Fluorescence In Situ Hybridization Research Articles

Targeted Therapy in Salivary Gland Cancer: Prevalence of a Selected Panel of Actionable Molecular Alterations in a German Tertiary Referral Center Patient Cohort.

Salivary gland carcinomas (SGC) are a heterogeneous group of malignancies, with 24 subtypes defined by the World Health Organization (WHO). The standard of therapy is surgical resection, with adjuvant radiotherapy in most cases. However, disease recurrence (R) or metastasis (M) is common and no active systemic therapies are currently available for RM-SGC resulting in a 5-year survival rate of only 20%. Overall, 55 SGC patients with seven different histological tumor subtypes were included in this study. formalin-fixed paraffin-embedded (FFPE) tissue samples were used for immunohistochemical (IHC) staining targeting HER2/neu, androgen receptor (AR), PD-L1, EGFR, panTRK, and TROP2. Fluorescence in situ hybridization (FISH) was performed for detecting HER2/neu amplifications and NTRK1/2/3 translocations in selected cases with relevant HER2/neu and panTRK protein expression, respectively. IHC and FISH results were correlated with patients' clinical and histopathological data. The overall prevalence of druggable molecular alterations, defined as an immunoreactive score ≥ 9 in at least one of the analyzed targets, was 54.4% with the highest percentage in oncocytic carcinomas (100%) and lowest percentage in acinic cell carcinomas (10%). EGFR overexpression proved to be the most common alteration (32.7% of cases) followed by overexpression of TROP2 (27.3%), AR (10.9%), HER2/neu (7.3%), PD-L1 (1.8%), and panTRK (1.8%). HER2/neu amplifications were found in 50% and NTRK translocations were found in 100% of all cases with elevated Her2/neu and panTRK protein expression, respectively. Our data indicate that targeted therapy using e.g., trastuzumab deruxtecan, bicalutamide, pembrolizumab, cetuximab, entrectinib or sacituzumab govitecan might be a promising option especially for a relevant subset of patients with RM-SGC not suitable for salvage surgery. However, evidence from clinical studies regarding response rates to these therapies remains sparse, which underlines the need of multicenter clinical trials.

Symbiont Acquisition Strategies in Post-Settlement Stages of Two Co-Occurring Deep-Sea Rimicaris Shrimp.

At deep-sea hydrothermal vents, deprived of light, most living communities are fueled by chemosynthetic microorganisms. These can form symbiotic associations with metazoan hosts, which are then called holobionts. Among these, two endemic co-occurring shrimp of the Mid-Atlantic Ridge (MAR), Rimicaris exoculata and Rimicaris chacei are colonized by dense and diversified chemosynthetic symbiotic communities in their cephalothoracic cavity and their digestive system. Although both shrimp harbor similar communities, they exhibit widely different population densities, distribution patterns at small scale and diet, as well as differences in post-settlement morphological modifications leading to the adult stage. These contrasting biological traits may be linked to their symbiotic development success. Consequently, key questions related to the acquisition of the symbiotic communities and the development of the three symbiotic organs are still open. Here we examined symbiotic development in juveniles of R. exoculata and R. chacei from TAG and Snake Pit using 16S metabarcoding to identify which symbiotic lineages are present at each juvenile stage. In addition, we highlighted the abundance and distribution of microorganisms at each stage using Fluorescence insitu Hybridization (FISH) and Scanning Electron Microscopy (SEM). For the first time, Candidatus Microvillispirillaceae family with Candidatus Rimicarispirillum spp. (midgut tube), Candidatus Foregutplasma rimicarensis and Candidatus BG2-rimicarensis (foregut) were identified in late juvenile stages. However, these lineages were absent in early juvenile stages, which coincides for the midgut tube with our observations of an immature tissue, devoid of microvilli. Conversely, symbiotic lineages from the cephalothoracic cavity were present from the earliest juvenile stages of both species and their overall diversities were similar to those of adults. These results suggest different symbiont acquisition dynamics between the cephalothoracic cavity and the digestive system, which may also involve distinct transmission mechanisms.

SRY+ Derivative X Chromosome in a Female With Apparently Typical Sexual Development.

When the SRY gene is present in a 46,XX fetus, some degree of testicular development is expected. Our laboratory performed prenatal genetic testing for a fetus that had screened positive for Y chromosome material by noninvasive prenatal screening (NIPS) but that had apparently typical female development by ultrasound imaging. The aim of this study was to determine the clinical relevance of the NIPS results. We analyzed fetal material obtained via amniocentesis procedure by G-banding, microarray, and fluorescence insitu hybridization (FISH). Optical genome mapping (OGM) was also performed. G-band analysis revealed a normal 46,XX karyotype. Microarray and FISH analyses together detected an SRY+ gain of 5.7 Mb from terminal Yp that was translocated to terminal Xq, with a loss of 1.6 Mb from terminal Xq. The final karyotype was 46,X,der(X)t(X;Y)(q28;p11.2). Prenatal ultrasound and postnatal physical examination revealed apparently typical female genitalia. The Xq deletion encompassed a gene, IKBKG, that is sensitive to loss of function, suggesting that preferential inactivation of the derivative X chromosome allowed for typical female development. OGM software did not directly identify this translocation. This case demonstrates how the SRY gene may be present in a 46,XX biological female without differences of sexual development.

Multinucleated tumor cells and micropapillary morphology appear to be predictors of poor prognosis in renal cell carcinoma with papillary and oncocytic features

Renal cell carcinoma with papillary and oncocytic features (RCC-PO) are poorly understood, partially due to conflicting results in multiple studies. The histological features that predict behavior of RCC-PO have not been elucidated. The aim is to review clinicopathologic features and to correlate clinical outcomes of patients with RCC-PO to further expand our knowledge on these heterogeneous tumors. An archival search was done for “RCC” and “papillary,” and tumors with >50% papillary and oncocytic features were included. Clinicopathologic data including tumor size, grade, stage, molecular and immunohistochemical testing when performed, and follow-up data were collected. Using multivariate analyses, correlation between histological features, tumor stage and prognosis were analyzed. Sixty-one patients with RCC-PO were identified of which 49 (80%) were male with a median age of 65 (range: 36–93) years, and a mean tumor size of 5.2 (range: 1–21.5) cm. Micropapillary features were seen in 4, bizarre nuclei (at least 3 times larger or with irregular shape) in 6, multinucleated tumor cells (MTC) in 15, single or small clusters (SSC) (made of 2–3 tumor cells) located away from areas of necrosis in 16, and striking eosinophilic cytoplasmic inclusions in 3 tumors, respectively. Thirty-six (59%) tumors were high-grade (WHO/ISUP grade 3–4), and 23 (38%) had a high stage (≥pT3 or pN1). Tumors were positive for AMACR (15/16) and CK7 (13/17), with preserved FH (7/7) staining and were all negative for CD117 (0/7), ALK, TFE3, cathepsin K, Melan A, and HMB45 (0/4, each). Three tumors underwent chromosomal microarray (CMA) plus gene fusion assay, and FISH and germline testing for FLCN and MET gene alterations by PCR were done on 1 each. Ten (16%) patients had a local recurrence (LR) or metastasis after nephrectomy; 4 died of disease (2 had tumors with micropapillary features), with a median follow-up of 7 (range: 0.01–19) years. Tumors with micropapillary features showed significantly higher RCC-PO-related mortality (50% vs. 3.5%, p < 0.001). In multivariable analysis, SSC correlated with a higher stage (HR: 11.95; p = 0.005); micropapillary features (HR: 18.42; p = 0.017) and MTC (HR: 180.22; p = 0.036) with presence of metastasis/LR; and micropapillary features with a higher RCC-PO-related mortality (HR: 60.35; p = 0.036). RCC-PO are cytogenetically heterogeneous with overlapping features of various renal neoplasms. Micropapillary features and MTC appear to be independent predictors of poor outcomes in these tumors.

Molecular and Clinicopathologic Characterization of HER2-overexpressed Squamous Cell Carcinoma of the Cervix.

HER2 amplification in cervical cancer has been associated with worse clinical prognosis and a potential favorable response to HER2 inhibitors. Immunohistochemistry for the HER2 receptor is a universally accepted surrogate test for HER2 amplification, but no standardized scoring system currently exists for cervical carcinomas. In this study, we investigated HER2 overexpression in cervical squamous cell carcinoma and correlated it with HER2 amplification using fluorescence in situ hybridization (FISH) and molecular methods. Seventy-two cases of human papillomavirus-associated cervical cancer were retrospectively reviewed, and at least 2 representative tumor sections were stained for HER2. HER2 scoring was performed using the 2018 American Society of Clinical Oncology/College of American Pathologist breast cancer criteria, and cases with equivocal (2+) to positive (3+) expression were analyzed for HER2 amplification using FISH and next-generation sequencing. The average patient age was 50yrs (range: 27-85yr), with most patients being African American (73.6%) and diagnosed at FIGO stage I (65.3%). Nineteen (26.4%) had equivocal HER2 expression and 4 (5.5%) showed positive expression. Three of the 4 cases with positive expression had enough tumors for FISH, and all 3 were amplified. Three cases with equivocal expression showed HER2 polysomy on FISH, and none showed HER2 amplification. Late clinical stage, high tumor grade, and regional lymph node metastasis were significantly correlated with HER2 overexpression and HER2 amplification. Next-generation sequencing of the 3 HER2-amplified tumors showed amplification of various genes, including CD274, JAK2, BIRC3, and ERBB2, and a PIK3CA missense mutation. In summary, HER2 immunohistochemistry is a reliable predictive marker of HER2 amplification in cervical cancer.

Prognostic implications and diagnostic significance of TFE3 rearrangement in renal cell carcinoma

ObjectivesTo investigate the impact of TFE3 rearrangement, analyzing clinicopathological features that influence renal cell carcinoma (RCC) recurrence, and clarify the role of immunohistochemistry (IHC) staining in diagnosis.MethodsWe screened patients diagnosed of clear cell RCC (ccRCC), fluorescence in situ hybridization (FISH) was performed on all TFE3 positive IHC tumors. Clinicopathological and survival features were collected for analysis.ResultsOut of 695 patients treated for renal tumors, 478 (68.7%) were ccRCC and 22 were suspected of TFE3 rearrangement based on IHC. Subsequent testing revealed 8 (1.15%) were positive in the FISH test (TFE3-rearranged-RCC) and 14 (2.01%) tested negative. No significant differences were noted in general characteristics among the three groups, except for age, TFE3-rearranged-RCC were younger than ccRCC (median age, 49 vs. 58 years, p=0.02). TFE3-rearranged-RCC exhibited a significant higher recurrence rate compared to ccRCC (50% vs 18.8%) and multivariate analysis revealed that TFE3 rearrangement, along with tumor size and metastasis, was an independent prognostic factor for recurrence (HR=4.6; 95% CI 1.1-21.2; p=0.05). Survival analysis demonstrated a significant shorter PFS (progression-free survival) for TFE3-rearranged-RCC compared to ccRCC.ConclusionsTFE3 rearrangement is an independent prognostic factor for recurrence and contributes to a worse PFS, suggesting the necessity of careful follow-up. Diagnosis should be confirmed using FISH due to low specificity of IHC. Further studies are needed to confirm TFE3 IHC staining as a prognostic factor.

Diagnostic Accuracy Performance of Fluorescence In Situ Hybridization (FISH) for Biliary Strictures: A Systematic Review and Meta-Analysis.

Background and Aims: This systematic review and meta-analysis aims to compare the performance of UroVysion® FISH based on the different definitions of a positive result used in published literature with the goal of determining the optimal FISH definition for detecting pancreaticobiliary malignancy. Methods: A systematic literature search identified studies from database inception to Sept 2024 that evaluated the diagnostic performance of FISH in determining malignancy among patients with biliary strictures. All thresholds for positive FISH, as defined by the individual study, were included in this review. Subgroup analysis was performed based on the definitions of positive FISH as follows: (1) polysomy only; (2) polysomy, tetrasomy, or trisomy; and (3) polysomy or 9p deletion. Results: Eighteen studies comprising 2516 FISH specimens were analyzed, including 1133 (45.0%) with malignancy. Using a threshold for positivity as defined in individual studies, the overall sensitivity of FISH was 57.6% (95% confidence interval [CI], 49.4-65.4%), and the overall specificity was 87.8% (95% CI, 79.2-93.2%). Subgroup analysis showed that polysomy as the threshold for positive FISH yielded a sensitivity of 49.4% (95% CI, 43.2-55.5%), with an increased specificity of 96.2% (95% CI, 92.7-98.1%), while polysomy + tetrasomy/trisomy as positive FISH resulted in an increased sensitivity of 64.3% (95% CI 55.4-72.2%) but a decreased specificity of 78.9% (95% CI 64.4-88.5%). The addition of 9p deletion to polysomy as the criteria for a positive test resulted in a non-significant increase in sensitivity (54.7% (95% CI 42.4-66.5%) while maintaining specificity (95.1% (95% CI 84.0-98.6%). Conclusions: Based on these findings, polysomy only or polysomy/9p deletion should be considered as the criterion for defining a positive FISH test to improve diagnostic sensitivity while maintaining high specificity.

Overexpression of Fibroblast Growth Factor 8 Is a Predictor of Impaired Survival in Esophageal Squamous Cell Carcinoma and Correlates with ALK/EML4 Alteration.

FGF8, ALK, and EML4 have been identified as promising biomarkers in a number of malignancies. The aim of this study was to examine the prognostic role of FGF8, ALK, and EML4 in esophageal squamous cell carcinoma (ESCC). Methods: Consecutive patients with ESCC who underwent upfront resection were included in this study. ALK and EML4 gene status was evaluated by fluorescence in situ hybridization (FISH) using a triple-color break-apart single-fusion probe and a probe against 2p11. FGF8, ALK, and EML4 protein expression was determined by immunohistochemistry. Results: A total of 122 patients were included in this study. Multivariate analysis revealed that FGF8 overexpression is an independent negative prognostic factor for patients' overall survival (OS) (p = 0.04). Furthermore, a significant correlation between the expression of FGF8, and ALK (p = 0.04) and EML4 (p = 0.01) alteration was found. Conclusions: FGF8 overexpression is an adverse independent prognostic factor in patients with upfront resected ESCC. Furthermore, FGF8 expression significantly correlates with ALK and EML4 amplification and may therefore qualify as a future therapeutic target.

Leveraging AI to Automate Detection and Quantification of Extrachromosomal DNA (ecDNA) to Decode Drug Responses.

Traditional drug discovery efforts have largely focused on targeting rapid, reversible protein-mediated adaptations to undermine cancer cells' resistance to therapy. However, cancer cells also exploit DNA-based strategies, typically viewed as slow, irreversible, and unpredictable changes like point mutations or the selection of drug-resistant clones. Contrary to this perception, extrachromosomal DNA (ecDNA) represents a form of DNA alteration that is rapid, reversible, and predictable, playing a crucial role in cancer's adaptive response. In this study, we present a novel post-processing pipeline for the automated detection and quantification of ecDNA in Fluorescence in situ Hybridization (FISH) images using the Microscopy Image Analyzer (MIA) tool. Our approach is particularly designed to monitor ecDNA dynamics during drug treatment, providing a quantitative framework to understand how ecDNA enables cancer cells to swiftly and reversibly adapt to therapeutic pressure. This pipeline not only offers a valuable resource for researchers aiming to automate ecDNA detection in FISH images but also sheds light on the adaptive mechanisms of ecDNA in response to epigenetic remodeling agents like JQ1.

Clinicopathologic Characteristics and Follow-Up Outcomes of Invasive Breast Carcinoma With Different Positive HER2 Fluorescence In Situ Hybridization Patterns: Experience From a Single Academic Institution

Human epidermal growth factor receptor 2 (HER2)-positive breast carcinoma (BC) encompasses a spectrum of molecular subtypes, characterized by varying HER2/CEP17 ratios and HER2 copy numbers, influencing responses to anti-HER2 therapy. This study stratified HER2 fluorescence in situ hybridization (FISH)-positive patients into 3 distinct groups—group 1 with high copy number (G1-HC: ratio ≥2, copy number ≥6), group 1 with low copy number (G1-LC: ratio ≥2, copy number ≥4 and <6), and group 3 (G3: ratio <2.0, copy number ≥6.0)—and evaluated their clinicopathologic features, response to anti-HER2 therapy, and outcomes. In a cohort of 2702 continuous primary BCs, G1-HC BCs accounted for 304 cases (11.3%), G1-LC for 37 cases (1.4%), and G3 for 75 cases (2.8%). G1-HC BCs were associated with younger age, higher tumor grade, and estrogen receptor negativity compared with G1-LC BCs. Furthermore, G1-HC BCs exhibited increased progesterone receptor negativity and HER2 immunohistochemistry 3+ compared with G1-LC and G3 BCs. Analysis of the subgroup of HER2 immunohistochemistry 2+-only cases (n = 166) showed similar results. Notably, G1-HC patients exhibited significantly enhanced responses to anti-HER2 neoadjuvant chemotherapy compared with G1-LC and G3 patients. Conversely, G1-LC patients displayed a lower likelihood of disease-free status compared with G1-HC and G3 patients, albeit with no significant differences in overall survival, distant metastasis, or local recurrence among the groups. These findings offer valuable clinicopathologic insights into different HER2 FISH positive subgroups, potentially informing future criteria for interpreting HER2 FISH results.

Application of FISH based G2-PCC assay for the cytogenetic assessment of high radiation dose exposures: Potential implications for rapid triage biodosimetry.

The main goal of this study is to test the utility of calyculin A induced G2-PCC assay as a biodosimetry triage tool for assessing a wide range of low and acute high radiation dose exposures of photons. Towards this initiative, chromosome aberrations induced by low and high doses of x-rays were evaluated and characterized in G2-prematurely condensed chromosomes (G2-PCCs) by fluorescence in situ hybridization (FISH) using human centromere and telomere specific PNA (peptide nucleic acid) probes. A dose dependent increase in the frequency of dicentric chromosomes was observed in the G2-PCCs up to 20 Gy of x-rays. The combined yields of dicentrics and rings in the G2-PCCs showed a clear dose dependency up to 20 Gy from 0.02/cell for 0.1 Gy to 14.98/cell for 20 Gy. Centric rings were observed more frequently than acentric ring chromosomes in the G2-PCCs at all the radiation doses from 1 Gy to 20 Gy. A head-to-head comparison was also performed by FISH on the yields of chromosome aberrations induced by different doses of x-rays (0 Gy -7.5 Gy) in colcemid arrested metaphase chromosomes and calyculin A induced G2-PCCs. In general, the frequencies of dicentrics, rings and acentric fragments were slightly higher in G2-PCCs than in colcemid arrested metaphase chromosomes at all the radiation doses, but the differences were not statistically significant. To reduce the turnaround time for absorbed radiation dose estimation, attempt was made to obtain G2-PCCs by reducing the culture time to 36 hrs. The absorbed doses estimated in x-rays irradiated (0,1,2 and 4 Gy) G2-PCCs after 36 hrs of culture were grossly like that of G2-PCCs and colcemid arrested metaphase chromosomes prepared after 48 hrs of culture. Our study indicates that the shortened version of calyculin A induced G2-PCC assay coupled with the FISH staining technique can serve as an effective triage biodosimetry tool for large-scale radiological/nuclear incidents.

Clinicopathologic characterization of secretory carcinoma of salivary gland

BackgroundTo investigate the clinicopathologic characteristics, therapeutic methods, and prognosis of secretory carcinoma of salivary gland (SCSG).MethodsThe clinicopathologic data of 13 patients with SCSG admitted to Shanxi Cancer Hospital from January 2018 to June 2023 were retrospectively analyzed, and a literature review was performed.ResultsA total of eight males and five females aged 22–78 years old were enrolled, and they commonly presented with painless masses in the parotid or submandibular gland. They all underwent surgical treatment, accompanied by typical pathological examinations postoperatively. Fluorescence in situ hybridization (FISH) was conducted in seven cases, the results were all positive, and no gene fusion other than ETV6-NTRK3 was found. Two patients developed local relapse during follow-up, both of which were in the surgical area. By the end of the follow-up, 12 patients survived and one patient died.ConclusionsSCSG is a rare low-grade malignancy with a good prognosis. Pathological and immunohistochemical characteristics are the key to secretory carcinoma (SC) diagnosis, and surgical excision is the major treatment means for SCSG. Whether to perform simultaneous cervical lymph node dissection and other adjuvant therapies should be determined based on the pathological stage and the presence or absence of high-risk factors.

SS18-SSX Expression and Clinicopathologic Profiles in a Contemporary Cohort of Primary Paratesticular Synovial Sarcoma: A Series of Fourteen Patients.

Synovial sarcoma (SS) is a rare genitourinary malignancy with a specific SS18::SSX 1/2 gene fusion in majority of the instances. The paratesticular location of this neoplasm is extremely rare and only 4 cases are reported in the literature. Herein, we describe the clinicopathologic features and molecular profile of paratesticular SS in the largest case series to date and to the best of our knowledge, and the only series to use novel SS18-SSX antibody for immunohistochemistry. Clinicopathologic, immunohistochemical (IHC), molecular, treatment, and follow-up data of the patients were analyzed. There were 14 patients, ranging from 15 to 47 years (mean: 30y). The tumor size ranged from 4​​​​​​ to 15cm. The tumors were unilateral, solid, and homogeneous tan-white with monomorphic spindle cell histology. All 14 tumors expressed SS18-SSX and TLE1 IHC and harbored SS18 rearrangement. In addition, the tumor with multifocal SS18-SSX expression had lower break-apart signals in the FISH assay (38% of the tumor cells; range: 29% to 85%). Radical orchiectomy was performed in all 14 patients and adjuvant chemotherapy was administered in 9 patients. Follow-up was available in 9 patients. The follow-up duration ranged from 5 to 24 months (median=10mo). Four patients died of metastatic disease (range: 5 to 16mo) and 2 patients who are alive had metastatic disease at the last follow-up. Based on our experience with the largest series to date and aggregate of the published data, paratesticular SS has a poor prognosis despite aggressive therapy. Owing to its rarity, the differential diagnosis is wide and requires a systematic approach for ruling out key morphologic mimics aided with SS18-SSX IHC and molecular confirmation because this distinction carries important therapeutic and prognostic implications. Due to the excellent concordance of SS18-SSX IHC results with FISH results as observed in our study, we would like to suggest inclusion of SS18-SSX in the diagnostic immunohistochemistry panel of all spindle cell sarcomas where synovial sarcoma is considered as a morphologic differential. SS18-SSX-positive staining may be used as a surrogate for FISH assay in a resource-limited setting where molecular assay is not available. Furthermore, IHC has a fairly shorter turn-around-time, is less complex, and of low cost.

Whole-chromosome oligo-painting in licorice unveils interspecific chromosomal evolutionary relationships and possible origin of triploid genome species.

Licorice is one of the most extensively studied medicinal plants in the world, whose roots and rhizomes have long been used as both a sweetener and an essential component in numerous herbal preparations. However, the genus Glycyrrhiza has a complex composition, and the interspecies chromosomal relationships, origin, and evolution are still largely unclear. Here, we develop a set of whole-chromosome painting probes that allowed identification of all eight chromosomes of licorice on same metaphase chromosomes. Comparative chromosome painting analyses in seven different Glycyrrhiza species revealed that the genus Glycyrrhiza maintained extraordinarily conserved chromosomal synteny after about 3-12 million years of divergence. No cytologically visible inter-chromosomal rearrangements were identified in any species. By comparative chromosomal karyotype analyses, we revealed interspecific chromosome evolutionary relationships and dramatic variable chromosomal karyotype after independent divergence and demonstrated that G. prostrate was the most closely related to the ancestral type among the seven Glycyrrhiza species. Furthermore, we also discovered a G. glandulosa seed with distinct triploid-genome for the first time in China, suggesting the existence of a polyploid evolutionary pathway in the genus Glycyrrhiza, which challenges the previous notion that only diploids of licorice existed in nature. This study expands our knowledge of the chromosome evolution of licorice and will lay an important foundation for the genome origin and evolution studies in the genus Glycyrrhiza.

MRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

LRRC75A-AS1 Drives the Epithelial-Mesenchymal Transition in Cervical Cancer by Binding IGF2BP1 and Inhibiting SYVN1-Mediated NLRP3 Ubiquitination.

Cervical cancer severely affects women's health with increased incidence and poor survival for patients with metastasis. Our study aims to investigate the mechanism by which lncRNA LRRC75A-AS1 regulates the epithelial-mesenchymal transition (EMT) of cervical cancer through modulating m6A and ubiquitination modification. In this study, tumor tissues were collected from patients to analyze the expression of LRRC75A-AS1 and SYVN1. Migratory and invasive capacities of HeLa and CaSki cells were evaluated with wound healing and transwell assays. CCK-8 and EdU incor-poration assays were employed to examine cell proliferation. The interaction between LRRC75A-AS1, IGF2BP1, SYVN1, and NLRP3 was evaluated through RNA immunoprecipitation, RNA pull-down, FISH, and coimmunoprecipitation assays, respectively. MeRIP-qPCR was applied to analyze the m6A modification of SYVN1 mRNA. A subcutaneous tumor model of cervical cancer was established. We showed LRRC75A-AS1 was upregulated in tumor tissues, and LRRC75A-AS1 enhanced EMT through activating NLRP3/IL1β/Smad2/3 signaling in cervical cancer. Furthermore, LRRC75A-AS1 inhibited SYVN1-mediated NLRP3 ubiquitination by destabilizing SYVN1 mRNA. LRRC75A-AS1 competitively bound to IGF2BP1 protein and subsequently impaired the m6A modification of SYVN1 mRNA and its stability. Knockdown of LRRC75A-AS1 repressed EMT and tumor growth via inhibiting NLRP3/IL-1β/Smad2/3 signaling in mice. In conclusion, LRRC75A-AS1 competitively binds to IGF2BP1 protein to destabilize SYVN1 mRNA, subsequently suppresses SYVN1-mediated NLRP3 ubiquitination degradation and activates IL1β/Smad2/3 signaling, thus promoting EMT in cervical cancer. Implication: LRRC75A-AS1 promotes cervical cancer progression, and this study suggests LRRC75A-AS1 as a new therapeutic target for cervical cancer.

Optical Genome Mapping for Detection of BCR::ABL1-Another Tool in Our Toolbox.

Background:BCR::ABL1 fusion is mostly derived from a reciprocal translocation t(9;22)(q34.1;q11.2) and is rarely caused by insertion. Various methods have been used for the detection of t(9;22)/BCR::ABL1, such as G-banded chromosomal analysis, fluorescence in situ hybridization (FISH), quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and optical genome mapping (OGM). Understanding the strengths and limitations of each method is essential for the selection of appropriate method(s) of disease diagnosis and/or during the follow-up. Methods: We compared the results of OGM, chromosomal analysis, FISH, and/or RT-PCR in 12 cases with BCR::ABL1. Results:BCR:ABL1 was detected by FISH and RT-PCR in all 12 cases. One case with ins(22;9)/BCR::ABL1 was cryptic by chromosomal analysis and nearly missed by OGM. Atypical FISH signal patterns were observed in five cases, suggesting additional chromosomal aberrations involving chromosomes 9 and/or 22. RT-PCR identified the transcript isoforms p210 and p190 in seven and five cases, respectively. Chromosomal analysis revealed additional chromosomal aberrations in seven cases. OGM identified extra cytogenomic abnormalities in 10 cases, including chromoanagenesis and IKZF1 deletion, which were only detected by OGM. Conclusions: FISH offers rapid and definitive detection of BCR::ABL1 fusion, while OGM provides a comprehensive cytogenomic analysis. In scenarios where OGM is feasible, chromosomal analysis and RT-PCR may not offer additional diagnostic value.

High prevalence of chromosome 17 in breast cancer micronuclei: a means to get rid of tumor suppressors?

Micronuclei (MN), defined as small extra-nuclear chromatin bodies enclosed by a nuclear envelope, serve as noticeable markers of chromosomal instability (CIN). The MN have been used for breast cancer (BC) screening, diagnosis, and prognosis. However, more recently they have gained attention as seats for active chromosomal rearrangements. BC subtypes exhibit differential CIN levels and aggressiveness. This study aimed to investigate MN chromosomal contents across BC subtypes, exploring its potential role in aggressiveness and pathogenesis. Immunostaining of BC cells was performed with anti-centromeric antibody followed by confocal microscopy. Further, fluorescence in situ hybridization (FISH) was done to check the presence of specific chromosomes in the MN. The real time PCR was also done from the RNA isolated from MN to check the expression of TP53 gene. BC cell lines (CLs) showed the presence of both centromere-positive ( +) and -negative ( -) MN, with significant variation in frequency among hormone and human epidermal growth factor receptor positive and triple-negative (TN) BC cells. FISH targeting chromosomes 1, 3, 8, 11, and 17 detected centromeric signals for all the above chromosomes in MN with a relatively higher prevalence of chromosome 17 in all the CLs. Out of all the CLs, TNBC cells demonstrated the highest frequency of centromere + and chromosome 17 + MN. TP53 expression could also be demonstrated inside the MN by FISH and real time PCR. Patient sample imprints also confirmed the presence of chromosome 17 in MN with polysomy of the same in corresponding nuclei. The high prevalence of chromosome 17 in BC MN may connote the importance of its rearrangements in the pathogenesis of BC. Further, the higher prevalence of chromosome 17 and 1 signals in TNBC MN point towards the significance of pathogenetic events involving the genes located in these chromosomes in evolution of this more aggressive phenotype.

Continuous nucleolar ribosomal RNA synthesis in differentiating lens fiber cells until abrupt nuclear degradation required for ocular lens transparency.

Cellular differentiation requires highly coordinate action of all three transcriptional systems to produce rRNAs, mRNAs, and various "short" and "long" non-coding RNAs by RNA Polymerase I, II, and III systems, respectively. The RNA Polymerase I catalyzes transcription of about 400 copies of rDNA genes generating 18S, 5.8S, and 28S rRNA molecules from the individual primary transcript. Lens fiber cell differentiation is a unique process to study transcriptional mechanisms of individual crystallin genes as their very high transcriptional outputs are directly comparable only to globin genes in erythrocytes. Importantly, both terminally differentiated lens fiber cells and mammalian erythrocytes degrade their nuclei though by different mechanisms. In lens, generation of organelle-free zone (OFZ) includes degradation of mitochondria, endoplasmic reticulum, Golgi apparatus, and nuclei; nevertheless, very little is known about their nucleoli and rRNA transcription. Here, using RNA fluorescence in situ hybridization (FISH) we evaluated nascent rRNA transcription during the entire process of lens fiber cell differentiation. The lens fiber cell nuclei undergo morphological changes prior their denucleation, including chromatin condensation; remarkably, the nascent rRNA transcription persists in all nuclei next to the OFZ. The changes in both nuclei and nucleoli shape and microarchitecture were evaluated by immunofluorescence to detect fibrillarin, nucleolin, UBF, and other nuclear proteins. These studies demonstrate for the first time that highly condensed lens fiber cell nuclei have the capacity to support rRNA transcription. Thus, "late" production of rRNA molecules and consequently the ribosomes contribute to the terminal translational mechanisms to produce maximal quantities of the crystallin proteins.

Impact of sex chromosome abnormalities in male chronic myeloid leukemia patients

IntroductionChronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the Philadelphia chromosome (Ph) resulting from the reciprocal translocation t(9;22)(q34;q11). Sex chromosomal abnormalities are found rarely in CML patients. Materials and methodsConventional and molecular cytogenetic analysis (Fluorescent In Situ Hybridization (FISH)) were performed to reveal the presence of Ph and additional chromosomal abnormalities (ACA). ResultsThree patients with abnormalities within sex chromosomes, one Klinefelter syndrome (KS) patient and two cases with LOY (Loss of Y) diagnosed with CML CP were included in the study. All three patients initially responded to the targeted therapy, became resistant to their treatment course and switched to 2nd line therapies. In the 15th month, the KS patient abruptly turned into BC and showed clonal evolution with four karyotypic patterns. According to Mitelman databases, this is the first KS-CML patient who showed three major route abnormalities, +8, i(17) and + der(22) in a single clone. Survival analysis showed one patient with LOY chromosome expired on the 48th month of diagnosis. ConclusionThe application of cytogenetic techniques in identifying the constitutional abnormality of KS, ACA at initial CML diagnosis, revealing of extra Ph during the treatment course and clonal evolution in BC (Blast Crisis) assist the proper monitoring of disease transformation and serves as a major diagnostic tool for CML patients thereby switching the treatment protocols. A systematic stratification of patients according to the chromosomal abnormalities is needed in CML patients.