Discordance between Immunohistochemistry and Erb-B2 Receptor Tyrosine Kinase 2 mRNA to Determine Human Epidermal Growth Factor Receptor 2 Low Status for Breast Cancer.

Novel human epidermal growth factor receptor 2 (HER2)-directed antibody-drug conjugates have demonstrated efficacy in HER2-low expressing breast cancers, which are currently defined as those with immunohistochemistry (IHC) scores of 1+ or 2+ with a negative in situ hybridization assay. However, current HER2 testing methods are designed to identify HER2-amplified tumors with high expression levels. The true definition of HER2-low expressing breast cancers remains controversial. Using quantitative molecular analysis of breast cancers based on RNA expression, the dynamic range of HER2 expression exceeds that detected by in situ IHC approaches. Erb-B2 receptor tyrosine kinase 2 (ERBB2) mRNA expression levels across IHC groups using patient samples derived from the Tamoxifen Exemestane Adjuvant Multicenter Trial were investigated. The standardized mean differences in ERBB2 mRNA scores in log base 2 are 0.47 (95% CI, 0.36-0.57), 0.58 (95% CI, 0.26-0.70), and 0.32 (95% CI, -0.12 to 0.75) when comparing IHC 0+ without staining versus IHC 0+ with some staining, IHC 0+ with some staining versus IHC 1+, and IHC 1+ versus IHC 2+/fluorescence in situ hybridization-negative, respectively. The results showed immunohistochemical methods have a comparatively limited dynamic range for measuring HER2 protein expression. The range of expression based on RNA abundance suggests a molecular method defining HER2-low cancers may better serve the treatment decision needs of this group. Indeed, the validity of RNA abundance to identify HER2-low cancers and predict treatment response needs to be further evaluated by prospective clinical trials.

FISH-Based Karyotype Analyses of Four Dracaena Species.

The genus Dracaena is the main source of dragon's blood, which is a plant resin and has been used as traditional medicine since ancient times in different civilizations. However, the chromosome numbers and karyotypes present in this genus remain poorly understood. In this study, fluorescence in situ hybridization (FISH) using oligonucleotide probes for ribosomal DNAs (5S and 45S rDNA) and telomeric repeats (TTTAGGG)3 was applied to analyze 4 related species: Dracaena terniflora Roxb., Dracaena cambodiana Pierre ex Gagnep., Aizong (Dracaena sp.), and Dracaena cochinchinensis (Lour.) S.C. Chen. In all 4 species, both 5S and 45S rDNA showed hybridization signals in the paracentromeric region of a pair of chromosomes; the sizes of the 45S rDNA signals were larger than those of the 5S rDNA. Importantly, the telomeric repeat signals were located in the telomeric regions of almost all chromosomes. The results indicated that the chromosome number of all 4 Dracaena species is 2n = 40, and the lengths of the mitotic metaphase chromosomes range from 0.99 to 2.98 µm. Our results provide useful cytogenetic information, which will be beneficial to future studies in genome structure of the genus Dracaena.

Integrative analysis of the human brain mural cell transcriptome.

Brain mural cells, including pericytes and vascular smooth muscle cells, are important for vascular development, blood-brain barrier function, and neurovascular coupling, but the molecular characteristics of human brain mural cells are incompletely characterized. Single cell RNA-sequencing (scRNA-seq) is increasingly being applied to assess cellular diversity in the human brain, but the scarcity of mural cells in whole brain samples has limited their molecular profiling. Here, we leverage the combined power of multiple independent human brain scRNA-seq datasets to build a transcriptomic database of human brain mural cells. We use this combined dataset to determine human-mouse species differences in mural cell transcriptomes, culture-induced dedifferentiation of human brain pericytes, and human mural cell organotypicity, with several key findings validated by RNA fluorescence in situ hybridization. Together, this work improves knowledge regarding the molecular constituents of human brain mural cells, serves as a resource for hypothesis generation in understanding brain mural cell function, and will facilitate comparative evaluation of animal and in vitro models.

Pan-Cancer Biomarkers: Changing the Landscape of Molecular Testing.

CONTEXT.­: The increasing use of large panel next-generation sequencing technologies in clinical settings has facilitated the identification of pan-cancer biomarkers, which can be diagnostic, prognostic, predictive, or most importantly, actionable. OBJECTIVE.­: To discuss recently approved and emerging pan-cancer and multihistology biomarkers as well as testing methodologies. DATA SOURCES.­: The US Food and Drug Administration approval documents, National Comprehensive Cancer Network guidelines, literature, and authors' own publications. CONCLUSIONS.­: Since 2017, the US Food and Drug Administration has approved genotype-directed therapies for pan-cancer biomarkers, including microsatellite instability, neurotrophic receptor kinases fusions, and high-tumor mutation burden. Both the importance and rarity of these biomarkers have increased the prevalence of genomic profiling across solid malignancies. As an integral part of the management team of patients with advanced cancer, pathologists need to be aware of these emerging biomarkers, the therapies for which they determine eligibility, and the strengths and pitfalls of the available clinical assays.

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

The hypothetical impact of Accelerate Pheno™ system on time to effective therapy and time to definitive therapy in an institution with an established antimicrobial stewardship programme currently utilizing rapid genotypic organism/resistance marker identification.

Background: Rapid organism identification and antimicrobial susceptibility testing (AST) can optimize antimicrobial therapy in patients with bacteraemia. The Accelerate Pheno™ system (ACC) can provide identification and AST results within 7 h of a positive culture. Objectives: To assess the hypothetical impact of ACC on time to effective therapy (TTET), time to definitive therapy (TTDT) and antimicrobial usage at the Detroit Medical Center (DMC). Methods: Patients with positive blood cultures from 29 March to 24 June 2016 were included. ACC was performed in parallel with normal laboratory procedures, but results were not made available to the clinicians. The potential benefit of having ACC results was determined if clinicians modified therapy based on actual AST results. Potential changes in TTET, TTDT and antibiotic usage were calculated. Results: One hundred and sixty-seven patients were included. The median TTET was 2.4 h (IQR 0.5, 15.1). Had ACC results been available, TTET could have been improved in four patients (2.4%), by a median decrease of 18.9 h (IQR 11.3, 20.4). The median TTDT was 41.4 h (IQR 21.7, 73.3) and ACC results could have improved TTDT among 51 patients (30.5%), by a median decrease of 25.4 h (IQR 18.7, 37.5). ACC implementation could have led to decreases in usage of cefepime (16% reduction), aminoglycosides (23%), piperacillin/tazobactam (8%) and vancomycin (4%). Conclusions: ACC results could potentially improve time to de-escalation and reduce use of antimicrobials. The impact of ACC on TTET was small, likely related to the availability of other rapid diagnostic tests at DMC.

Disponibilidad de técnicas moleculares para niños con tumores de difícil caracterización. Un problema en la Oncopatología pediátrica. Aplicación y difusión de técnicas de diagnóstico complementarias en el ámbito público / Availability of molecular techniques for children with difficult-to-characterize tumors. A problem in pediatric oncopathology. Application and diffusion of complementary diagnostic techniques in the public setting

La revolución de la biología molecular y el desarrollo de la investigación biomédica básica para el diagnóstico y posterior manejo del cáncer infantil han llevado a la necesidad de organización de grupos interdisciplinarios de profesionales, los cuales se encargan de afrontar los nuevos desafíos diagnósticos y terapéuticos. Los sarcomas indiferenciados pediátricos constituyen un grupo heterogéneo de neoplasias malignas de aspecto primitivo y polifenotípico. La categorización de gran parte de este tipo de tumores es posible gracias a la aplicación de técnicas moleculares complementarias al estudio histopatológico. El objetivo del presente estudio fue recategorizar sarcomas indiferenciados mediante la implementación de una nueva metodología diagnóstica. Se efectuaron técnicas de inmunohistoquimica (IHQ), FISH de interfase y RT-PCR a partir de tejido fijado en formol e incluido en parafina en 144 casos de sarcomas indiferenciados. Se logró la recategorización del 95.1% de los casos, arribando a 24 diagnósticos diferentes. Sólo un 4.9% permanece aún como sarcoma indiferenciado o inclasificable. Los resultados alcanzados por este estudio demuestran la importancia de contar con nuevas herramientas diagnósticas a nivel molecular y recursos humanos especializados que posibiliten su correcta implementación para el diagnóstico de neoplasias de difícil caracterización (AU)

The revolution of molecular biology and the development of basic medical research for the diagnosis and subsequent management of childhood cancer have led to a need to organize interdisciplinary groups of professionals in charge of facing new diagnostic and treatment challenges. Childhood undifferentiated sarcomas are a heterogeneous group of malignant neoplasms that are primitive in appearance and have polyphenotypic features. Categorization of a large part of this type of tumor has become possible with molecular techniques as a complement to histopathological studies. The aim of this study was to categorize undifferentiated sarcomas using new diagnostic tools. Immunohistochemistry (IHC), interfase FISH, and RT-PCR techniques were used on formalin-fixed and paraffin-embedded tissues of 144 cases of undifferentiated sarcomas. Overall, 95.1% of the cases could be recategorized resulting in 24 different diagnoses. In only 4.9% the diagnosis of undifferentiated or unclassifiable sarcoma was maintained. These results emphasize the importance of the availability of new diagnostic tools at the molecular level and specialized human resources enabling adequate implementation for the diagnosis of difficult-to-characterize neoplasms (AU)

Visualisation of the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans in co-cultures with micro-algae by CARD-FISH.

Some studies have described the isolation and 16S rRNA gene sequence-based identification of hydrocarbon-degrading bacteria living associated with marine eukaryotic phytoplankton, and thus far the direct visual observation of these bacteria on micro-algal cell surfaces ('phycosphere') has not yet been reported. Here, we developed two new 16S rRNA-targeted oligonucleotide probes, PCY223 and ALGAR209, to respectively detect and enumerate the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). To enhance the hybridization specificity with the ALGAR209 probe, a competitor probe was developed. These probes were tested and optimized using pure cultures, and then used in enrichment experiments with laboratory cultures of micro-algae exposed to phenanthrene, and with coastal water enriched with crude oil. Microscopic analysis revealed these bacteria are found in culture with the micro-algal cells, some of which were found attached to algal cells, and whose abundance increased after phenanthrene or crude oil enrichment. These new probes are a valuable tool for identifying and studying the ecology of P. algicola and A. aromaticivorans in laboratory and field samples of micro-algae, as well as opening new fields of research that could harness their ability to enhance the bioremediation of contaminated sites.

Rapid and Efficient FISH using Pre-Labeled Oligomer Probes.

Fluorescence in situ hybridization (FISH) is used to visualize the distribution of DNA elements within a genome. Conventional methods for FISH take 1-2 days. Here, we developed a simplified, rapid FISH technique using pre-labeled oligonucleotide probes (PLOPs) and tested the procedure using 18 PLOPs from 45S and 5S rDNA, Arabidopsis-type telomere, and newly-identified Panax ginseng-specific tandem repeats. The 16 developed rDNA PLOPs can be universally applied to plants and animals. The telomere PLOPs can be utilized in most plants with Arabidopsis-type telomeres. The ginseng-specific PLOP can be used to distinguish P. ginseng from related Panax species. Differential labeling of PLOPs allowed us to simultaneously visualize different target loci while reducing the FISH hybridization time from ~16 h to 5 min. PLOP-FISH is efficient, reliable, and rapid, making it ideal for routine analysis, especially of newly sequenced genomes using either universal or specific targets, such as novel tandem repeats identified from whole-genome sequencing data.

Identification of fluorescence in situ hybridization assay markers for prediction of disease progression in prostate cancer patients on active surveillance.

BACKGROUND: Prostate Cancer (PCa) is the second most prevalent cancer among U.S. males. In recent decades many men with low risk PCa have been over diagnosed and over treated. Given significant co-morbidities associated with definitive treatments, maximizing patient quality of life while recognizing early signs of aggressive disease is essential. There remains a need to better stratify newly diagnosed men according to the risk of disease progression, identifying, with high sensitivity and specificity, candidates for active surveillance versus intervention therapy. The objective of this study was to select fluorescence in situ hybridization (FISH) panels that differentiate non-progressive from progressive disease in patients with low and intermediate risk PCa. METHODS: We performed a retrospective case-control study to evaluate FISH biomarkers on specimens from PCa patients with clinically localised disease (T1c-T2c) enrolled in Watchful waiting (WW)/Active Surveillance (AS). The patients were classified into cases (progressed to clinical intervention within 10 years), and controls (did not progress in 10 years). Receiver Operating Characteristic (ROC) curve analysis was performed to identify the best 3-5 probe combinations. FISH parameters were then combined with the clinical parameters ─ National Comprehensive Cancer Network (NNCN) risk categories ─ in the logistic regression model. RESULTS: Seven combinations of FISH parameters with the highest sensitivity and specificity for discriminating cases from controls were selected based on the ROC curve analysis. In the logistic regression model, these combinations contributed significantly to the prediction of PCa outcome. The combination of NCCN risk categories and FISH was additive to the clinical parameters or FISH alone in the final model, with odds ratios of 5.1 to 7.0 for the likelihood of the FISH-positive patients in the intended population to develop disease progression, as compared to the FISH-negative group. CONCLUSIONS: Combinations of FISH parameters discriminating progressive from non-progressive PCa were selected based on ROC curve analysis. The combination of clinical parameters and FISH outperformed clinical parameters alone, and was complimentary to clinical parameters in the final model, demonstrating potential utility of multi-colour FISH panels as an auxiliary tool for PCa risk stratification. Further studies with larger cohorts are planned to confirm these findings.

Comparative Oligo-FISH Mapping: An Efficient and Powerful Methodology To Reveal Karyotypic and Chromosomal Evolution.

Developing the karyotype of a eukaryotic species relies on identification of individual chromosomes, which has been a major challenge for most nonmodel plant and animal species. We developed a novel chromosome identification system by selecting and labeling oligonucleotides (oligos) located in specific regions on every chromosome. We selected a set of 54,672 oligos (45 nt) based on single copy DNA sequences in the potato genome. These oligos generated 26 distinct FISH signals that can be used as a "bar code" or "banding pattern" to uniquely label each of the 12 chromosomes from both diploid and polyploid (4× and 6×) potato species. Remarkably, the same bar code can be used to identify the 12 homeologous chromosomes among distantly related Solanum species, including tomato and eggplant. Accurate karyotypes based on individually identified chromosomes were established in six Solanum species that have diverged for >15 MY. These six species have maintained a similar karyotype; however, modifications to the FISH signal bar code led to the discovery of two reciprocal chromosomal translocations in Solanum etuberosum and S. caripense We also validated these translocations by oligo-based chromosome painting. We demonstrate that the oligo-based FISH techniques are powerful new tools for chromosome identification and karyotyping research, especially for nonmodel plant species.

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing.

Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background-from off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.

Study on the homology of the genomes of tetraploid Asiatic lilies (Lilium) using FISH.

Asiatic lily cultivars, bred by hybridization and (or) chromosome doubling of species of section Sinomartagon of Lilium, are diploid, triploid, or tetraploid, but the homology of the genomes among species of section Sinomartagon and Asiatic lilies remains unclear. In the present research, two tetraploid Asiatic cultivars were analyzed, using 45S rDNA as probe, for their FISH karyotypes and their chromosomal association, anaphase I, telophase II, and pollen viability were surveyed to assess the multivalent segregation. Chromosomal assortment of six progenies of the two tetraploid cultivars were also investigated. The results showed that the tetraploid cultivars had similar FISH karyotypes, they predominantly formed multivalents, and these were equally separated because their anaphase I, telophase II, and pollen viability were similar to those of diploid species. Apart from minor variations, FISH karyotypes of progenies were similar to each other and to their parents. Based on these results and considering the high crossability among species of section Sinomartagon and (or) Asiatic lilies, we concluded that species of section Sinomartagon and their resulting cultivars share a common genome; thus, polyploidy Asiatic lilies are autopolyploid.

Clinicopathological features of double-hit B-cell lymphomas with MYC and BCL2, BCL6 or CCND1 rearrangements.

Double-hit (DH) lymphomas are B-cell lymphomas characterized by chromosomal rearrangements, specifically of MYC and either BCL2, BCL6 or CCND1. We reviewed 22 cases of DH lymphomas. BCL2/MYC DH lymphomas constituted the majority of these DH lymphomas (17 cases; 77%), followed by BCL6/MYC (2 cases; 9%) lymphomas. Assessing morphological features using the 2008 World Health Organization classification system, 15 cases (68%) were determined to be B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BCLU) (10 cases; 45%), or as DLBCL (5 cases; 23%), and 2 cases (9%) were classified as morphologically untransformed follicular lymphoma. Burkitt lymphoma was rare (1 case; 5%) among DH lymphomas. Nineteen cases were treated with R-CHOP or a high dose chemotherapy regimen. After a median follow-up of 11 months, 7 patients had died, and the 1-year survival rate was 62.5%. High dose chemotherapy did not improve the outcome. We suggest that screening of genetic variations to detect DH lymphomas is required in diagnosing all lymphomas, even those determined morphologically to be follicular lymphoma.

Tyramide-FISH mapping of single genes for development of an integrated recombination and cytogenetic map of chromosome 5 of Allium cepa.

Chromosome 5 of onion carries major quantitative trait loci (QTL) that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors, all of which are of interest to breeders. SNPs, SSRs, and RFLPs in expressed regions of the onion genome have been genetically mapped, and we used these clones and sequences from the NCBI database to develop DNA probes for in situ hybridization to integrate the genetic and physical maps of onion chromosome 5. We produced genomic amplicons from expressed regions of the onion genome that carried both exons and introns in order to increase the hybridization specificity of the probes and to enlarge the target DNA sizes. Tyramide-FISH technique was used to increase the detection sensitivity of relatively short target DNA regions, which range from 950 to 2100 bp. Through the integration of genetic and chromosomal maps, we were able to estimate the distribution of recombination events along onion chromosome 5. We demonstrated the efficiency of chromosomal in situ mapping of exon-intron genomic clones for the extremely large genome of onion.

Analysis of receptor tyrosine kinase gene amplification on the example of FGFR1.

FISH (fluorescent in situ hybridization) is a molecular cytogenetic method to detect large-scale genetic alterations in tissue and/or cells. Numerical aberrations (deletions and amplifications) and structural aberrations (translocations and fusions) are detectable. Probes bind complementary to the DNA strand of the region of interest. Subsequently, the probes are detected via fluorochromes and appear as colored dots that can be assessed under the fluorescence microscope.In situ hybridization is divided into three steps: pretreatment, hybridization, and posthybridization. Pretreatment opens up the cell membranes for hybridization, so that the probe can bind to the complementary DNA target. Posthybridization includes washing steps to remove excessive probes and detection of probes via secondary marked fluorochromes. DAPI stains nuclei and serves as mounting media.

Application of flow-FISH for dynamic measurement of telomere length in cell division.

This method makes it possible to measure the fluorescence of a DNA probe in cells with known division number and targeted surface antigen. In fact, this method is a combination or consistent application of three other methods: cell tracking by vital dye, surface immunophenotyping, and flow-FISH. The idea in developing this method was to study telomere length changes in cells with known surface antigen after every new cell division. First, the in vitro cell culturing and staining with CFSE vital dye are performed. Then, cells are stained with surface MAbs labeled with biotin, followed by incubation with streptavidin-labeled fluorochrome. After that, cells are fixed with BS(3) reagent followed by the flow-FISH procedure with PNA-probe complementary to telomere DNA repeats. Finally, in one tube, it is possible to determine telomere length in surface antigen-labeled cells that have made the exact same number of divisions after incubation.

Microscopic analysis of chromatin localization and dynamics in C. elegans.

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.

Visualising single molecules of HIV-1 and miRNA nucleic acids.

BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

Cytomolecular identification of individual wheat-wheat chromosome arm associations in wheat-rye hybrids.

Chromosome pairing in the meiotic metaphase I of wheat-rye hybrids has been characterized by sequential genomic and fluorescent in situ hybridization allowing not only the discrimination of wheat and rye chromosomes, but also the identification of the individual wheat and rye chromosome arms involved in the chromosome associations. The majority of associations (93.8%) were observed between the wheat chromosomes. The largest number of wheat-wheat chromosome associations (53%) was detected between the A and D genomes, while the frequency of B-D and A-B associations was significantly lower (32 and 8%, respectively). Among the A-D chromosome associations, pairing between the 3AL and 3DL arms was observed with the highest frequency, while the most frequent of all the chromosome associations (0.113/cell) was found to be the 3DS-3BS. Differences in the pairing frequency of the individual chromosome arms of wheat-rye hybrids have been discussed in relation to the homoeologous relationships between the constituent genomes of hexaploid wheat.