characterize these organisms in situ. FISH also suggests that immigrating Chloroflexi species die off in the anaerobic digester environment and their high abundance in anaerobic digesters, observed with DNA based sequencing surveys, was quite possibly due to the persistence of their DNA after their death. This observation is important for the interpretation of popular DNA-based sequencing methods applied for the characterisation of communities with substantial immigration rates, such as anaerobic digesters.

A Diagnostic Algorithm Combining Immunohistochemistry and Molecular Cytogenetics to Diagnose Challenging Melanocytic Tumors

Some melanocytic tumors are diagnostic challenges and require ancillary tools in helping the pathologists to determine their potential of malignancy. We intend to propose a diagnostic algorithm in helping to classify challenging melanocytic tumors combining histology, immunohistochemistry, and cytogenetics. We report on 24 spitzoid and/or misdiagnosed melanocytic tumors studied with a triple p16, Ki-67, and HMB45 immunohistochemistry score, fluorescent in situ hybridization (FISH) with melanoma-dedicated and non-melanoma-dedicated probes and comparative genomic hybridization on DNA microarray (CGH array). Melanoma-dedicated FISH probe classified as favor malignant 8/8 melanomas, 1/2 atypical spitzoid tumor, and 4/14 nevi with polyploidy. Only 10 CGH array assays were contributive and concluded in complex chromosomal patterns as hallmarks of malignancy in 5 melanomas, single isolated imbalances in 3 nevi, and no chromosomal gain or loss in 2 nevi. The p16-Ki-67-HMB45 immunohistochemistry score was favor benign (ie, 0 to 3) in 13/14 nevi and in the favor benign atypical spitzoid tumor according to FISH analyses. The FISH-favor malignant atypical spitzoid tumor, 8/8 melanomas, and 1 tumor initially diagnosed as a Spitz nevus had favor malignant p16-Ki-67-HMB45 immunohistochemistry scores (ie, 4 to 9). Additional FISH analyses detected a 9p21/CDKN2A double deletion, frequently reported in melanomas but not in nevi, in the tumor initially diagnosed as a Spitz nevus with a favor malignant p16-Ki-67-HMB45 score. To conclude, in our opinion, histology and p16-Ki-67-HMB45 immunohistochemistry could consist in first-line tools to diagnose a difficult melanocytic tumor, followed by cytogenetics analyses in cases of discrepancies between histology and immunohistochemistry.

High-resolution chromosome painting with repetitive and single-copy oligonucleotides in Arachis species identifies structural rearrangements and genome differentiation

BackgroundArachis contains 80 species that carry many beneficial genes that can be utilized in the genetic improvement of peanut (Arachis hypogaea L. 2n = 4x = 40, genome AABB). Chromosome engineering is a powerful technique by which these genes can be transferred and utilized in cultivated peanut. However, their small chromosomes and insufficient cytological markers have made chromosome identification and studies relating to genome evolution quite difficult. The development of efficient cytological markers or probes is very necessary for both chromosome engineering and genome discrimination in cultivated peanut.ResultsA simple and efficient oligonucleotide multiplex probe to distinguish genomes, chromosomes, and chromosomal aberrations of peanut was developed based on eight single-stranded oligonucleotides (SSONs) derived from repetitive sequences. High-resolution karyotypes of 16 Arachis species, two interspecific F1 hybrids, and one radiation-induced M1 plant were then developed by fluorescence in situ hybridization (FISH) using oligonucleotide multiplex, 45S and 5S rDNAs, and genomic in situ hybridization (GISH) using total genomic DNA of A. duranensis (2n = 2x = 20, AA) and A. ipaënsis (2n = 2x = 20, BB) as probes. Genomes, chromosomes, and aberrations were clearly identifiable in the established karyotypes. All eight cultivars had similar karyotypes, whereas the eight wild species exhibited various chromosomal variations. In addition, a chromosome-specific SSON library was developed based on the single-copy sequence of chromosome 6A of A. duranensis. In combination with repetitive SSONs and rDNA FISH, the single-copy SSON library was applied to identify the corresponding A3 chromosome in the A. duranensis karyotype.ConclusionsThe development of repetitive and single-copy SSON probes for FISH and GISH provides useful tools for the differentiation of chromosomes and identification of structural chromosomal rearrangement. It facilitates the development of high-resolution karyotypes and detection of chromosomal variations in Arachis species. To our knowledge, the methodology presented in this study demonstrates for the first time the correlation between a sequenced chromosome region and a cytologically identified chromosome in peanut.

Quality Assurance/Quality Control of Fluorescence in Situ Hybridization Tests in Hematologic Malignancies

Because of its’ simplicity, reliability and cost-effectiveness, fluorescence in situ hybridization (FISH) is a major technology that is widely applied in clinical diagnosis, especially for hematologic malignancies, even in the era of next-generation sequencing (NGS) [1-4]. In the Clinical Cytogenetics Laboratory at MD Anderson Cancer Center, over 15,000 FISH tests are performed each year, including approximately 1,000 BCR-ABL1 and 500 MYC FISH tests, respectively. In this chapter, we introduce the quality assurance/quality control (QA/QC) measurements applied for FISH tests by following the American College of Medical Genetics and Genomics (ACMGG) technical standards and guidelines [5]. We believe that these measurements are essential for high-quality clinical diagnosis services provided by a clinical laboratory. The BCR-ABL1 FISH test using a commercial fusion probe set on cell suspensions of bone marrow (BM) and peripheral blood (PB) specimen and MYC FISH test using a commercial break-apart probe on paraffin-embedded tissue specimen are shown here as examples. For the use of home-brew FISH probes and FDA-approved FISH test kits, some different QA/QC measurements need be taken and are not included in this chapter.

P1.09-22 Detection of ALK Gene Rearrangement in FFPE Tissues of Non-Small Cell Lung Cancer Using in Situ RNA Hybridization

Approximately 4-7% of non-small cell lung cancer (NSCLC) harbor anaplastic lymphoma kinase (ALK) gene rearrangements which lead to overexpression of ALK fusion protein and constitutive activation of the kinase. The tumor with ALK gene rearrangements is sensitive to its inhibitor, so identification of this molecular feature from the patients accurately is important. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are conventional methods for testing ALK gene rearrangement and protein overexpression respectively; however it is challenge for distinguishing some rare variations by FISH, or determining the equivocal stain by IHC. A morphology related method under bright field about rearranged mRNA expression (RNAscope) of ALK gene fusion was developed, which is helpful to realize the biological behavior of ALK gene, together with FISH (break apart FISH probe kit, Abbott) and IHC (D5F3 clone, Ventana). We explored 7 cases of formalin-fixed paraffin-embedded (FFPE) samples from NSCLC patients by RNAscope® 2.5 Red assay Three of seven samples were found with ALK positive by RNAscope, which matched with FISH and IHC results. In these 3 ALK positive samples, exon 19-29 of ALK gene transcribed RNA signals were revealed by probe-Hs-ALK E1-E18 (score 1 to 2 in >50% cells), whereas exon 1-18 transcribed RNA signals were not shown by probe-Hs-ALK E19-E29, which confirmed by FISH results (with break apart signals). In 2 of 4 ALK RNAscope negative cases, ALK RNA signals were detected by both probe-Hs-ALK E1-E18 (score 2) and probe-Hs-ALK E19-E29 (score 2) in small regions (showed a small number of signals), which may indicate that ALK gene amplification and also confirmed by FISH results (with more than 2 fusion signals). And these 2 typical ALK RNAscope negative samples were considered close to be disomy by FISH. Our results demonstrate that RNAscope® 2.5 Red assay is a reliable and precise method to detect ALK mRNA expression caused by ALK gene fusion and also a one to discover the ALK gene amplification as well in FFPE samples of NSCLC, even though latter’s clinical significant is not clear. RNAscope® 2.5 Red assay is a both sensitive and specific method to provide information on the spatial distribution of ALK gene status and morphologic characteristic of tumors simultaneously under the bright field microscope.

Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection

An enrichment culture of Candidatus Brocadia fulgida was identified by three independent methods: analysis of autofluorescence using different microscope filter blocks and a fluorescence spectrometer, fluorescence in situ hybridization (FISH) with anammox-specific probes and partial sequencing of the 16S rDNA, hydrazine synthase hzsA and hydrazine oxidoreductase hzo. The filter block BV-2A (400–440, 470 LP, Nikon) was suitable for preliminary detection of Ca. B. fulgida. An excitation-emission matrix revealed three pairs of excitation-emission maxima: 288–330 nm, 288–478 nm and 417–478 nm. Several autofluorescent cell clusters could not be stained with DAPI or by FISH, suggesting empty but intact cells (ghost cells) or inhibited permeability. Successful staining of autofluorescent cells with the FISH probes Ban162 and Bfu613, even at higher formamide concentrations, suggested insufficient specificity of Ban162. Under certain conditions, Ca. B. fulgida lost its autofluorescence, which reduced the reliability of autofluorescence for identification and detection. Non-fluorescent Ca. Brocadia cells could not be stained with Ban162, but with Bfu613 at higher formamide concentrations, suggesting a dependency between both parameters. The phylogenetic analysis showed only good taxonomical clustering of the 16S rDNA and hzsA. In conclusion, careful consideration of autofluorescent characteristics is recommended when analysing and presenting FISH observations of Ca. B. fulgida to avoid misinterpretations and misidentifications.

Use of fluorescence in situ hybridization to detect Felis catus papillomavirus type 2 in feline Bowenoid in situ carcinomas.

Papillomaviruses (PVs) are ubiquitous host- and site-specific viruses. PV infections in cats are associated with oral papillomas, viral plaques, Bowenoid in situ carcinomas (BISCs), squamous cell carcinomas and sarcoids; this association is primarily based on PCR detection of PV DNA within said lesions. PV DNA is frequently detectable on normal feline skin; thus, it is possible that some of the implicated DNA is commensal rather than associated with lesion formation. Therefore, the aim of the present study was to use fluorescence in situ hybridization (FISH) to localize PV DNA within feline BISCs, to provide additional evidence that PV infection may influence the development of these neoplasms. FISH probes targeting Felis catus papillomavirus type 2 (FcaPV2) DNA were used to localize FcaPV2 DNA within 42 BISCs from which FcaPV2 DNA had previously been amplified via PCR. Fifteen of 42 BISC lesions (35.7%) demonstrated intralesional FcaPV2 using FISH. Probe annealing was predominantly located within the nuclei of koilocytes found in the upper strata of the epidermis. Probes were typically scattered multifocally within the lesions; most commonly this was near the periphery of the BISCs. These results confirm that a proportion of BISCs contain FcaPV2 DNA. These results further support a causative association between FcaPV2 and BISCs in cats.

Physical mapping of the blue-grained gene from Thinopyrum ponticum chromosome 4Ag and development of blue-grain-related molecular markers and a FISH probe based on SLAF-seq technology.

A Thinopyrum ponticum chromosome 4Ag physical map was constructed, the blue-grained gene was localized, and related specific markers and a FISH probe were developed by SLAF-seq. Decaploid Thinopyrum ponticum (2n = 10x = 70) serves as an important gene pool for wheat improvement. The wheat-Th. ponticum 4Ag (4D) disomic substitution line Blue 58, derived from a distant hybridization between Th. ponticum and common wheat (Triticum aestivum L.), bears blue coloration in the aleurone layer. To map the blue-grained gene, eight wheat-Th. ponticum 4Ag translocation lines with different chromosomal segment sizes were obtained from Blue 58 using 60Co-γ ray irradiation and were characterized using cytogenetic and molecular marker analysis. A small-segment blue-grained wheat translocation line L13, accounting for one-fifth of 4AgL, was obtained. A physical map of chromosome 4Ag was constructed containing 573 specific-locus amplified fragment sequencing (SLAF-seq) markers, including three bins with 223 markers on 4AgS and eight bins with 350 markers on 4AgL. The blue-grained gene in three blue-grained translocation lines L5, L9, and L13, was located on bin 4AgL-6 with FL 0.75-0.89. Moreover, 89 blue-grain-related molecular markers and one fluorescence in situ hybridization (FISH) probe, pThp12.19, were identified in this bin. The newly developed translocation lines and the molecular markers and FISH probe will facilitate the application of the Th. ponticum-origin blue-grained characteristic in wheat breeding.

A comparative cytogenetic study of 17 Avena species using Am1 and (GAA)6 oligonucleotide FISH probes

Fluorescence in situ hybridization (FISH) was performed to explore the genomic and species relationships among 17 taxa using Am1 (oligo-Am1) and (GAA)6 oligonucleotide probes. Oligo-Am1 (51 bp) hybridized strongly over almost the entire length of all chromosomes in the C genome. Six translocations between the A and C genomes were found in AACC tetraploids and AACCDD hexaploids, four minor translocations between the C and D genomes were found in AACCDD hexaploids, and two large translocations between the C and D genomes were found in A. sativa. In the 17 Avena species, (GAA)6 regions mainly appear as sharp, thin bands at pericentromeric positions in the A, B, and C genomes and at termini in the B genome. However, no (GAA)6 signal loci were observed in the D genome. The (GAA)6 signal number was constant in both AA and CC diploids, though with different signal intensities. The (GAA)6 signal pattern was diverse in AABB, AACC, and AACCDD polyploids, with each species exhibiting one signal pattern. The (GAA)6 signal number was consistent in diploids and varied in polyploids, describing an intragenomic relationship among Avena species. This study is the first to test these two oligonucleotides, which are based on synthesized repeat units (18–51 bp), in the genus Avena. Our approach paves the way for future studies in which FISH probes can be used to assign other landmark genomic sequence oligonucleotides to physical chromosomes.

PO-269 Development of a fluorescence in situ hybridization (FISH) method for detection of intra-tumour bacteria involved in pancreatic cancer chemoresistance

IntroductionPancreatic cancer (PC) is projected to become the second leading cause of cancer-related deaths in 2020. Recent studies demonstrate that bacteria are a component of pancreatic tumour microenvironment, and they may also play a critical role in mediating resistance to chemotherapy.1 Remarkably, a prospective study showed that the presence of two oral pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, was associated with an elevated risk of developing PC.2Material and methodsThis study aimed at developing a novel fluorescence in situ hybridization (FISH) based method to specifically detect oral bacteria in formalin-fixed paraffin-embedded (FFPE) sections from PC patients. Multi-wavelength confocal spinning disk microscopy was used to discriminate with high spatial resolution and sensitivity the tissue high autofluorescence signal from even low number of emitters/bacteria. Bacterial FISH probes were primers for the 16 s rRNA molecule.Results and discussionsFISH allowed the in situ localisation and the study of spatial organisation of bacterial cells as they occur in PC. FISH results were always definitive and well-detectable both in in 4 and 20 µm FFPE sections. The intensity of the fluorescence was a direct measure for the activity of the bacterial cells, whereas inactive cells were recognised by their low intensity fluorescence. Thus, our method gives a optimal sensitivity of fluorescence and low background by subtracting autofluorescence components.Our FISH preliminary data confirmed the presence of bacteria in the pancreatic tumour microenvironment and support further studies aimed at elucidating how oral bacteria are involved in PC aetiology.It is well-known that the chemoresistance (intrinsic and acquired) has been a major problem for PC treatment3 and a recent study showed the role of bacteria in resistance to gemcitabine.1 To this end, the establishment of our reliable FISH protocol allows us to further investigate the potential role of oral bacteria in the failure of standard treatments for PC.ConclusionOur new robust methodology will contribute to obtain meaningful data and to unravel the complex interaction between oral bacteria and pancreatic cancer chemoresistance in clinically well-annotated tissues, ultimately identifying specific bacteria as potential biomarkers for early detection/treatment.References. Geller, et al. Science2017;357:1156–1160.. Farrell JJ, et al. Gut2012; 61:582–588.. Coppola, et al. Drug Resist. Updates 2017;31:43–51.

Abstract 2076: Identification of novel fusion genes and expression variants in primary and patient-derived xenograft samples of pediatric leukemia using error-corrected RNA sequencing

Abstract Introduction Chromosomal rearrangements generating gene fusions are more common in pediatric malignancies compared to adults, and possess diagnostic, prognostic and therapeutic value. Traditionally chromosomal rearrangements including structural variants (SVs) are identified using karyotyping and fluorescence in situ hybridization (FISH); however, these methods are not sensitive for small rearrangements and single nucleotide variants (SNVs). The need to detect these cryptic SV and SNVs in pediatric cancers demands the development of assays to analyze complex RNA molecules. Methods Primary bone marrow samples obtained from Nemours Biobank were used for RNA isolation. Targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was conducted on 30 primary pediatric leukemia samples and their corresponding mouse passaged xenograft samples. RNA data (fastq) was analyzed via a custom cloud environment leveraging ArcherDx Version 5.1.3 software. The gene fusion data produced by the Archer panel was initially correlated with diagnostic FISH data available for each primary sample. Results Ten out of 30 primary bone marrow samples possessed gene fusions detected by routinely tested FISH probes for diagnostic purposes, and including ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and KMT2A. In addition, this approach detected cryptic gene fusions in 10 samples that were negative for chromosomal rearrangements via FISH, including SPTAN1-ABL1, RUNX1-MKL1, NUP98-NSD1, P2RY8-CRLF2 and TCF3-HLF. The remaining 10 samples, which did not possess detectable gene fusions, showed abnormal exon usage and domain duplications for several, key oncogenes along with novel mutations. A comparison of the primary sample and mouse passaged xenograft sample revealed that majority of gene fusions representing the abundant clone remained consistent between the primary and xenograft sample in secondary and tertiary passages. Certain gene fusions representing minor clones appeared and disappeared in xenograft samples and subsequent passages in mice in comparison to the primary patient sample, highlighting the heterogeneity of the disease. Thus, the presence of major driver mutations at similar allelic frequencies in xenografts compared to primary samples and over multiple passages confirms the utility of xenograft models for preclinical drug testing. Discussion Using a novel approach that utilizes targeted error-corrected sequencing, all the aberrations detected by clinical diagnostic testing were verified, plus several novel fusion events were identified with high confidence. This method also validated the concordance between primary and xenograft samples. Characterization of these novel cryptic fusions and exonal variants in leukemogenesis will enable identification of new drug targets and prognostic factors for pediatric leukemia. Citation Format: Sonali P. Barwe, Anilkumar Gopalakrishnapillai, Nitin Mahajan, Todd Druley, E. Anders Kolb, Erin L. Crowgey. Identification of novel fusion genes and expression variants in primary and patient-derived xenograft samples of pediatric leukemia using error-corrected RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2076.

Development of a Fluorescence in Situ Hybridization Probe for Detecting IKZF1 Deletion Mutations in Patients with Acute Lymphoblastic Leukemia

Intragenic deletion of IKZF1 is a recurrent genomic alteration in acute lymphoblastic leukemia. The deletions are mediated by illegitimate variable(diversity)joining recombination via cryptic recombination signal sequences (RSSs). We developed a fluorescence in situ hybridization (FISH) probe set that can detect any type of IKZF1 deletion, including the commonly deleted exon 4 to 7 region. The probe set consists of a designed probe for the commonly deleted region (Cy3; red) and a bacterial artificial chromosomes clone probe for detecting the 3' flanking region (Spectrum Green). Intact IKZF1 showed a fusion signal, and the deleted allele showed loss of the red signal (0R1G1F). The FISH probes worked correctly for human leukemic cell lines and clinical samples. One case showed an atypical break-apart signal (1R1G1F). Inverse PCR of the case revealed rearrangement of the excised IKZF1 fragment into a legitimate RSS site at Ig κ on chromosome 2, suggesting a pathogenic role of this recombination-activating gene 1/2-mediated event. In this study, we established FISH probe detecting IKZF1 deletion in a quick, quantitative, and cost-effective manner, and the results provided a novel insight into B-cell receptor editing by rearrangement of a cryptic RSS-mediated genomic fragment in acute lymphoblastic leukemia pathology.

PO-025 Antiproliferative effects of rosemary (rosmarinus officinalis, L.) extract in colon cancer cells lines

IntroductionInfections by oncoviruses are an important risk factor for the development of neoplasms. Particularly, anogenital cancers are strongly associated with persistent infections by high-risk human papillomaviruses (HPV). In the case of cervical cancer the physical state of the virus seems to be an important prognostic marker. Especially the integration of viral DNA into the host cell genome bears the risk of following degenerations. Therefore, the early and sensitive detection is of high interest. Besides Pap-test and PCR, fluorescence in situ hybridization (FISH) is used for a quantitative and qualitative detection of HPV infections and risk assessment.Material and methodsFor analysis high-risk HPV FISH probes should be developed for standardised, type-specific and quantitative detection of HPV nucleic acid in host cells. Due to the short viral DNA sequence, present with just few copies, conventional FISH probes have little sensitivity. Therefore, we tested specific FISH probes targeting different regions of the HPV genome in combination with signal amplification. PCR-produced FISH probes were biotin labelled by direct incorporation of biotinylated nucleotides or nick translation. Our biotinylated HPV probes were tested on HPV-positive and –negative cells and detected using fluorescence labelled streptavidin or subsequent tyramide signal amplification. In addition to FISH, probes were used for chromogenic in situ hybridization. All analysis were performed with our fully automatized multicolor fluorescence imaging platform VideoScan.Results and discussionsThe presence of HPV DNA in cell lines was confirmed by (digital) PCR. The protocol for synthesis of biotinylated probes using HPV sequence specific primers and genomic DNA as template was successful. Biotin incorporation was checked with agarose gel electrophoresis and modified dot blot hybridization. In in situ hybridization experiments, our probes showed high signals in HPV high-copy number CaSki cells and a tissue model, compared to HPV negative samples.ConclusionHighly sensitive and specific FISH probes offer a great potential for the detection of HPV DNA in patient samples and thereby for the assessment of tumour risks. Using our probes, we got positive results in hybridization experiments using chromogenic and fluorescent detection methods with or without further amplification steps. Next, comparative FISH experiments will be performed to check probe sensitivity in cells containing few HPV copies and their practicability in biopsy derived tissue samples.

Fluorescent In Situ Hybridization for TP53 in the Diagnosis of Pediatric Osteogenic Sarcoma.

Osteogenic sarcoma (OS) is the most common malignant bone tumor in children and adolescents. Despite advances in molecular genetic characterization of pediatric and adult tumors, the diagnosis of OS still depends almost entirely on light microscopy. The lack of consistent genetic changes in OS has greatly hindered the development of any diagnostic molecular test. Recently, whole-genome sequencing has shown that ~50% of cases of OS have a translocation involving the TP53 gene with breakpoints confined to the first intron. We developed a 2 color break-apart fluorescent in situ hybridization (FISH) probe for intron 1 of TP53 and applied it to an archived series to assess its diagnostic utility. The study group included 37 cases of OS (including osteoblastic, chondroblastic, and fibroblastic), as well as 53 cases of non-OS pediatric sarcomas (including Ewing sarcoma, rhabdomyosarcoma, undifferentiated small cell sarcoma, CCNB3-BCOR sarcoma, CIC-DUX sarcoma, synovial sarcoma, and malignant peripheral nerve sheath tumor) and 27 cases of benign bone lesions (including osteoblastoma, chondromyxoid fibroma, fibrous dysplasia, and fibro-osseous dysplasia). A rearranged signal was found in 20/37 cases (54%) of OS and in none of the other sarcomas or benign bone lesions, giving the FISH test 100% specificity for a diagnosis of OS. p53 immunostaining was generally not predictive of the results obtained by FISH and could not substitute for this test. This FISH probe offers a simple and specific genetic test to aid in the diagnosis of OS, despite the genetic complexity of this tumor.