Fluorescence In Situ Hybridization Research Articles

BIOM-69. THE ONCOGENIC FLIP IN PATIENTS WITH LEPTOMENINGEAL METASTATIC DISEASE (LMD): LONGITUDINAL DETECTION IN CEREBROSPINAL FLUID TUMOR CELLS (CSF-TCS) REVEALS IMPLICATIONS FOR DIFFERENTIAL TREATMENT OF THE LMD TUMOR

Abstract INTRODUCTION Patients with LMD have poor prognosis and limited treatment options. Oncogene amplification of primary, metastatic, and CNS metastatic tumors can be heterogeneous. Therefore, patients with LMD may benefit from assessment of clinically relevant biomarkers in CSF, which may guide the choice of a targeted therapy specifically for the LMD tumor. CNSide is a CLIA-validated laboratory-developed test ordered commercially at the discretion of physicians for CSF-TC enumeration, immunocytochemical (ICC) and fluorescence in situ hybridization (FISH) analysis of oncogene amplification. We longitudinally analyze oncogenes in CSF-TCs in patients with LMD of various primary cancers. METHODS CSF was collected from patients with suspected or confirmed LMD; 613 tests were ordered on 218 individual patients with breast (N=105 patients), lung (N=65), gastrointestinal (N=10), and other cancers. Using CNSide, CSF-TCs were isolated and tested via ICC (ER, PD-L1, and PR), and FISH (ALK, cMET, cMyc, EGFR, FGFR1, HER2, NTRK1, NTRK3, PTEN, RET, and ROS1). RESULTS In patients with lung cancer, ALK was detected in 14% (17/118) of samples, CMET in 61% (78/128), HER2 in 73% (16/22), and RET in 4% (4/90). In patients with breast cancer, HER2 was detected in 39% (65/168) of samples, FGFR1 in 32% (19/60), ER in 26% (44/168) and PR in 4% (5/120). 66 patients underwent 2+ CSF draws; 24 underwent 5+. Among these, there were 13 ICC flips (7 acquired mutations) and 58 FISH probe detection flips (26 acquired mutations). 20/66 patients (30.3%) had at least one flip in their ordered biomarkers. CONCLUSION CNSide can be used to detect oncogene amplification on CSF-TCs of patients with LMD, and mutational status of the LMD tumor may differ from the original tumor biopsy. CSF-TC analysis may provide therapeutic insights that vary from the original tumor and could open the door to additional treatment targets for patients struck with LMD.

Deep-Learning-Based Approach in Cancer-Region Assessment from HER2-SISH Breast Histopathology Whole Slide Images

Fluorescence in situ hybridization (FISH) is widely regarded as the gold standard for evaluating human epidermal growth factor receptor 2 (HER2) status in breast cancer; however, it poses challenges such as the need for specialized training and issues related to signal degradation from dye quenching. Silver-enhanced in situ hybridization (SISH) serves as an automated alternative, employing permanent staining suitable for bright-field microscopy. Determining HER2 status involves distinguishing between “Amplified” and “Non-Amplified” regions by assessing HER2 and centromere 17 (CEN17) signals in SISH-stained slides. This study is the first to leverage deep learning for classifying Normal, Amplified, and Non-Amplified regions within HER2-SISH whole slide images (WSIs), which are notably more complex to analyze compared to hematoxylin and eosin (H&E)-stained slides. Our proposed approach consists of a two-stage process: first, we evaluate deep-learning models on annotated image regions, and then we apply the most effective model to WSIs for regional identification and localization. Subsequently, pseudo-color maps representing each class are overlaid, and the WSIs are reconstructed with these mapped regions. Using a private dataset of HER2-SISH breast cancer slides digitized at 40× magnification, we achieved a patch-level classification accuracy of 99.9% and a generalization accuracy of 78.8% by applying transfer learning with a Vision Transformer (ViT) model. The robustness of the model was further evaluated through k-fold cross-validation, yielding an average performance accuracy of 98%, with metrics reported alongside 95% confidence intervals to ensure statistical reliability. This method shows significant promise for clinical applications, particularly in assessing HER2 expression status in HER2-SISH histopathology images. It provides an automated solution that can aid pathologists in efficiently identifying HER2-amplified regions, thus enhancing diagnostic outcomes for breast cancer treatment.

TMOD-26. GENERATION OF A CHROMOSOME 8 GAIN;NF1-/- HIPSC-DERIVED SCHWANN CELL PRECURSOR MODEL

Abstract Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome caused by a germline mutation in the NF1 gene. The loss of the second copy of NF1 within Schwann cell precursors (SCPs) contributes to the formation of plexiform neurofibromas (PNs) found in 30-50% of NF1 patients. PNs can progress into malignant peripheral sheath tumors (MPNSTs), an aggressive form of cancer and a leading cause of mortality in NF1 patients. Our lab previously demonstrated frequent chromosome 8 (Chr8) gain in NF1-MPNST patient-derived xenografts (PDX) and patient tumors. To investigate Chr8 gain’s role, we created isogenic NF1-/- iPSC cell lines with and without Chr8 gain. Trisomy 8 fibroblasts were obtained and single-cell cloned to establish wildtype (WT) and Chr8 gain populations. Washington University’s Genome Engineering & Stem Cell Center (GESC@MGI) reprogrammed these cells into human induced pluripotent stem cells (hiPSCs). Using synthetic gRNA and CRISPR/Cas9, NF1 knockout lines were generated. Four iPSC cell lines were differentiated into SCPs, and cell survival and proliferation were assessed using Incucyte cell survival and CellTiter-Glo® Assay. Fluorescence in situ hybridization (FISH) and karyotyping validated the human iPSCs. qPCR revealed high expression of iPSC markers OCT3/4 and SOX2 in all iPSC lines. After differentiation, SCP markers (GAP43 and ITGA4) were upregulated in WT, NF1-/-, and Chr8 gain;NF1-/- SCP lines, demonstrating successful differentiation. Furthermore, Chr8 gain; NF1-/- SCPs displayed enhanced proliferation and increased cell survival compared to WT and NF1-/- SCPs, suggesting improved characteristics in the presence of Chr8 gain. In summary, our research provides insights into Chr8 gain and NF1 loss effects on SCP differentiation, proliferation, and survival. Understanding these molecular mechanisms may contribute to developing targeted therapies for NF1.

BIOM-70. CSF TUMOR CELL (CSF-TC) DETECTION, QUANTIFICATION AND BIOMARKER ASSESSMENT HELPS IN CLINICAL MANAGEMENT OF BREAST CANCER AND NON-SMALL CELL LUNG CANCER PATIENTS HAVING LEPTOMENINGEAL DISEASE (FORESEE STUDY, NCT05414123)

Abstract Leptomeningeal metastases (LM) or the presence of cancer cells in the cerebrospinal fluid (CSF)/pia remains a challenging disease to diagnosis and treat. Current standard of care methods to diagnose or assess treatment response of LM (Clinical Evaluation, MRI and Cytology) have limited sensitivity and specificity and are poorly quantifiable. This creates challenges for physicians to manage LM or determine the best course of treatment. The FORESEE Study was a multi-center, prospective clinical trial evaluating a novel diagnostic platform, CNSide, that aimed to overcome these challenges. The FORESEE study enrolled patients with breast or non-Small Cell Lung Cancer (NSCLC) with suspicion of or confirmed LM. CNSide can detect and quantify tumor cells in the CSF from patients with breast cancer or NSCLC having a suspicious or confirmed LM. This platform also is able to identify actionable mutations in the CSF via Fluorescent In Situ Hybridization (FISH) and next-generation sequencing. The primary end point of the FORESEE study was clinical utility and the impact of CNSide on physician treatment decisions. Secondary endpoints included evaluating the clinical performance of CNSide vs. cytology in tumor cell detection (sensitivity, specificity, PPV and NPV). The study enrolled 40 patients (22 breast cancer and 18 NSCLC). Each patient underwent standard of care diagnostic evaluations at baseline and at three follow up timepoints through their treatment. CNSide aided clinical decision making in 93% (50/54) of the clinical decisions. At baseline 39 patients were assessed and confirmed a positive LM diagnosis in 15% (6/39), a negative LM diagnosis in 8% (3/39), progression in 15% (6/39), resolution in 15% (6/39) and no progression, or resolution in 36% (14/39) of the patients. CNSide informed the specific drug selected for treatment in 26% (10/39) patients at baseline and demonstrated clinical high utility. The FORESEE data is undergoing ongoing analysis; updated analysis will be presented at the meeting.

A Rare Case of Ewing Sarcoma of the Left Ethmoid Sinus With Orbital Extension

ABSTRACTBackgroundEwing sarcoma (ES) of the ethmoid sinus with orbital involvement in an adult is very rare, with 16 reported cases in the literature. Immunohistochemical studies show small blue round cells positive for CD99 and fluorescence in situ hybridization (FISH) testing reveals positivity for the EWSR1 gene.MethodsA 38‐year‐old male with a diagnosis of ES of the ethmoid sinus presented with left‐sided periorbital pain and edema, rhinorrhea, and proptosis. The patient underwent neoadjuvant chemotherapy, surgical resection of the left skull base, and postoperative proton radiotherapy.ResultsThe patient tolerated chemotherapy, surgical resection, and adjuvant proton radiotherapy well with resolution of proptosis, diplopia, and pain. Due to local recurrence, he is currently undergoing adjuvant chemotherapy.ConclusionOur findings provide insight on the clinical presentation and appropriate management of extraosseous ES, specifically in the ethmoid sinus in the adult population.

FISH combined with RT-PCR facilitates classification of Chinese adult patients with B-other ALL through improved identification of ZNF384 rearrangement

ZNF384 gene rearrangements are a distinct subtype of adult B cell acute lymphoblastic leukemia (B-ALL). We screened 46 B-other ALL patients for ZNF384 fusions using fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR). Clinical data, treatment response, and minimal residual disease (MRD) status were analyzed. Ten (21.7%) patients were ZNF384-r positive (nine by FISH, nine by RT-PCR, eight by both). FISH showed atypical signals, including 3’ signal gain and 5’ signal deletion. EP300 was the main fusion partner (n = 5). TAF15::ZNF384, SYNRG::ZNF384, CREBBP::ZNF384, and TCF3::ZNF384 fusions were found in one patient each; one case’s partner gene is unknown. One patient was MRD-negative at the end of the first induction, lower than in patients without ZNF384-r. ZNF384-r incidence matched B-other ALL incidence in Chinese patients. Combined FISH and RT-PCR improved detection. ALL with ZNF384-r has unique features, and lower MRD-negative response may indicate a negative impact on traditional treatments.

Deep learning method for detecting fluorescence spots in cancer diagnostics via fluorescence in situ hybridization

Fluorescence in Situ Hybridization (FISH) is a technique for macromolecule identification that utilizes the complementarity of DNA or DNA/RNA double strands. Probes, crafted from selected DNA strands tagged with fluorophore-coupled nucleotides, hybridize to complementary sequences within the cells and tissues under examination. These are subsequently visualized through fluorescence microscopy or imaging systems. However, the vast number of cells and disorganized nucleic acid sequences in FISH images present significant challenges. The manual processing and analysis of these images are not only time-consuming but also prone to human error due to visual fatigue. To overcome these challenges, we propose the integration of medical imaging with deep learning to develop an automated detection system for FISH images. This system features an algorithm capable of quickly detecting fluorescent spots and capturing their coordinates, which is crucial for evaluating cellular characteristics in cancer diagnosis. Traditional models struggle with the small size, low resolution, and noise prevalent in fluorescent points, leading to significant performance declines. This paper offers a detailed examination of these issues, providing insights into why traditional models falter. Comparative tests between the YOLO series models and our proposed method affirm the superior accuracy of our approach in identifying fluorescent dots in FISH images.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Detection of Alternative Lengthening of Telomeres via Chromogenic in situ Hybridization (ALT-CISH) for the Prognostication of PanNETs and Other Neoplasms

Molecular studies have shown ALT to be an important prognostic biomarker of shorter relapse-free survival (RFS) for patients with pancreatic neuroendocrine tumors (PanNETs) and other neoplasms. However, the preferred method of detecting ALT in tissue is by fluorescence in situ hybridization (FISH), which has several clinical limitations. These issues necessitate the creation of a chromogenic ALT assay that can be easily implemented into routine practice. A CISH assay was developed using genetically modified osteosarcoma cell lines, 20 normal pancreata, 20 ALT-positive PanNETs, and 20 ALT-negative PanNETs. Thereafter, it was validated on a multi-institutional cohort of 360 surgically resected PanNETs and correlated with multiple clinicopathologic features, RFS, and FISH results. Separately, 109 leiomyosarcomas (LMS) were evaluated by both CISH and FISH, and, similarly, the prognostic significance of ALT status was assessed. Upon optimization, ALT-CISH was identified in 112 of 360 (31%) primary PanNETs and was 100% concordant with FISH testing. ALT correlated with several adverse prognostic findings and distant metastasis (all p<0.004). The 5-year RFS for patients with ALT-positive PanNETs was 35% as compared to 94% for ALT-negative PanNETs. By multivariate analysis, ALT was an independent prognostic factor for shorter RFS. Similarly, ALT was associated with shorter RFS in LMS patients and, analogous to PanNETs, a negative, independent prognostic factor. ALT-CISH was developed and validated in not only PanNETs, but also sarcomas, specifically LMS. CISH testing has multiple advantages over FISH that facilitate its widespread clinical use in the detection of ALT and prognostication of patients with diverse neoplasms.

Quercetin inhibits endothelial & hepatocellular carcinoma cell crosstalk via reducing extracellular vesicle-mediated VEGFR2 mRNA transfer.

This study aims to investigate the regulatory effects of quercetin extracellular vesicles (EVs)-mediated expression of vascular endothelial growth factor receptor 2 (VEGFR2) in hepatocellular carcinoma (HCC)-derived circulating tumor cells (CTCs) and the underlying mechanisms. CTCs were isolated from patients with pathologically diagnosed HCC, with VEGFR2 expression visualized by fluorescence in situ hybridization (FISH). The human HCC cell line Huh-7 and SK-HEP-1 were used for in vitro studies to assess EVs uptake, VEGFR2 mRNA transfer, invasion, migration, cancer stem cell (CSC) properties, and VEGF secretion. Results showed that VEGFR2 mRNA was commonly expressed in HCC-CTCs, with a higher incidence in biphenotypic CTCs. Its expression was limited in HCC cell lines, but present in certain liver cells. In vitro experiments confirmed that VEGFR2 mRNA could be transferred to HCC cells via EVs from primary tumor endothelial cells (PTECs), which was impaired by quercetin treatment. Quercetin significantly reduced VEGFR2 mRNA and protein expression in HCC cells, weakened their invasive and metastatic capacities, and diminished VEGFR2-mediated CSC properties. In vivo, quercetin reduced VEGF secretion, impaired angiogenesis, slowed tumor growth, and decreased the number and proportion of VEGFR2-positive CTCs. In summary, VEGFR2 mRNA is present in HCC-CTCs, potentially sourced from PTECs-derived EVs. Quercetin effectively inhibits VEGFR2 expression, impacting HCC cell invasion, metastasis, and CSC characteristics. Besides, it reduces VEGFR2-positive CTCs in vivo. These effects support its therapeutic potential in HCC treatment by targeting the angiogenesis and tumor dissemination pathway.

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

ABSTRACTBackgroundWhen 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.MethodsWe analyzed fetal material obtained via amniocentesis procedure by G‐banding, microarray, and fluorescence in situ hybridization (FISH). Optical genome mapping (OGM) was also performed.ResultsG‐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.ConclusionThis case demonstrates how the SRY gene may be present in a 46,XX biological female without differences of sexual development.

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.

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.

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.