Sequential Fluorescence In Situ Hybridization Research Articles

Development and application of whole-chromosome painting of chromosomes 7A and 8A of Arachis duranensis based on chromosome-specific single-copy oligonucleotides.

For peanut, the lack of stable cytological markers is a barrier to tracking specific chromosomes, elucidating the genetic relationships between genomes and identifying chromosomal variations. Chromosome mapping using single-copy oligonucleotide (oligo) probe libraries has unique advantages for identifying homologous chromosomes and chromosomal rearrangements. In this study, we developed two whole-chromosome single-copy oligo probe libraries, LS-7A and LS-8A, based on the reference genome sequences of chromosomes 7A and 8A of Arachis duranensis. Fluorescence in situ hybridization (FISH) analysis confirmed that the libraries could specifically paint chromosomes 7 and 8. In addition, sequential FISH and electronic localization of LS-7A and LS-8A in A. duranensis (AA) and A. ipaensis (BB) showed that chromosomes 7A and 8A contained translocations and inversions relative to chromosomes 7B and 8B. Analysis of the chromosomes of wild Arachis species using LS-8A confirmed that this library could accurately and effectively identify A genome species. Finally, LS-7A and LS-8A were used to paint the chromosomes of interspecific hybrids and their progenies, which verified the authenticity of the interspecific hybrids and identified a disomic addition line. This study provides a model for developing specific oligo probes to identify the structural variations of other chromosomes in Arachis and demonstrates the practical utility of LS-7A and LS-8A.

Prognostic value of clonal evolution identified by sequential FISH in untreated chronic lymphocytic leukaemia

Aim: The aim of the current study was to evaluate the potential clinical impact of clonal evolution detected by fluorescence in situ hybridization (FISH) in untreated chronic lymphocytic leukaemia (CLL) patients managed with a watch-and-wait strategy. Methods: We performed both overall survival (OS) and time to first treatment (TTFT) analysis. For the first one, we exploited a real-life cohort of 123 consecutive CLL patients followed at our institution, for which at least a second FISH evaluation during watch and wait was available. For TTFT analysis, we considered only patients treated after the second FISH sample (n = 69). Results: Considering the original cohort, patients who acquired a FISH abnormality displayed a worse outcome with a median OS of 91.9 months compared to 147.3 months for patients who did not acquire any FISH abnormalities (P = 0.007). Unmutated immunoglobulin heavy chain gene (IGHV) genes were associated with a higher probability of acquiring a FISH abnormality (P = 0.04). Turning to TTFT analysis, patients who gained at least one FISH abnormality (n = 7, 10%) were characterised by an earlier treatment requirement with a median TTFT of 1.1 months, compared to 2.7 months in patients who did not acquire any FISH abnormalities (n = 62, 90%) (P = 0.025). Conclusions: The dynamic acquisition of karyotypic abnormalities by FISH predicts poor outcomes and early treatment requirement in CLL patients. Our results suggest that FISH analysis could be integrated with other clinical and biological features to obtain dynamic scores that are able to predict outcomes at different phases of disease history.

Subcellular spatially resolved gene neighborhood networks in single cells

Image-based spatial omics methods such as fluorescence in situ hybridization (FISH) generate molecular profiles of single cells at single-molecule resolution. Current spatial transcriptomics methods focus on the distribution of single genes. However, the spatial proximity of RNA transcripts can play an important role in cellular function. We demonstrate a spatially resolved gene neighborhood network (spaGNN) pipeline for the analysis of subcellular gene proximity relationships. In spaGNN, machine-learning-based clustering of subcellular spatial transcriptomics data yields subcellular density classes of multiplexed transcript features. The nearest-neighbor analysis produces heterogeneous gene proximity maps in distinct subcellular regions. We illustrate the cell-type-distinguishing capability of spaGNN using multiplexed error-robust FISH data of fibroblast and U2-OS cells and sequential FISH data of mesenchymal stem cells (MSCs), revealing tissue-source-specific MSC transcriptomics and spatial distribution characteristics. Overall, the spaGNN approach expands the spatial features that can be used for cell-type classification tasks.

Molecular cytogenetics and development of St-chromosome-specific molecular markers of novel stripe rust resistant wheat\u2013Thinopyrum intermedium and wheat\u2013Thinopyrum ponticum substitution lines

BackgroundOwing to their excellent resistance to abiotic and biotic stress, Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Th. ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for transmission of novel genes, fluorescence in situ hybridization (FISH) karyotype construction and specific molecular marker development.ResultsSix wheat–Thinopyrum DSLs derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), were characterized by sequential FISH–genome in situ hybridization (GISH), multicolor GISH (mc-GISH), and an analysis of the wheat 15 K SNP array combined with molecular marker selection. ES-9 (DS2St (2A)) and ES-10 (DS3St (3D)) are wheat–Th. ponticum DSLs, while ES-23 (DS2St (2A)), ES-24 (DS3St (3D)), ES-25(DS2St (2B)), and ES-26 (DS2St (2D)) are wheat–Th. intermedium DSLs. ES-9, ES-23, ES-25 and ES-26 conferred high thousand-kernel weight and stripe rust resistance at adult stages, while ES-10 and ES-24 were highly resistant to stripe rust at all stages. Furthermore, cytological analysis showed that the alien chromosomes belonging to the same homoeologous group (2 or 3) derived from different donors carried the same FISH karyotype and could form a bivalent. Based on specific-locus amplified fragment sequencing (SLAF-seq), two 2St-chromosome-specific markers (PTH-005 and PTH-013) and two 3St-chromosome-specific markers (PTH-113 and PTH-135) were developed.ConclusionsThe six wheat–Thinopyrum DSLs conferring stripe rust resistance can be used as bridging parents for transmission of valuable resistance genes. The utility of PTH-113 and PTH-135 in a BC1F2 population showed that the newly developed markers could be useful tools for efficient identification of St chromosomes in a common wheat background.

Chromosomal Characterization of Tripidium arundinaceum Revealed by Oligo-FISH.

Sugarcane is of important economic value for producing sugar and bioethanol. Tripidium arundinaceum (old name: Erianthus arundinaceum) is an intergeneric wild species of sugarcane that has desirable resistance traits for improving sugarcane varieties. However, the scarcity of chromosome markers has hindered the cytogenetic study of T. arundinaceum. Here we applied maize chromosome painting probes (MCPs) to identify chromosomes in sorghum and T. arundinaceum using a repeated fluorescence in situ hybridization (FISH) system. Sequential FISH revealed that these MCPs can be used as reliable chromosome markers for T. arundinaceum, even though T. arundinaceum has diverged from maize over 18 MYs (million years). Using these MCPs, we identified T. arundinaceum chromosomes based on their sequence similarity compared to sorghum and labeled them 1 through 10. Then, the karyotype of T. arundinaceum was established by multiple oligo-FISH. Furthermore, FISH results revealed that 5S rDNA and 35S rDNA are localized on chromosomes 5 and 6, respectively, in T. arundinaceum. Altogether, these results represent an essential step for further cytogenetic research of T. arundinaceum in sugarcane breeding.

A universal karyotypic system for hexaploid and diploid Avena species brings oat cytogenetics into the genomics era

BackgroundThe identification of chromosomes among Avena species have been studied by C-banding and in situ hybridization. However, the complicated results from several cytogenetic nomenclatures for identifying oat chromosomes are often contradictory. A universal karyotyping nomenclature system for precise chromosome identification and comparative evolutionary studies would be essential for genus Avena based on the recently released genome sequences of hexaploid and diploid Avena species.ResultsTandem repetitive sequences were predicted and physically located on chromosomal regions of the released Avena sativa OT3098 genome assembly v1. Eight new oligonucleotide (oligo) probes for sequential fluorescence in situ hybridization (FISH) were designed and then applied for chromosome karyotyping on mitotic metaphase spreads of A. brevis, A. nuda, A. wiestii, A. ventricosa, A. fatua, and A. sativa species. We established a high-resolution standard karyotype of A. sativa based on the distinct FISH signals of multiple oligo probes. FISH painting with bulked oligos, based on wheat-barley collinear regions, was used to validate the linkage group assignment for individual A. sativa chromosomes. We integrated our new Oligo-FISH based karyotype system with earlier karyotype nomenclatures through sequential C-banding and FISH methods, then subsequently determined the precise breakage points of some chromosome translocations in A. sativa.ConclusionsThis new universal chromosome identification system will be a powerful tool for describing the genetic diversity, chromosomal rearrangements and evolutionary relationships among Avena species by comparative cytogenetic and genomic approaches.

A Quick Immuno-FISH Protocol for Detecting RNAs, Proteins, and Chromatin Modifications.

Immunofluorescence and fluorescence in situ hybridization (FISH) are widely used cytogenetic techniques for visualization of protein and RNA/DNA molecules. Here, we describe an experimental procedure for quick sequential immunofluorescence and RNA FISH (immuno-FISH), which enables the simultaneous detection of proteins, chromatin modifications, and RNAs on the inactive X-chromosome (Xi) using female mouse embryonic fibroblast (MEF) and tail-tip 3T3 cell lines. Using a pooled array of oligonucleotides labeled with a single fluorophore as an RNA FISH probe, we can reduce the time for RNA FISH from an overnight process to 1-2h without losing its sensitivity. This protocol could be applied to visualization of various protein and RNA molecules, and chromatin modifications.

ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful method to visualize the spatial positions of specific genomic loci and RNA species. Recent technological advances have leveraged FISH to visualize these features in a highly multiplexed manner. Notable examples include chromatin tracing, RNA multiplexed error-robust FISH (MERFISH), multiplexed imaging of nucleome architectures (MINA), and sequential single-molecule RNA FISH. However, one obstacle to the broad adoption of these methods is the complexity of the multiplexed FISH probe design. In this paper, we introduce an easy-to-use, versatile, and all-in-one application called ProbeDealer to design probes for a variety of multiplexed FISH techniques and their combinations. ProbeDealer offers a one-stop shop for multiplexed FISH design needs of the research community.

Development of a set of chromosome-specific oligonucleotide markers and karyotype analysis in the Japanese morning glory Ipomoea nil

The phylogenetic relationships for Ipomoea species are incongruent in previous studies. Comparative karyotype analysis can provide valuable information for phylogenetic relationships among species. A reliable and efficient system for chromosome identification is the foundation for karyotype analysis. However, for the Ipomoea species, individual chromosomes were not identified in all previous reported karyotypes due to their small size, high number and similar morphology. Fluorescence in situ hybridization (FISH) using oligonucleotides (oligos) as probes is a new strategy for chromosome identification and karyotype analysis. Here, we developed the first set of oligo-based probes based on the reference genome of Ipomoea nil, a model species in the genus Ipomoea. In all, we developed four oligo-FISH probes. By a combined use of four oligo probes, sequential FISH analysis were conducted on the same metaphase cells with each round including two probes, which permitted simultaneous identification of all I. nil chromosomes and anchoring 15 pseudochromosomes to individual cytological chromosomes. Moreover, 45S and 5S rDNA were mapped to specific chromosomes. A karyotype based on individually identified chromosomes was established, which was the first FISH-based molecular cytogenetic karyotype of I. nil. Our study has created the basis for studying chromosome variation and evolution in genus Ipomoea by comparative karyotype analysis using this set of oligo probes.

Characterization and Evaluation of Resistance to Powdery Mildew of Wheat-Aegilops geniculata Roth 7Mg (7A) Alien Disomic Substitution Line W16998.

Aegilops geniculata Roth has been used as a donor of disease-resistance genes, to enrich the gene pool for wheat (Triticum aestivum) improvement through distant hybridization. In this study, the wheat–Ae. geniculata alien disomic substitution line W16998 was obtained from the BC1F8 progeny of a cross between the common wheat ‘Chinese Spring’ (CS) and Ae. geniculata Roth (serial number: SY159//CS). This line was identified using cytogenetic techniques, analysis of genomic in situ hybridization (GISH), functional molecular markers (Expressed sequence tag-sequence-tagged site (EST–STS) and PCR-based landmark unique gene (PLUG), fluorescence in situ hybridization (FISH), sequential fluorescence in situ hybridization–genomic in situ hybridization (sequential FISH–GISH), and assessment of agronomic traits and powdery mildew resistance. During the anaphase of meiosis, these were evenly distributed on both sides of the equatorial plate, and they exhibited high cytological stability during the meiotic metaphase and anaphase. GISH analysis indicated that W16998 contained a pair of Ae. geniculata alien chromosomes and 40 common wheat chromosomes. One EST–STS marker and seven PLUG marker results showed that the introduced chromosomes of Ae. geniculata belonged to homoeologous group 7. Nullisomic–tetrasomic analyses suggested that the common wheat chromosome, 7A, was absent in W16998. FISH and sequential FISH–GISH analyses confirmed that the introduced Ae. geniculata chromosome was 7Mg. Therefore, W16998 was a wheat–Ae. geniculata 7Mg (7A) alien disomic substitution line. Inoculation of isolate E09 (Blumeria graminis f. sp. tritici) in the seedling stage showed that SY159 and W16998 were resistant to powdery mildew, indeed nearly immune, whereas CS was highly susceptible. Compared to CS, W16998 exhibited increased grain weight and more spikelets, and a greater number of superior agronomic traits. Consequently, W16998 was potentially useful. Germplasms transfer new disease-resistance genes and prominent agronomic traits into common wheat, giving the latter some fine properties for breeding.

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications

BackgroundClementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine.ResultsA graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin.ConclusionsThe composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Programmable Chromosome Painting with Oligopaints.

Current methods for chromosome painting via fluorescence in situ hybridization (FISH) are costly, time-consuming, and limited in complexity. In contrast to conventional sources of probe, Oligopaints are computationally designed, synthesized on microarrays, and amplified by PCR. This approach allows for precise control over the sequences they target, which can range from a few kilobases to entire chromosomes with the same basic protocol. We have utilized the flexibility and scalability of Oligopaints to generate low-cost and renewable chromosome paints for Drosophila, mouse, and human chromosomes. These Oligopaint libraries can be customized to label any genomic feature(s) in a chromosome-wide manner. Additionally, this method is compatible with sequential FISH to label entire genomes with a single denaturation step. Here, we outline a protocol and considerations to scale the Oligopaint technology for fluorescent labeling of whole chromosomes.

Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut

Interspecific hybridization is an important approach to improve cultivated peanut varieties. Cytological markers such as tandem repeats will facilitate alien gene introgression in peanut. Telomeric repeats have also been frequently used in chromosome research. Most plant telomeric repeats are (TTTAGGG)n that are mainly distributed at the chromosome ends, although interstitial telomeric repeats (ITRs) are also commonly identified. In this study, the telomeric repeat was chromosomally localized in 10 Arachis species through sequential GISH (genomic in situ hybridization) and FISH (fluorescence in situ hybridization) combined with 4’,6-diamidino-2-phenylindole (DAPI) staining. Six ITRs were identified such as in the centromeric region of chromosome Bi5 in Arachis ipaënsis, pericentromeric regions of chromosomes As5 in A. stenosperma, Bho7 in A. hoehnei and Av5 in A. villosa, nucleolar organizer regions of chromosomes As3 in A. stenosperma and Adi3 in A. diogoi, subtelomeric regions of chromosomes Bho9 in A. hoehnei and Adu7 in A. duranensis, and telomeric region of chromosome Es7 in A. stenophylla. The distributions of the telomeric repeat, 5S rDNA, 45S rDNA and DAPI staining pattern provided not only ways of distinguishing different chromosomes, but also karyotypes with a higher resolution that could be used in evolutionary genome research. The distribution of telomeric repeats, 5S rDNA and 45S rDNA sites in this study, along with inversions detected on the long arms of chromosomes Kb10 and Bho10, indicated frequent chromosomal rearrangements during evolution of Arachis species.

A functional centromere lacking CentO sequences in a newly formed ring chromosome in rice

An awned rice (Oryza sativa) plant carrying a tiny extra chromosome was discovered among the progeny of a telotrisomic line 2n+4L. Fluorescence in situ hybridization (FISH) using chromosome specific BAC clones revealed that this extra chromosome was a ring chromosome derived from part of the long arm of chromosome 4. So the aneuploidy plant was accordingly named as 2n+4L ring. We did not detect any CentO FISH signals on the ring chromosome, and found only the centromeric probe Centromeric Retrotransposon of Rice (CRR) was co-localized with the centromere-specific histone CENH3 as revealed by sequential FISH after immunodetection. The extra ring chromosome exhibited a unique segregation pattern during meiosis, including no pairing between the ring chromosome and normal chromosome 4 during prophase I and pre-separation of sister chromatids at anaphase I.

Dense transcript profiling in single cells by image correlation decoding.

Recent work in sequential fluorescent in-situ hybridization (FISH) has demonstrated the ability to uniquely encode a large number of molecular species in single cells. However, the multiplexing capacity is practically limited by the density of the barcoded objects in the cell. Here, we present a general method using image correlation to resolve the temporal barcodes in sequential hybridization experiments, allowing high density objects to be decoded. Using this correlation FISH (corrFISH) approach, we profiled the gene expression of ribosomal proteins in single cells in cell cultures and in mouse thymus tissue sections. In tissues, corrFISH revealed cell type specific gene expression of ribosomal proteins. The combination of sequential barcoding FISH and correlation analyses provides a general strategy for multiplexing a large number of RNA molecules and potentially other high copy number molecules in single cells.

Molecular cytogenetic characterization of a new wheat-rye 1BL•1RS translocation line expressing superior stripe rust resistance and enhanced grain yield.

A new wheat-rye 1BL•1RS translocation line, with the characteristics of superior stripe rust resistance and high thousand-kernel weight and grain number per spike, was developed and identified from progenies of wheat-rye- Psathyrostachys huashanica trigeneric hybrids. The wheat-rye 1BL•1RS translocation line from Petkus rye has contributed substantially to the world wheat production. However, due to extensive growing of cultivars with disease resistance genes from short arm of rye chromosome 1R and coevolution of pathogen virulence and host resistance, these cultivars successively lost resistance to pathogens. In this study, a new wheat-rye line K13-868, derived from the progenies of wheat-rye-Psathyrostachys huashanica trigeneric hybrids, was identified and analyzed using fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH), acid polyacrylamide gel electrophoresis (A-PAGE), and molecular markers. Cytological studies indicated that the mean chromosome configuration of K13-868 at meiosis was 2n=42=0.14 I+18.78 II (ring)+2.15 II (rod). Sequential FISH and GISH results demonstrated that K13-868 was a compensating wheat-rye 1BL•1RS Robertsonian translocation line. Acid PAGE analysis revealed that clear specific bands of rye 1RS were expressed in K13-868. Simple sequence repeat (SSR) and rye 1RS-specific markers ω-sec-p1/ω-sec-p2 and O-SEC5'-A/O-SEC3'-R suggested that the 1BS arm of wheat had been substituted by the 1RS arm of rye. At the seedling and adult growth stage, compared with its recurrent wheat parent SM51 and six other wheat cultivars containing the 1RS arm in southwestern China, K13-868 showed high levels of resistance to stripe rust (Puccinia striiformis f. sp. tritici, Pst) pathogens prevalent in China, which are virulent to Yr10 and Yr24/Yr26. In addition, K13-868 expresses higher thousand-kernel weight and more grain number per spike than these controls in two growing seasons, suggesting that this line may carry yield-related genes of rye. This translocation line, with significant characteristics of resistance to stripe rust and high thousand-kernel weight and grain number per spike, could be utilized as a valuable germplasm for wheat improvement.

Biallelic losses of 13q do not confer a poorer outcome in chronic lymphocytic leukaemia: analysis of 627 patients with isolated 13q deletion

Losses in 13q as a sole abnormality confer a good prognosis in chronic lymphocytic leukaemia (CLL). Nevertheless, its heterogeneity has been demonstrated and the clinical significance of biallelic 13q deletions remains controversial. We compared the clinico-biological characteristics of a series of 627 patients harbouring isolated 13q deletions by fluorescence in situ hybridization (FISH), either monoallelic (13q × 1), biallelic (13q × 2), or the coexistence of both clones (13qM). The most frequent 13q deletion was 13q × 1 (82·1%), while 13q × 2 and 13qM represented 8·6% and 9·3% of patients respectively. The median percentage of altered nuclei significantly differed across groups: 55%, 72·5% and 80% in 13q × 1, 13q × 2 and 13qM (P < 0·001). However, no significant differences in the clinical outcome among 13q groups were found. From 84 patients with sequential FISH studies, eight patients lost the remaining allele of 13q whereas none of them changed from 13q × 2 to the 13q × 1 group. The percentage of abnormal cells detected by FISH had a significant impact on the five-year cumulative incidence of treatment and the overall survival, 90% being the highest predictive power cut-off. In conclusion, loss of the remaining 13q allele is not enough to entail a worse prognosis in CLL. The presence of isolated 13q deletion can be risk-stratified according to the percentage of altered cells.

Molecular cytogenetic monitoring from CD34+ peripheral blood cells in myelodysplastic syndromes: First results from a prospective multicenter German diagnostic study

The gold standard of cytogenetic analysis in myelodysplastic syndromes (MDS) is conventional chromosome banding (CCB) analysis of bone marrow (BM) metaphases. Most aberrations can also be detected by fluorescence-in situ-hybridization (FISH). For this prospective multicenter German diagnostic study (www.clinicaltrials.gov: #NCT01355913) 360 patients, as yet, were followed up to 3 years by sequential FISH analyses of immunomagnetically enriched CD34+ peripheral blood (PB) cells using comprehensive FISH probe panels, resulting in a total number of 19,516 FISH analyses. We demonstrate that CD34+ PB FISH correlates significantly with CCB analysis and represents a feasible method for a reliable non-invasive cytogenetic monitoring from PB.

Accumulation of numerical and structural chromosome imbalances in spermatozoa from reciprocal translocation carriers

Is there a relationship between the occurrence of specific segregation modes and the production of additional numerical abnormalities in spermatozoa from reciprocal translocation carriers? The production of aneuploid and diploid spermatozoa tends to be associated with an unbalanced segregation outcome of the rearranged chromosomes. Carriers of reciprocal translocations have an increased genetic reproductive risk as a consequence of producing higher numbers of unbalanced spermatozoa. These imbalances can originate during the segregation of the rearranged chromosomes and also from the occurrence of interchromosomal effects (ICEs). Usually, the outcome of both events is studied independently by means of sperm fluorescent in situ hybridization (FISH). We designed a sequential FISH protocol based on two successive hybridization rounds to study the segregation outcome of the rearranged chromosomes and the presence of additional numerical abnormalities in the same sperm nuclei. The study was performed between February 2010 and February 2012. Sperm samples from eight reciprocal translocation carriers were processed for FISH analysis. Numerical abnormalities for chromosomes X, Y, 13, 18 and 21 were evaluated in the first hybridization round. The aneuploid and diploid nuclei were relocated and analysed for the segregation outcome of the rearranged chromosomes in the second hybridization round. In every carrier, another population of non-selected spermatozoa was also analysed with the aim of defining the general segregation outcome of each reorganization event. Overall, the selected population of aneuploid and diploid spermatozoa showed significant increased frequencies of unbalanced segregation modes of the rearranged chromosomes (3:1, 4:0 and 'other') when compared with the non-selected population of spermatozoa. A P-value of <0.05 was chosen to determine if differences observed were statistically significant. FISH only allows the analysis of a limited number of chromosomes. Information about the content of additional chromosomes would have been useful in order to broaden the number of aneuploid spermatozoa population, and to infer a more accurate possible mechanism for generating chromosomal imbalances. There was no previous data about a relationship between chromosomal numerical abnormalities and segregation of rearranged chromosomes. Our findings are consistent with a possible gathering of chromosomal abnormalities in a given nucleus. This information can be used towards a better understanding of the meiotic mechanisms involved in non-disjunction events in gametes from reciprocal translocation carriers. Also, it would help to provide a better reproductive genetic risk assessment in these patients. This work was supported by funding of projects SAF2010-2241 (Ministerio de Ciencia e Innovación, Spain), SGR2009-282 (Generalitat de Catalunya, Spain) and UAB CF-180034 (Universitat Autònoma de Barcelona, Spain). The authors declare the lack of competing interests in this study.

Clonal Evolution Predicts Poor Prognosis in Chronic Lymphocytic Leukaemia and Is Possibly a Worse Prognostic Factor Than 17p Deletion: A Population Based Cohort study of FISH Testing in British Columbia, Canada

AbstractAbstract 1401▪▪This icon denotes a clinically relevant abstract Introduction:CLL has been considered a genetically stable disease; however, recent evidence suggests that clonal evolution (CE) occurs at a higher rate than previously known. The clinical significance of CE in terms of CLL progression, requirement for and response to therapy (tx), overall (OS) and treatment-free survival (TFS) is not clear. The relationship between CE and tx and to the pathophysiology of CLL is also currently unknown. There is a need for improved understanding of CE in CLL and its possible impact on prognosis, tx, and outcomes. We performed a population-based analysis of all patients (pts) referred for CLL fluorescence in situ hybridization (FISH) testing in BC, Canada who had 32 FISH studies done to determine the incidence of CE and its association with clinical outcomes. Methods:FISH testing has been standard of care for CLL pts in BC since 2004 and includes minimally evaluation of 4 prognostically significant recurrent cytogenetic (CG) abnormalities (abn): deletion 13q (13q-), deletion 11q (11q-), trisomy 12 (+12) and 17p- as well as IGH abn in a pt subset. Complete clinical and pathological data has been systematically retrospectively collected on all pts who have had CLL FISH testing performed. We included all pts who had 32 sequential CLL FISH tests 33 months apart from 2004-Dec 2011. Primary endpoints were (a) incidence of CE and (b) overall survival. Results:Of 861 pts screened, 134 met inclusion criteria. 21 pts (16%) had CE with median (med) age 57 yrs (range 45–80) at dx and 63 yrs (49–83) at time CE detected; 16 (77%) were male; Rai stage was low in 7 pts (33%), int in 11 (52%) and high in 3 (14%). At med follow-up of 7 yrs (1–37), 19/21 (91%) of CE pts and 99/113 (88%) non-CE pts received tx. Tx included nucleoside analog (NA) in 3 (14%) vs 23 (20%) pts, NA + rituximab (R) in 8 (38%) vs 53 (47%) pts, chlorambucil in 5 (24%) vs 14 (12%) pts and cyclophosphamide based in 3 (14%) vs 8 (7%) pts in the CE and non-CE groups respectively. 9 (43%) CE vs 36 (32%) non-CE pts underwent stem cell transplant (HSCT). 2 untx pts developed CE. CG abns detected on initial FISH test, classified by Dohner hierarchy were: 13q- (32%), +12 (22%), 17p- (12%), 11q- (11%), none of the 4 abns (23%). A total of 28 abns defining CE were detected in the 21pts and the most common were: 11q- (10 pts, 48%), 13q (10 pts, 47%). Predictors of CE on univariate analysis included lymphocyte count at dx (OR 1.01 per increase in lymphs by 1 ×109/L, 95% CI 1.00–1.02, p=0.048) and presence of 13q- at 1st FISH testing (OR 0.4, 95% CI 0.14–0.98, p=0.047); however these were not predictive of CE on multivariate analysis (MVA) after controlling for covariates (p=0.13 and p=0.19 respectively). No other baseline characteristics were predictive for CE including gender (p=0.31), age (p=0.38), WBC at dx (p=0.07), and CD38 positivity (p=0.56). OS was markedly reduced in pts who developed CE compared to those who did not: HR 5.9, median OS 8.2 yrs vs not reached, p=<0.001 (Fig 1). On MVA, CE retained its negative effect with HR 6.3 (95% CI 2.2–17.6), p<0.001, after adjusting for sex, age, Rai stage and baseline CG abn. [Display omitted] [Display omitted] Conclusion:CE is more common than previously recognized and carries a poor prognosis. In this study pts with CE fared worse than those with 17p-. OS of pts with CE in this cohort is in keeping with the med OS for 11q- and 17p- reported in the literature, indicating that presence of CE signifies a new poor prognostic indicator. These observations should prompt FISH testing in all pts at diagnosis and relevant time points in follow-up, such as with change in clinical behaviour and prior to new tx. Furthermore, as CE was thought to be tx related, the fact that 2 untx pts developed CE is a novel finding and provides further evidence of the clinical importance of CE in CLL. It is unclear whether CE pts should be considered for more aggressive tx, including HSCT. Results of our study provide a better understanding of the relationship of CE and disease progression in CLL, allowing for improved pt management and tx of this clinically heterogeneous and incurable disease. Disclosures:No relevant conflicts of interest to declare.