For Fluorescence In Situ Hybridization Research Articles

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

Abstract PS09-07: SPAG5 as a companion prognostic and predictive test for management of early stage HER2 positive breast cancer (BCs)

Abstract Background: SPAG5 is a novel oncogene that is amplified in 40% of HER2+BC. Herein, we evaluated the prognostic and predictive significance of SPAG5 in HER2+ BC Methods: This retrospective cohort study included 2454 patients with HER2+BC derived from 6 cohorts in adjuvant (Adj) and neoadjuvant (NACT) settings. Adj cohorts: The association between SPAG5 and survivals were evaluated in 1354 HER2+ BC who received Adj therapy: 1) HER2+ Adj transcript cohort (n=448): 31% (137/448) did not receive Anthracycline (AC), Taxane (TX), Trastuzumab (TZ) or Pertuzuman (PZ) whereas 20% (89/448) had received AC alone and 35% (155/448) received TZ + AC +/- TX (Median follow- up (MFU): 60 months (m). 2) HER2+ Adj Molecular Taxonomy of BC International Consortium cohort (n=240): patients with ER- and lymph node (LN) + received Adj chemotherapy but none received TZ (MFU: 109 m). 3) HER2+ Adj Historical cohort (n=206): no TZ, AC or TX was received (MFU: 143 m). 4) HER2+/ER- cohort (n=101): AC alone was received; MFU: 62 m. 5) HER+ Adj TZ cohort (n=359): patients received TZ +TX+/-AC (MFU: 90 m). Neo-Adjuvant cohort: the associations of SPAG5 protein expression with pathological complete response (pCR) was evaluated in 1100 BCs (MFUT: 67 m) including 200 local cases participated in ROSCO trial. In this cohort; 73% (803/1100) received AC+TX. AC alone was given to 187/1100 (17%) whereas 88/1100 (8%) received TX alone. Of HER2+ BC, 14% (54/399) received AC +/- TX and 63% received TZ + TX +/-AC and 23% (92/399) received TZ + PZ + TX +/- AC. Sections were IHC profiled for HER2, ER, PR, Ki67 and SPAG5 and stained for Fluorescent in situ hybridisation (FISH) using a novel triple colour probe (HER2/SPAG5/Ch17). ASCO guidelines recommendations were followed. Results: In univariate survival analysis, High SPAG5 (+) transcript was associated with shorter 5- year distant metastases free survival (5-y DRFS); p< 0.0001, compared to SPAG5 low (-) in HER2+ patients who did not receive TZ or AC whereas in those who received AC alone, SPAG5+ was associated with lower risk of 5-y DRFS than SPAG5- (p < 0.001). Multivariable Cox regression analysis showed that SPAG5+ transcript ; as well as LN status, was independently associated with poor prognosis (HR (95% CI): 4.9 (2.0-12.0), p< 0.001). The interaction term between SPAG5 and AC was significantly associated with lower risk of DR. Multivariable Cox regression analysis for 10-year relapse free survival (10y-RFS) in HER2+ patients who did not receive AC or TZ confirmed that SPAG5+ protein expression ; as well as LN status and tumour size, were independently associated with poor prognosis (HR (95% CI): 2.1 (1.3, 3.5), p=0.002). SPAG5+ predicted response for NACT +/- HER2 targeting agents. For instance; None of HER2 IHC 2+ with SPAG5- expression/(0/191) either with HER2 FISH- (0/121) or HER2 FISH+ (0/70) achieved pCR whereas 51% (48/95) of those of HER2 IHC 2+/SPAG5+ achieved pCR (p < 0.0001). Similarly patients with HER2+ IHC 3+/SPAG5+ achieved higher pCR rate compared to those with SPAG5- expression [73% (107/146) vs., 10% (14/135); respectively]; p< 0.0001. Noteworthy, after receiving TZ+ PZ +Texans +/- AC, patients with SPAG5+ achieved higher pCR rate compared to those with SPAG5- expression [85% (50/59) vs., 0% (0/51); respectively, p< 0.001]. Similarly in HER2 + BC patients who received TZ + TX +/- AC, SPAG5+ expression was associated with higher pCR [67% (67/101)] compared to those with SPAG5- [10% (11/107)]; p< 0.001. In HER2+ who received AC alone, SPAG5+ cases achieved higher pCR compared to those with SPAG5- [56% (20/36) vs., 6% (3/47); p< 0.001; respectively]. Multivariable logistic regression analysis showed that SPAG5+ expression as well as ER, HER2 IHC, grade, clinical TNM and HER2 targeting agents were independent predictors for pCR (OR (95% CI: 13.74 (5.76-32.79; p< 0.001). Conclusions SPAG5 is an independent poor prognostic factor in HER2+ BC and could help in distinguishing those who would and would not benefit from NACT and HER2 targeting agents. Citation Format: TAREK ABDEL-Fatah, Graham Ball, Daniel Yeo, Graham Hickman, Arlene Chan, Ian Ellis, Stephen Chan. SPAG5 as a companion prognostic and predictive test for management of early stage HER2 positive breast cancer (BCs) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS09-07.

Prenatal diagnosis of Freeman-Sheldon syndrome using ultrasound and genetic testing. Case report

To describe a case of prenatal diagnosis of Freeman-Sheldon syndrome based on ultrasound findings and complete fetal exome sequencing. A 33-year-old patient currently on treatment for hypothyroidism in whom a 19-week detailed anatomical ultrasound scan showed fetal deformities in more than two body areas (upper and lower limbs), suggesting a diagnosis of arthrogryposis. Genetic counseling was provided and amniocentesis was performed at 20 weeks for fluorescence in situ hybridization (FISH) analysis and complete fetal exome sequencing, with the latter allowing the identification of a heterozygous pathogenic variant of the MYH3 gene which is associated with type 2A distal arthrogryposis. Complete fetal exome sequencing was a key factor in identifying the MYH3 gene mutation and confirmed that the deformities seen on ultrasound were associated with type 2A distal arthrogryposis. It is important to perform complete fetal exome sequencing in cases of joint malformations seen on prenatal ultrasound.

Characterization of DNA Degrading Microorganisms from Dewar Creek Hot Springs in Western Canada

Historically, discovery and subsequent characterization of microbial species relied on pure cultures. Some challenges associated with creating pure cultures have been overcome with advances in culture independent and DNA-based molecular methods such as single-cell genomics, metagenomics, or large scale amplicon sequencing. With these advances, the rate of discovery of new species from genomic data has quickly outpaced the number of organisms with cultured representatives. As description and characterization still rely on cultures, our understanding of yet uncultured species is greatly lacking. Major lineages in the bacterial domain equivalent to phyla that lack any cultured representatives are termed “candidate phyla.” Candidate phyla are found across the bacterial tree of life, and many uncultured organisms are found to be dominant in understudied environments. Extreme environments such as thermal springs are an example of understudied environments, making them excellent environments for studying novel microbial lineages. The objective of this research is to characterize uncultured bacterial lineages from Dewar Creek hot spring in Western Canada, with a focus on DNA and protein metabolizing bacteria. Based on previous genomic data from organisms in this hot spring, we hypothesize that the candidate phylum S2R-29, with extremely low GC content, metabolizes extracellular DNA or protein Dewar Creek is a thermal spring located in the Purcell Wilderness Conservancy in British Colombia, Western Canada. It is one of Canada’s hottest springs, reaching temperatures of up to 83˚C. Samples of DNA extracted from the Dewar Creek hot spring were PCR-amplified with primers for the v3v4 region of the 16S rRNA gene to detect S2R-29, and then sequenced on an Illumina Miseq. S2R-29 was found in samples ranging in temperature from 60˚C to 77 ˚C. In addition to samples from Dewar Creek, samples from other thermal springs in Canada, as well as samples from springs in New Zealand are also being sequenced with the same primers to determine the prevalence of this candidate phylum in other similar environments. This will give a better idea of the growth conditions and range of this organism. Previously, S2R-29 single amplified genomes (SAGs) were generated from Dewar Creek samples. Analysis of these SAGs suggests that S2R-29 has the potential to use peptides and DNA as carbon sources (Fig. 1). To test the potential to metabolize DNA and protein, enrichments of samples with 13C labelled dNTPs or 13C labelled protein have been started. These enrichments will be used for stable isotope probing (SIP) to determine if any organisms in Dewar Creek are metabolizing dNTPs or protein. In addition to SIP, primers specific for S2R-29 for quantitative PCR (qPCR) have been designed and will be run on the enrichments to determine if there are any changes in the abundance of S2R-29 over time in these enrichments, further testing the metabolic potential of these organisms. Finally, probes for fluorescence in situ hybridization (FISH) will be designed for S2R-29. These will be used to perform FISH with samples from Dewar Creek in order to visualize this candidate phylum. With this research, we hope to characterize this novel phylum, and possibly discover other novel lineages of DNA- and protein- metabolizing bacteria in Dewar Creek. This research will help better understand these processes in understudied environments as well as aid our understanding of the roles that bacterial metabolisms might play in biogeochemical cycles such as biodegradation of organic matter. In all, we hope that this research gives insight into the kinds of lifestyles bacteria have evolved in order to thrive in high temperature environments.

Translational practice of fluorescence in situ hybridisation to identify neuroblastic tumours with TERT rearrangements.

Recently, telomerase reverse transcriptase (TERT) gene rearrangements have been identified in neuroblastoma (NB), the typical pathological type of neuroblastic tumours (NTs); however, the prevalence of TERT rearrangements in other types of NT remains unknown. This study aimed to develop a practical method for detecting TERT defects and to evaluate the clinical relevance of TERT rearrangements as a biomarker for NT prognosis. A TERT break-apart probe for fluorescence in situ hybridisation (FISH) was designed, optimised, and applied to assess the genomic status of TERT in Chinese children with NTs at the Beijing Children's Hospital from 2016 to 2019. Clinical, histological, and genetic characteristics of TERT-rearranged NTs were further addressed. Genomic TERT rearrangements could be effectively detected by FISH and were mutually exclusive with MYCN amplification. TERT rearrangements were identified in 6.0% (38/633) of NTs overall, but 12.4% (31/250) in high-risk patients. TERT rearrangements identified a subtype of aggressive NTs with the characteristics of Stage 3/4, high-risk category, over 18 months old, and presenting all histological subtypes of NB and ganglioneuroblastoma nodular. Moreover, TERT rearrangements were significantly associated with elevated TERT expression levels and decreased survival chances. Multivariable analysis confirmed that it was an independent prognostic marker for NTs. FISH is an easily applicable method for evaluating TERT defects, which define a subgroup of NTs with unfavourable prognosis. TERT rearrangements would contribute to characterising NT molecular signatures in clinical practice.

A unified pipeline for FISH spatial transcriptomics

High-throughput spatial transcriptomics has emerged as a powerful tool for investigating the spatial distribution of mRNA expression and its effects on cellular function. There is a lack of standardized tools for analyzing spatial transcriptomics data, leading many groups to write their own in-house tools that are often poorly documented and not generalizable. To address this, we have expanded and improved the starfish library and used those tools to create PIPEFISH, a semi-automated and generalizable pipeline that performs transcript annotation for fluorescence in situ hybridization (FISH)-based spatial transcriptomics. We used this pipeline to annotate transcript locations from three real datasets from three different common types of FISH image-based experiments, MERFISH, seqFISH, and targeted in situ sequencing (ISS), and verified that the results were high quality using the internal quality metrics of the pipeline and also a comparison with an orthogonal method of measuring RNA expression. PIPEFISH is a publicly available and open-source tool.

Abstract A049: Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer

Abstract Introduction: ETS gene rearrangements involving ERG, ETV1 and ETV4, as well as PTEN deletions are among the most common genomic alterations in prostate cancer. Although these events are found to be mutually exclusive, the multifocality of prostate cancer suggests that they may co-occur in some tumors. The immunohistochemistry (IHC) detection of both ERG overexpression and PTEN protein loss are reliable substitutes for fluorescence in situ hybridization (FISH) assay to probe genomic alterations. The lack of specific antibodies that can discriminate between multiple ETS factors, however, has limited the evaluation for the co-occurrence of these genomic alterations in prostate cancer. Improving our ability to identify the multifocality or inter-focal heterogeneity of tumors can enhance our understanding of biological mechanisms of tumor initiation and progression, and help stratify patients for treatment. Methods: Using monoclonal antibodies (mAbs) that we developed against ETV1 and ETV4, we examined their expression in relation to ERG and PTEN expression by IHC using two TMAs constructed from primary prostate cancer specimens of independent patient cohorts: one was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men; the other, from a single 2 mm core of individual tumors from a cohort of 152 AA and 304 CA men. Positive expression for each ETS factor and PTEN were evaluated for association with race and clinico-pathologic features that include PSA at diagnosis, Gleason score, pathologic T stage, biochemical recurrence, and metastasis. Univariate analysis of BCR free survival as a function of ERG, ETV1 and ETV4 and PTEN expression was performed for both patient cohorts. Results: ERG(+) expression alone or in combination with PTEN loss were significantly higher in CA men in both patient cohorts. When expression of ETS factors were evaluated together with PTEN status, positive expression of ETS factors and PTEN loss were significantly more prevalent among CA men in both patient cohorts ETV1 and ETV4 expression were detected at 5% or less in both patient cohorts. Cases harboring tumor foci that were positive for more than one marker evaluated frequently showed either intra-tumor or inter-tumor heterogeneity. Univariate analysis of PTEN-ETS protein expression status and BCR free survival showed that patients with loss of PTEN expression trends towards poorer BCR free survival regardless of ETS expression status. Conclusion: We showed that the IHC detection of multiple aberrantly expressed proteins such as ERG, ETV1, ETV4 and PTEN in prostate tumor specimens using highly specific mAbs can reveal the heterogeneity and multi-focality in prostate cancer, which could be used to enhance the stratification of patients for novel treatment strategies. Citation Format: Shyh-Han Tan, Cara Schafer, Denise Young, Jiji Jiang, Yingjie Song, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn. Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A049.

Prognosis of patients with HER2-low breast cancer brain metastasis: A single-institution experience.

e13109 Background: Many patients with breast cancer were reported to suffer from brain metastasis in the advanced setting. Traditionally, the HER2-low entity was classified as HER2-negative when evaluating prognosis and administrating treatment in patients with breast cancer brain metastasis (BCBM). Trastuzumab deruxtecan (T-DXd) was proved to have potent efficacy in HER2-low breast cancer, even with brain metastasis, which made this subtype a distinct entity. However, the evidence regarding HER2-low BCBM is scarce. Methods: Based on the clinical database of our institution, the BCBM patients with available HER2 status between January 2010 and July 2021 were selected. The enrolled patients with HER2 1/2+ in immunohistochemistry and negative for fluorescence in situ hybridization (FISH) were classified as HER2-low. The comparisons were conducted between HER2-low and HER2-zero category. We predefined the primary endpoint as the overall survival (OS) after the diagnosis of BCBM, which were further analyzed basing the status of hormone receptor (HR). The Kaplan–Meier curves with log-rank test and Cox proportional hazards model were utilized to analyze the survival outcomes. Results: A total of 272 cases were included in the database, among which the patients with HER2-zero (n=64) and HER2-low (n=71) subtypes were analyzed eventually. The OS of patients with HER2-low BCBM was better than the HER2-zero counterparts in the overall population (p=0.002) and the HR-negative group (p=0.008), while the similar tendency was not observed in the HR-positive patients. Moreover, the Cox regression analysis demonstrated that the HER2-low status was a significant prognostic factor for the better clinical outcome in the HR-negative patients (p=0.046 for multivariate analysis). Conclusions: The patients with HER2-low BCBM had better prognosis than the HER2-zero counterparts, especially in the HR-positive subgroup. The low expression of HER2 is supposed to be related to the long-term survival of BCBM patients.

No NFATC2 fusion in simple bone cyst of the jaw.

Simple Bone Cysts (SBCs) predominantly occur in long bones and 59% harbour NFATC2 rearrangements. Jaw SBC is rare and was previously referred to as traumatic bone cyst. It can rarely occur in association with cemento-osseous dysplasia (COD). To determine whether jaw SBCs represent the same entity as SBC of the long bones, or if they have a different molecular signature, we collected 48 jaw SBC cases of 47 patients to assess NFATC2 rearrangement. Out of the 48 cases, 36 could be used for fluorescence in-situ hybridization (FISH), of which nine (two of which associated with COD) were successful using an NFATC2 split probe. The remaining cases failed to show adequate FISH signals. All nine cases lacked NFATC2 rearrangement and five of these showed no detectable gene fusions using Archer FusionPlex. In our study, NFATC2 rearrangement is absent in solitary jaw SBC (n = 7) and COD-associated SBC (n = 2). Our findings suggest that SBC presenting in the jaw is molecularly different from SBC in long bones. Future molecular studies may confirm the absence of clonal molecular aberrations in SBC of the jaw which would support a non-neoplastic, reactive origin.

Preparation of Male Meiotic Chromosomes for Fluorescence In Situ Hybridization and Immunodetection with Major Focus on Dogroses.

Meiosis is a very essential cell division resulting in the formation of four haploid gametes in plants. The preparation of meiotic chromosomes is a key step in plant meiotic research. Well-spread chromosomes, low background signal, and effective cell wall elimination give the best hybridization results. Dogroses (Rosa, section Caninae) are allopolyploids and frequently pentaploids (2n=5x=35) with asymmetrical meiosis. Their cytoplasm is enriched with organic compounds such as vitamins, tannins, phenols, essential oils, and many more. The cytoplasm is often a huge problem, avoiding successful cytogenetic experiments using fluorescence staining techniques. Here, we present a protocol with modifications for the preparation of male meiotic chromosomes suitable for fluorescence in situ hybridization (FISH) and immunolabeling with a major focus on dogroses.

Development and applications of a collection of single copy gene-based cytogenetic DNA markers in garden asparagus.

Garden asparagus (Asparagus officinalis, 2n = 2x = 20 chromosomes) is an important dioecious vegetable crop and a model species for studying sex chromosome formation and evolution. However, few molecular cytogenetic studies on garden asparagus have been reported because of its small metaphase chromosomes, the scarcity of distinguished cytogenetic markers, and the high content of repetitive sequences. In this study, a set of single copy genes free of repetitive sequences with sizes ranging from 4.3 kb to 8.2 kb were screened and used as probes for fluorescence in situ hybridization (FISH) to identify individual chromosomes of garden asparagus. The chromosome-specific signal distribution patterns of these probes enabled the distinguishment of each pair of chromosomes. The sequence assembly and cytogenetic map were successfully integrated, and the results confirmed that the chromosome 1 representing the sex chromosome in the genome assembly is chromosome 5 in the karyotype analysis. The cytogenetic identification of the male-specific region of the Y chromosome (MSY) was implemented using a mixed probe derived from a number of MSY-specific single copy sequences. In addition, the chromosome orthologous relationship between garden asparagus (A1–A10, karyotypic analysis) and its hermaphrodite close relative, A. setaceus (B1–B10, karyotypic analysis), was analyzed using this collection of chromosome-specific cytological markers. The results showed that B3 is the ortholog of sex chromosome A5 and thus may represent the ancestral autosome of the current sex chromosome in garden asparagus. Chromosomes B5, B4, B1, B8, B7, and B9 are the orthologs of A2, A3, A4, A7, A8, and A10, respectively. The chromosome identification, cytogenetic recognition of MSY, and the orthologous relationship analysis between garden asparagus and A. setaceus are valuable for the further investigation of the sex chromosome emergence and evolutionary mechanism of garden asparagus and genome structure evolution in the Asparagus genus.

Obtaining Polytene, Meiotic, and Mitotic Chromosomes from Mosquitoes for Cytogenetic Analysis.

Chromosome visualization is a key step for developing cytogenetic maps and idiograms, analyzing inversion polymorphisms, and identifying mosquito species. Three types of chromosomes-polytene, mitotic, and meiotic-are used in cytogenetic studies of mosquitoes. Here, we describe a detailed method for obtaining high-quality polytene chromosome preparations from the salivary glands of larvae and the ovaries of females for Anopheles mosquitoes. We also describe how to obtain mitotic chromosomes from imaginal discs of fourth-instar larvae and meiotic chromosomes from the testes of male pupae for all mosquitoes. These chromosomes can be used for fluorescence in situ hybridization (FISH), a fundamental technique in cytogenetic research that is used for physical genome mapping, detecting chromosomal rearrangements, and studying chromosome organization.

Diversity and Ecophysiology of the Genus OLB8 and Other Abundant Uncultured Saprospiraceae Genera in Global Wastewater Treatment Systems.

The Saprospiraceae family within the phylum Bacteroidota is commonly present and highly abundant in wastewater treatment plants (WWTPs) worldwide, but little is known about its role. In this study, we used MiDAS 4 global survey with samples from 30 countries to analyze the abundance and distribution of members of Saprospiraceae. Phylogenomics were used to delineate five new genera from a set of 31 high-quality metagenome-assembled genomes from Danish WWTPs. Newly designed probes for fluorescence in situ hybridization (FISH) revealed rod-shaped morphologies for all genera analyzed, including OLB8, present mostly inside the activated sludge flocs. The genomes revealed potential metabolic capabilities for the degradation of polysaccharides, proteins, and other complex molecules; partial denitrification; and storage of intracellular polymers (glycogen, polyphosphate, and polyhydroxyalkanoates). FISH in combination with Raman microspectroscopy confirmed the presence of intracellular glycogen in Candidatus Brachybacter, Candidatus Parvibacillus calidus (both from the former genus OLB8), and Candidatus Opimibacter, and the presence of polyhydroxyalkanoates in Candidatus Defluviibacterium haderslevense and Candidatus Vicinibacter. These results provide the first overview of the most abundant novel Saprospiraceae genera present in WWTPs across the world and their potential involvement in nutrient removal and the degradation of macromolecules.

Qualitative investigation of µ- and κ-opioid receptor distribution in the brains of budgerigars (Melopsittacus undulatus).

To perform a qualitative analysis of the distribution of µ- and κ-opioid receptor mRNA in the forebrain and midbrain of budgerigars (Melopsittacus undulatus). 8 brains of male budgerigars. Custom-made RNA hybridization probes (RNAscope; Advanced Cell Diagnostics Inc) were used for fluorescent in situ hybridization (FISH) assays performed on selected fresh frozen prepared sections of brain tissue to identify µ- and κ-opioid receptor mRNA. There was κ-opioid receptor mRNA present in the nucleus dorsomedialis posterior thalami, lateral striatum, mesopallium, tractus corticohabenularis et corticoseptalis, griseum et fibrosum, stratum griseum centrale, medial striatum, and area parahippocampalis. There was µ-opioid receptor mRNA present in the stratum griseum centrale, stratum opticum, dorsomedialis posterior thalami, area parahippocampalis, medial striatum, and nidopallium intermedium. Consistent with previous studies in pigeons and domestic chicks, κ-opioid receptors were more abundant than µ-opioid receptors in the samples of the present study. The results of this study may also help explain the hyperexcitability or lack of response that can occur with administration of pure µ-opioid receptor agonists, but not κ-opioid receptor agonists. This study was not quantitative, so further research should endeavor to compare the various regions of the brain using FISH technology.

O-034 Xenotransplantation of ovarian cortex tissue from young girls with Turner Syndrome in a mice model: is normal follicular development possible?

Abstract Study question Does aneuploidy of granulosa and stromal cells affect folliculogenesis of small follicles in girls with mosaic Turner Syndrome (TS)? Summary answer Our results showed that despite high level aneuploidy in granulosa cells of small follicles and stromal cells folliculogenesis was not affected in mosaic TS patients. What is known already In 2018 the TurnerFertility study was initiated to explore if ovarian tissue cryopreservation (OTC) is a viable option for TS patients to preserve their fertility at an early age. In this study, karyotyping of ovarian cells in 7 mosaic TS patients showed that the majority of oocytes had a normal X chromosomal content, while granulosa and stromal cells were aneuploid. Until now, it remains unclear if follicular development is affected, and consequently the success rate of autotransplantation after OTC in girls with TS. Study design, size, duration A collaboration project was initiated between Radboudumc, The Netherlands and UCLouvain, Belgium to assess folliculogenesis of small follicles in TS patients by using a xenograft mice model. Ovarian cortex tissue was obtained from 18 mosaic TS patients and 13 age-matched controls. Participants/materials, setting, methods After unilateral ovariectomy, one fragment of the ovarian tissue was used for research. One part of this fragment was used for fluorescence in situ hybridization (FISH) to determine the X chromosomal content of ovarian cells in non-grafted tissue. The other part was xenografted into severe combined immunodeficient mice. After 5 months, grafts were retrieved and analysed for aneuploidy by FISH. The expression of 6 proteins essential for folliculogenesis was analysed by immunohistochemistry and immunofluorescence. Main results and the role of chance The mean follicle density in ovarian tissue of TS patients before grafting was significantly lower compared to controls. FISH analysis showed that 97% of the oocytes in non-grafted tissue had a normal X chromosomal content. Follicles of TS patients contained mainly or exclusively 45,X granulosa cells, but different levels of X chromosome mosaicism between TS patients and between follicles of the same patient were observed. In total, 12/18 grafts contained follicles after 5 months xenografting. Follicle density of both TS patients and controls decreased significantly after xenotransplantation. Despite the presence of high level aneuploidy in granulosa and stromal cells in the tissue before grafting, secondary and even antral follicles were observed after xenotransplantation. Remarkably, a shift from high to low percentage of 45,X granulosa cells was observed during folliculogenesis. Immunohistochemistry showed that proliferating cell nuclear antigen (PCNA) positive follicles from TS patients increased during grafting to almost 100%. Secretion of anti-Müllerian hormone by granulosa cells was impaired before grafting in peri/postpubertal TS girls, but recovered after grafting. Expression of c-kit receptor and bone morphogenetic protein 15 (BMP15) in peri/postpubertal TS patients remained abnormal after xenotransplantation, while secretion of growth differentiation factor 9 (GDF9) and kit ligand was similar to controls. Limitations, reasons for caution In this study, only ovarian tissue of mosaic TS patients was examined, because the chances of finding ovarian follicles in this subgroup is considerably higher than in other TS patients. Subtle effects of reduced expression of c-kit receptor and BMP15 on folliculogenesis might have gone unnoticed. Wider implications of the findings Small follicles of mosaic TS patients are able to grow to secondary and antral follicles, despite the presence of aneuploid granulosa and stromal cells before grafting. Therefore, OTC could be a realistic option for young mosaic TS patients to preserve their fertility provided that sufficient numbers of follicles are present. Trial registration number NCT03381300

Abstract 3412: Liquid biopsy for t(11;14)/blc2 identification in multiple myeloma

Abstract Background: Multiple myeloma (MM) is a spatially heterogeneous plasma cell malignancy of the bone marrow (BM). Venetoclax is a potent inhibitor of the anti-apoptotic protein BCL2 with evidence in MM that t(11;14) and high BCL2 expression confers greater sensitivity to venetoclax. BM biopsy is required for fluorescent in-situ hybridisation (FISH) detection of t(11;14) and the evaluation of MM cell BCL2 expression, with success being entirely dependent on the quality of the BM sample procured. We hypothesised that a liquid biopsy strategy to enable interrogation of cell-free circulating tumour (ct)DNA and extra-cellular (ex)RNA would represent a more practical and less invasive alternative strategy to BM biopsy for the detection of t(11;14) and the determination of MM cell BCL2 expression, respectively. Methods: Blood and BM were obtained from 24 FISH characterised MM patients - normal karyotype (n=9), t(11;14) (n=11) and t(4;14) (n=4). Plasma derived ctDNA and exRNA was extracted utilising the QIAamp circulating nucleic acid kit and matched PBMC DNA was obtained for germ line controls. BM CD138+ MM cells for DNA and RNA extraction were selected with Miltenyi Biotec CD138 microbeads. A low density custom panel of unique capture probes (SSEL XT HS2, SureSelect Custom Tier 2, Agilent Technology), spanning approximately 10% of the CCND1 and IGH loci involved in t(11;14), was used for sequencing with approximately 5 million reads per sample with adapter sequences trimmed from the reads and aligned to the hg38 using bwa mem algorithm. Structural variant caller, Lumpy was used for identifying translocations, with low confidence reads excluded with a defined Z-score threshold. Droplet digital PCR (ddPCR) for BCL2 was performed on exRNA. Results: The low-density capture seq identified t(11;14) in 6 of 11 (55%) of the CD138+ BM samples, but not in paired germ-line samples nor in the non-t(11;14) cohort BM samples. Interrogation of ctDNA identified t(11;14) in 1 of 11 samples but also in ctDNA from a patient labelled as being t(4;14) positive raising questions about the validity of the FISH attribution. We are now utilising a modification of the assay using ultra-high density capture probes with coverage approaching 100% of the IGH region and higher ctDNA input to detect translocations involving the IGH region on chromosome 14 with any partner chromosome, including translocations undetectable by FISH. BCL2 exRNA transcripts were detected in all patients with a range of 2-120 copies/ml of plasma with 79% of patients classified as ‘high’ BCL2 expression (>40 copies/ml of plasma) compared to 59% of the matched CD138+ BM samples. Conclusion: We provide proof-of-concept of the potential of ctDNA and exRNA for t(11;14) detection and ddPCR quantitation of BCL2 expression, respectively, providing a rationale for further exploration of ctDNA and exRNA liquid biopsy testing as a strategy for identifying patients with t(11;14) and/or high BCL2. Citation Format: Andrew Spencer, Sridurga Mithraprabhu, Jaynish Shah, Kawa Choi, Ashley George, Carlos Hader, Tiffany Khong. Liquid biopsy for t(11;14)/blc2 identification in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3412.

Abstract P3-08-03: HOXB13 is coamplified with HER2 in a subset of breast cancers

Abstract Background: The Breast Cancer Index (BCI) is an algorithmic gene expression-based signature comprised of two functional biomarker panels, the Molecular Grade Index (MGI) and the two-gene ratio, HOXB13/IL17BR. The HOXB13/IL17BR ratio has been shown to predict endocrine response across several different treatment scenarios, and the homeobox factor, HOXB13, is the primary determinant of endocrine benefit and response. Previously, we found that HOXB13 gene expression was positively correlated with HER2 in estrogen receptor-positive (ER+) but not ER-negative (ER-) tumors. To validate these findings, we examined HOXB13 gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. In addition, considering that the HOXB13 gene locus resides 9 megabases from the HER2 locus at chromosome 17q21-22, we examined whether HOXB13 was coamplified with HER2 in HER2-positive breast cancers. Methods: Messenger RNA (mRNA) sequencing (mRNAseq) data from the TCGA breast cancer dataset were used to correlate HOXB13 gene expression with immunohistochemical (IHC)-based HER2 expression in ER+ (ER+PR+, ER+PR-) and ER- (ER-PR+ and ER-PR-) breast cancers. Thresholded copy number data from GISTIC output and mRNAseq data from the TCGA datasets were used to correlate HER2 and HOXB13 gene amplification (at least 4 inferred copies) with HOXB13 gene expression in IHC-based ER+ (n= 562) and ER- (n=163) breast cancer subsets. Formalin-fixed paraffin embedded (FFPE) samples from 80 consecutive HER2-amplified invasive breast carcinomas (64 ER+, 16 ER-) diagnosed between 2010 and 2013 were retrospectively collected from the MGH clinical archives and processed for fluorescence in situ hybridization (FISH) for HER2 (spectrum orange) HOXB13 (spectrum green) and CEP17 (spectrum blue). A ratio of > 2.0 HER2 and HOXB13 to CEP17 signals in at least 60 interphase tumor cell nuclei was considered as amplification of HER2 and HOXB13.Results: In the TCGA breast tumors, HOXB13 gene expression was significantly higher in HER2 positive (by IHC status) tumors in both ER+ (p= 3.7 x10-5) and ER- (p= 4.7 x10-4) tumors compared to HER2-negative samples. In HER2+ TCGA breast tumors, genomic analysis revealed co-amplification of HOXB13 with HER2 in 27% of all tumors, and 25.3% and 31.7% in the ER+ and ER- subsets, respectively. Amplification of the HOXB13 locus alone was observed in 2.4% of HER2-non amplified ER+ and ER- TCGA tumors. In the TCGA cohort, median HOXB13 gene expression was not significantly different when comparing HER2-amplified and HER2/HOXB13 coamplified ER+ (p= 0.33) and ER- (p=0.052) tumors, HER2-amplified and HER2-nonamplified ER+ (p=0.42) and ER- (p=0.67) tumors, and HER2/HOXB13 coamplified and HER2-nonamplified ER+ (p=0.50) and ER- (p=0.11) tumors. Lastly, in the consecutive case series of 80 HER2-amplified breast cancers from MGH, HOXB13 was coamplified in 23% of all tumors, and 23.4% and 18.75% in the ER+ and ER- subsets, respectively. Conclusion: HOXB13 was coamplified with HER2 in a substantial subset of TCGA and MGH primary breast cancers irrespective of ER status. Citation Format: Dennis Sgroi, Maristela Onozato, Irene Mitsiades, Ryan McMullin, Daniel Hicks, Angela Volorio, Esther Rheinbay. HOXB13 is coamplified with HER2 in a subset of breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-08-03.

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

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

Feasibility study for ex vivo fluorescence confocal microscopy (FCM) on diagnostic prostate biopsies.

Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. The method has been well established for the diagnosis of tumours in dermatological specimens. Preliminary results found good feasibility when this technique was used to examine prostate cancer (PCa) specimens. We report on the application of FCM in magnet resonance imaging (MRI)-fused prostate biopsies (10 patients, total number of biopsy specimens: n=121) and compare the results to conventional histology. Specific structures of the prostatic tissue were very well represented in the FCM images comparable to conventional histology. Prostate carcinoma was diagnosed with good sensitivity (79/68%) and high specificity (100%) by two pathologists with substantial/almost perfect levels of agreement with the results of conventional histology (kappa 0.79/0.86). Depending on the quality of the scans, malignant lesions of 1.8 mm and more in diameter were reliably diagnosed. Smaller lesions were rated as suspect for malignancy, but could not be consistently differentiated from reactive changes. Optimal image qualities were achieved in focus depths of up to 50 µm, whereas deeper scans led to insufficient representation of cytological features. Pre-treatment with acridine orange (AO) did not alter immunoreactivity of the tissue or its feasibility for fluorescence in situ hybridization (FISH) analyses and adequate amounts of DNA could be extracted for further polymerase chain reaction (PCR)-based examinations. FCM seems to be a promising tool for the timely diagnosis in cases of PCa in patients requiring therapy. In particular, this technique is a material-sparing method that conserves the biopsies as unfixed material for further analysis such as molecular tumour companion diagnosis.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds.

Simple SummaryAnopheles darlingi mosquitoes are the main vectors of malaria in the Brazilian Amazon. To assign genomic DNA sequences to chromosomes of this species, we performed fluorescence in situ hybridization of DNA probes with salivary glands polytene chromosomes. We compared the physical locations of the An. darlingi probes with homologous sequences in other Anopheles species, namely Anopheles albimanus and Anopheles gambiae. The results demonstrated that substantial genome rearrangements occurred throughout the evolutionary history of these mosquitoes. The physical mapping data can be useful for improving the structural accuracy of the An. darlingi genome assembly and for understanding the chromosomal evolution of these mosquitoes.The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.