Harnessing immune cells to leverage PARP inhibitors.

Homologous-recombination deficiency in DNA repair characterizes a unique group of cancers that are vulnerable to PARP inhibitors and cytotoxic chemotherapy. In this issue of Cell, Luo et al., demonstrated that this genetic attribute in cancer cells may reprogram tumor immune microenvironment and show promise of targeting effector-Treg cells.

Cationic Foldamer-Catalyzed Asymmetric Synthesis of Inherently Chiral Cages.

The stereochemistry of shape-persistent molecular cages, particularly those resembling prisms, exerts significant influence on their application-specific functionalities. Although methods exist for fabricating inherently chiral prism-like cages, strategies for catalytic asymmetric synthesis of these structures in a diversity-oriented fashion remain unexplored. Herein, we introduce an unprecedented organocatalytic desymmetrization approach for the generation of inherently chiral prism-like cages via phosphonium-containing foldamer-catalyzed SNAr reactions. This methodology establishes a topological connection, enabling the facile assembly of a wide range of versatile stereogenic-at-cage building blocks possessing two highly modifiable groups. Furthermore, subsequent stereospecific transformations of the remaining chlorides and/or ethers afford convenient access to numerous functionally relevant chiral-at-cage molecules.

Synergistically activating nucleophile strategy enabled organocatalytic asymmetric P-addition of cyclic imines.

Herein, we present an attractive organocatalytic asymmetric addition of P-nucleophiles to five-membered cyclic N-sulfonyl imines facilitated by phosphonium salt catalysis, enabling the highly enantioselective synthesis of tri- and tetra-substituted cyclic phosphorus-containing benzosultams. With this protocol, various cyclic α-aminophosphonates were efficiently synthesized with high yields and exceptional enantioselectivities (up to >99% ee) under mild reaction conditions. The utility and practicality of this method were demonstrated through gram-scale reactions and straightforward elaborations. Notably, the success of this approach relies on the deliberate selection of a synergistic organocatalytic system, which helps circumvent foreseeable side effects while handling secondary phosphine oxides (SPOs). Systematic mechanistic studies, incorporating experiments and DFT calculations, have revealed the critical importance of judiciously selecting bifunctional phosphonium salt catalysts for effectively activating P-nucleophiles while stereoselectively controlling the P-attack process.

Dual Inhibition of SYK and EGFR Overcomes Chemoresistance by Inhibiting CDC6 and Blocking DNA Replication.

Targeting multiple signaling pathways has been proposed as a strategy to overcome resistance to single-pathway inhibition in cancer therapy. A previous study in epithelial ovarian cancers identified hyperactivity of spleen tyrosine kinase (SYK) and epidermal growth factor receptor (EGFR), which mutually phosphorylate and activate each other. Given the potential for pharmacologic inhibition of both kinases with clinically available agents, this study aimed to assess the antitumor efficacy of both pharmacologic and genetic SYK and EGFR co-inhibition using a multifaceted approach to analyze the global phosphoproteome and chemoresistant ovarian cancer cell lines, patient-derived organoids, and xenograft models. Dual inhibition of SYK and EGFR in chemoresistant ovarian cancer cells elicited a highly synergistic antitumor effect. Notably, the combined inhibition strategy activated the DNA damage response, induced G1 cell cycle arrest, and promoted apoptosis. The phosphoproteomic analysis revealed that perturbation of SYK and EGFR signaling induced a significant reduction in both phosphorylated and total protein levels of cell division cycle 6 (CDC6), a crucial initiator of DNA replication. Together, this study offers preclinical evidence supporting dual inhibition of SYK and EGFR as a promising treatment for chemoresistant ovarian cancer that disrupts DNA synthesis by impairing formation of the prereplication complex. These findings warrant further clinical investigation to explore the potential of this combination therapy in overcoming drug resistance and improving patient outcomes.

Isolation and identification of specific Enterococcus faecalis phage C-3 and G21-7 against Avian pathogenic Escherichia coli and its application to one-day-old geese.

Colibacillosis caused by Avian pathogenic Escherichia coli (APEC), including peritonitis, respiratory tract inflammation and ovaritis, is recognized as one of the most common and economically destructive bacterial diseases in poultry worldwide. In this study, the characteristics and inhibitory potential of phages were investigated by double-layer plate method, transmission electron microscopy, whole genome sequencing, bioinformatics analysis and animal experiments. The results showed that phages C-3 and G21-7 isolated from sewage around goose farms infected multiple O serogroups (O1, O2, O18, O78, O157, O26, O145, O178, O103 and O104) Escherichia coli (E.coli) with a multiplicity of infection (MOI) of 10 and 1, respectively. According to the one-step growth curve, the incubation time of both bacteriophage C-3 and G21-7 was 10 min. Sensitivity tests confirmed that C-3 and G21-6 are stable at 4 to 50 °C and pH in the range of 4 to 11. Based on morphological and phylogenetic analysis, phages C-3 and G21-7 belong to Enterococcus faecalis (E. faecalis) phage species of the genus Saphexavirus of Herelleviridae family. According to genomic analysis, phage C-3 and G21-7 were 58,097 bp and 57,339 bp in size, respectively, with G+C content of 39.91% and 39.99%, encoding proteins of 97 CDS (105 to 3,993 bp) and 96 CDS (105 to 3,993 bp), and both contained 2 tRNAs. Both phages contained two tail proteins and holin-endolysin system coding genes, and neither carried resistance genes nor virulence factors. Phage mixture has a good safety profile and has shown good survival probability and feed efficiency in both treatment and prophylaxis experiments with one-day-old goslings. These results suggest that phage C-3 and G21-7 can be used as potential antimicrobials for the prevention and treatment of APEC.

Single-molecule epiallelic profiling of DNA derived from routinely collected Pap specimens for noninvasive detection of ovarian cancer.

Recent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour-specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low-cost methods remains challenging, especially in early-stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian-cancer-specific aberrations of DNA methylation from routinely-collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high-resolution melt to analyze locus-specific DNA methylation patterns on a molecule-by-molecule basis at or near single CpG-site resolution at a fraction (< 1/10th) of the cost of next-generation sequencing techniques. We demonstrate the feasibility of the platform by assessing intermolecular heterogeneity of DNA methylation in a panel of methylation biomarker loci using DNA derived from Pap specimens obtained from a cohort of 43 women, including 18 cases with ovarian cancer and 25 cancer-free controls. PapDREAM leverages systematic multidimensional bioinformatic analyses of locus-specific methylation heterogeneity to improve upon Pap-specimen-based detection of ovarian cancer, demonstrating a clinical sensitivity of 50% at 99% specificity in detecting ovarian cancer cases with an area under the receiver operator curve of 0.90. We then establish a logistic regression model that could be used to identify high-risk patients for subsequent clinical follow-up and monitoring. The results of this study support the utility of PapDREAM as a simple, low-cost screening method with the potential to integrate with existing clinical workflows for early detection of ovarian cancer. KEY POINTS: We present a microfluidic platform for detection and analysis of rare, heterogeneously methylated DNA within Pap specimens towards detection of ovarian cancer. The platform achieves high sensitivity (fractions <0.00005%) at a suitably low cost (∼$25) for routine screening applications. Furthermore, it provides molecule-by-molecule quantitative analysis to facilitate further study on the effect of heterogeneous methylation on cancer development.

PAI-1 uncouples integrin-ß1 from restrain by membrane-bound ß-catenin to promote collagen fibril remodeling in obesity-related neoplasms.

The paracrine actions of adipokine plasminogen activator inhibitor-1 (PAI-1) are implicated in obesity-associated tumorigenesis. Here, we show that PAI-1 mediates extracellular matrix (ECM) signaling via epigenetic repression of DKK1 in endometrial epithelial cells (EECs). While the loss of DKK1 is known to increase ß-catenin accumulation for WNT signaling activation, this epigenetic repression causes ß-catenin release from transmembrane integrins. Furthermore, PAI-1 elicits the disengagement of TIMP2 and SPARC from integrin-ß1 on the cell surface, lifting an integrin-ß1-ECM signaling constraint. The heightened interaction of integrin-ß1 with type 1 collagen (COL1) remodels extracellular fibrillar structures in the ECM. Consequently, the enhanced nanomechanical stiffness of this microenvironment is conducive to EEC motility and neoplastic transformation. The formation of extensively branched COL1 fibrils is also observed in endometrial tumors of patients with obesity. The findings highlight PAI-1 as a contributor to enhanced integrin-COL1 engagement and extensive ECM remodeling during obesity-associated neoplastic development.

Identification of EGFR R776H germline mutations in a patient with multifocal lung adenocarcinoma: A case report and literature review.

The advancement of molecular pathology techniques has led to the discovery of rare EGFR mutations for targeted therapy in lung cancer. Additionally, a substantial body of evidence indicates a connection between the development of lung cancer and genetic variations in the EGFR gene. Here, we present a case report of a patient with multifocal lung adenocarcinoma who possessed a rare germline mutation, EGFR R776H. An investigation into the family history of the patient exposed the notable incidence of lung adenocarcinoma, indicating a plausible genetic vulnerability to the ailment. To be specific, the patient's older brother and sister both suffered from lung cancer, which underlines the hereditary predisposition. Furthermore, it should be noted that the patient's daughter has inherited the germline mutation and also presented with multiple lung ground-glass nodules, emphasizing the clinical importance of this genetic variation. Following the lobectomy, the patient received treatment with almonertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), and at the latest follow-up, the patient has achieved partial remission. This case highlights the significance of taking into account germline possibilities when multiple lesions carry the same mutation. It stresses the importance of acquiring a comprehensive family history and performing genetic testing on leukocytes. Moreover, for the infrequent EGFR R776H mutation, third generation EGFR-TKIs may be a viable option.

Organocatalyzed Asymmetric Conjugate Addition of Alcohols to ß-Fluoroalkyl Vinylsulfones by Bifunctional Phosphonium Salt Catalyst.

Chiral secondary alcohols, serving as essential structural motifs, hold significant potential for diverse applications. The exploration of effective synthetic strategies toward these compounds is both attractive and challenging. Herein, we present an asymmetric oxa-Michael reaction involving aliphatic alcohols as nucleophiles and ß-fluoroalkyl vinylsulfones catalyzed by bifunctional phosphonium salt (BPS), achieving high yields and excellent enantioselectivities (up to 98 % yield and 98 % ee). Additionally, a sequential process including asymmetric oxa-Michael and debenzylation, facilitated by BPS/Lewis acid cooperation, was revealed for synthesizing diverse chiral secondary alcohol compounds in high yields (81-88 %) with consistent stereoselectivities. Furthermore, mechanistic explorations and subsequent results unveiled that the enantioselectivity originates from hydrogen-bonding and ion-pair interactions between the BPS catalyst and the substrates.

Organocatalytic skeletal reorganization for enantioselective synthesis of S-stereogenic sulfinamides.

The enantioselective synthesis of S-stereogenic sulfinamides has garnered considerable attention due to their structural and physicochemical properties. However, catalytic asymmetric synthesis of sulfinamides still remains daunting challenges, impeding their broad application in drug discovery and development. Here, we present an approach for the synthesis of S-stereogenic sulfinamides through peptide-mimic phosphonium salt-catalyzed asymmetric skeletal reorganization of simple prochiral and/or racemic sulfoximines. This methodology allows for the facile access to a diverse array of substituted sulfinamides with excellent enantioselectivities, accommodating various substituent patterns through desymmetrization or parallel kinetic resolution process. Mechanistic experiments, coupled with density functional theory calculations, clarify a stepwise pathway involving ring-opening and ring-closing processes, with the ring-opening step identified as crucial for achieving stereoselective control. Given the prevalence of S-stereogenic centers in pharmaceuticals, we anticipate that this protocol will enhance the efficient and precise synthesis of relevant chiral molecules and their analogs, thereby contributing to advancements in drug discovery.

Case report: Acquired resistance to crizotinib from a MET Y1230H mutation in a patient with non-small cell lung cancer and KIF5B-MET fusion.

Background: The c-met proto-oncogene (MET) serves as a significant primary oncogenic driver in non-small cell lung cancer (NSCLC) and has the potential to fuse with other genes, such as KIF5B, although it occurs infrequently. Only a limited number of reported cases have examined the clinical efficacy of crizotinib in patients with KIF5B-MET gene fusion, with no known data regarding acquired resistance to crizotinib and its potential mechanisms. In this report, we present the clinical progression of a female patient diagnosed with NSCLC and harboring a KIF5B-MET gene fusion. Case description: The patient initially exhibited partial response to first-line crizotinib treatment, albeit for a short duration and with limited efficacy. Subsequent disease progression revealed the emergence of a secondary MET mutation, specifically MET Y1230H, leading to acquired resistance to crizotinib. Conclusion: The reporting of this case is imperative for informing clinical practice, given the uncommon occurrence of NSCLC with MET fusion, displaying responsiveness to MET tyrosine kinase inhibitor therapy, as well as the emergence of the secondary Y1230H alteration as a potential resistance mechanism.

Effects of fermented sweet potato residue on nutrient digestibility, meat quality, and intestinal microbes in broilers.

This study aimed to investigate the effects of different proportions of dietary fermented sweet potato residue (FSPR) supplementation as a substitute for corn on the nutrient digestibility, meat quality, and intestinal microbes of yellow-feathered broilers. Experiment 1 (force-feeding) evaluated the nutrient composition and digestibility of mixtures with different proportions of sweet potato residue (70%, 80%, 90%, and 100%) before and after fermentation. In Experiment 2 (metabolic growth), a total of 420 one-day-old yellow-feathered broilers were randomly allocated to 4 groups and fed corn-soybean meal-based diets with 0, 5%, 8%, and 10% FSPR as a substitute for corn. The force-feeding and metabolic growth experiments were performed for 9 and 70 d, respectively. The treatment of 70% sweet potato residue (after fermentation) had the highest levels of crude protein, ether extract, and crude fiber and improved the digestibility of crude protein and amino acids (P < 0.05). Although dietary FSPR supplementation at different levels had no significant effect on growth performance and intestinal morphology, it improved slaughter rate, half-chamber rate, full clearance rate, and meat color, as well as reduced cooking loss in the breast and thigh muscles (P < 0.05). Dietary supplementation with 8% and 10% FSPR increased the serum immunoglobulin M and immunoglobulin G levels in broilers (P < 0.05). Furthermore, 10% FSPR increased the Shannon index and Ruminococcaceae_UCG-014, Ruminococcaceae_UCG-010 and Romboutsia abundances and decreased Sutterella and Megamonas abundances (P < 0.05). Spearman's correlation analysis showed that meat color was positively correlated with Ruminococcaceae_UCG-014 (P < 0.05) and negatively correlated with Megamonas (P < 0.05). Collectively, 70% sweet potato residue (after fermentation) had the best nutritional value and nutrient digestibility. Dietary supplementation with 8% to 10% FSPR as a substitute for corn can improve the slaughter performance, meat quality, and intestinal microbe profiles of broilers. Our findings suggest that FSPR has the potential to be used as a substitute for corn-soybean meals to improve the meat quality and intestinal health of broilers.

Salpingectomy for ectopic pregnancy reduces ovarian cancer risk-a nationwide study.

Recent studies propose fallopian tubes as the tissue origin for many ovarian epithelial cancers. To further support this paradigm, we assessed whether salpingectomy for treating ectopic pregnancy had a protective effect using the Taiwan Longitudinal National Health Research Database. We identified 316 882 women with surgical treatment for ectopic pregnancy and 3 168 820 age- and index-date-matched controls from 2000 to 2016. In a nested cohort, 91.5% of cases underwent unilateral salpingectomy, suggesting that most surgically managed patients have salpingectomy. Over a follow-up period of 17 years, the ovarian carcinoma incidence was 0.0069 (95% confidence interval [CI] = 0.0060 to 0.0079) and 0.0089 (95% CI = 0.0086 to 0.0092) in the ectopic pregnancy and the control groups, respectively (P < .001). After adjusting the events to per 100 person-years, the hazard ratio (HR) in the ectopic pregnancy group was 0.70 (95% CI = 0.61 to 0.80). The risk reduction occurred only in epithelial ovarian cancer (HR = 0.73, 95% CI = 0.63 to 0.86) and not in non-epithelial subtypes. These findings show a decrease in ovarian carcinoma incidence after salpingectomy for treating ectopic pregnancy.

Reconstructing atmospheric 129I deposition over 170 years with the varved sediment in the Sihailongwan Maar Lake, northeast China.

Long-term deposition of atmospheric radioactive iodine-129 (129I) is important for assessing the impact of human nuclear activities (HNAs), but still not well understood in East Asia. In this study, we quantitatively reconstructed the deposition history of airborne 129I using varved sediment from Sihailongwan Maar Lake (SHLW) in northeast China. Our results revealed significant increases in 129I concentrations and 129I/127I atomic ratios since the 1950s, indicating the influence of HNAs on the environment and marking the onset of the Anthropocene. The variation of 129I in the investigated site can be primarily attributed to the global fallout of ANWT as well as nuclear fuel reprocessing in Europe, Russia and the USA. Notably, neither the Chernobyl nor the Fukushima nuclear accidents have had any discernable impact on the SHLW Lake. Over the past 170 years (1846-2021), the reconstructed fluxes indicate a rapid increase in 129I deposition from the early 1950s until the 1970s followed by dramatic changes thereafter. The measured 129I fluxes range between (1.26-349) × 109 atoms m-2 yr-1 in the SHLW Lake, which are consistent with similar latitude zones across East Asia, but differ significantly from those observed in high-elevation glaciers within the Northern Hemisphere due to prevailing atmospheric circulation patterns. The total 129I inventory was calculated to be 11.9 × 1012 atoms m-2, with natural and anthropogenic 129I accounting for 2.86 % and 97.1 %, respectively, suggesting an overwhelming artificial contribution. The reconstructed fluxes and inventory of atmospheric 129I deposition quantitatively distinguish the natural and artificial contributions, and provide a novel insight into the historical environmental impact of HNAs in East Asia and the characteristics of the Anthropocene.

Novel Stereo-Induction Pattern in Pudovik Addition/Phospha-Brook Rearrangement Towards Chiral Trisubstituted Allenes.

Despite the significance of chiral allene skeletons in catalysis, organic synthesis and medicinal chemistry et al., there is a scarcity of reports on axially chiral allenyl phosphorus compounds. Here, we disclosed an efficient and straightforward cascade reaction between ethynyl ketones and phosphine oxides, resulting in a broad array of trisubstituted allenes incorporating a phosphorus moiety in high yields with excellent stereoselectivities facilitated by peptide-mimic phosphonium salt (PPS) catalysis, Additionally, comprehensive series of mechanistic experiments have been conducted to elucidate that this cascade reaction proceeds via an asymmetric Pudovik addition reaction followed by a subsequent phospha-Brook rearrangement that occurs concomitantly with kinetic resolution, representing a stereospecific rearrangement and protonation process facilitating central-to-axial chirality transfer in a cascade manner. We anticipate that our research will pave the way for a promising exploration of novel stereo-induction pattern in the Pudovik addition/phospha-Brook rearrangement cascade reaction.

PBX1 as a novel master regulator in cancer: Its regulation, molecular biology, and therapeutic applications.

PBX1 is a critical transcription factor at the top of various cell fate-determining pathways. In cancer, PBX1 stands at the crossroads of multiple oncogenic signaling pathways and mediates responses by recruiting a broad repertoire of downstream targets. Research thus far has corroborated the involvement of PBX1 in cancer proliferation, resisting apoptosis, tumor-associated neoangiogenesis, epithelial-mesenchymal transition (EMT) and metastasis, immune evasion, genome instability, and dysregulating cellular metabolism. Recently, our understanding of the functional regulation of the PBX1 protein has advanced, as increasing evidence has depicted a regulatory network consisting of transcriptional, post-transcriptional, and post-translational levels of control mechanisms. Furthermore, accumulating studies have supported the clinical utilization of PBX1 as a prognostic or therapeutic target in cancer. Preliminary results showed that PBX1 entails vast potential as a targetable master regulator in the treatment of cancer, particularly in those with high-risk features and resistance to other therapeutic strategies. In this review, we will explore the regulation, protein-protein interactions, molecular pathways, clinical application, and future challenges of PBX1.

Morphologic and Molecular Heterogeneity of High-grade Serous Carcinoma Precursor Lesions.

Serous tubal intraepithelial carcinoma (STIC) is the fallopian tube precursor lesion for most cases of pelvic high-grade serous carcinoma (HGSC). To date, the morphologic, molecular, and clinical heterogeneity of STIC and a less atypical putative precursor lesion, termed serous tubal intraepithelial lesion, has not been well characterized. Better understanding of precursor heterogeneity could impact the clinical management of women with incidental STICs (without concurrent carcinoma) identified in cases of prophylactic or opportunistic salpingectomy. This study analyzed morphologic and molecular features of 171 STICs and 21 serous tubal intraepithelial lesions. We assessed their histologic features, Ki-67 and p53 staining patterns, and genome-wide DNA copy number alterations. We classified all precursor lesions into 2 morphologic subtypes, one with a flat surface (Flat) and the other characterized by budding, loosely adherent, or detached (BLAD) morphology. On the basis of pathology review by a panel of 8 gynecologic pathologists, we found 87 BLAD, 96 Flat, and 9 indeterminate lesions. As compared with Flat lesions, BLAD lesions were more frequently diagnostic of STIC ( P <0.0001) and were found concurrently with HGSC ( P <0.0001). BLAD morphology was also characterized by higher Ki-67 proliferation index ( P <0.0001), presence of epithelial stratification ( P <0.0001), and increased lymphocyte density ( P <0.0001). BLAD lesions also exhibited more frequent DNA copy number gain/amplification at the CCNE1 or CMYC loci canonical to HGSCs ( P <0.0001). Both BLAD morphology and STIC diagnoses are independent risk factors for an elevated Ki-67 proliferation index. No correlation was observed between BLAD and Flat lesions with respect to patient age, presence of germline BRCA1/2 mutation, or p53 staining pattern. These findings suggest that tubal precursor lesions are morphologically and molecularly heterogeneous, laying the foundation for further studies on the pathogenesis of HGSC initiation and identifying histologic features predictive of poor patient outcomes.

Machine learning to detect the SINEs of cancer.

We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a machine learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to 7615 samples from 5178 individuals, 2073 with solid cancer and the remainder without cancer. Samples from patients with cancer and controls were prespecified into four cohorts used for model training, analyte integration, and threshold determination, validation, and reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the validation cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and eight common protein biomarkers detected 51% of the cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature-the global reduction of AluS subfamily elements in the circulating DNA of patients with solid cancer. We confirmed this reduction through the analysis of another independent dataset obtained with a different approach (whole-genome sequencing). The evaluation of Alu elements may therefore have the potential to enhance the performance of several methods designed for the earlier detection of cancer.

Aneuploidy Landscape in Precursors of Ovarian Cancer.

PURPOSE: Serous tubal intraepithelial carcinoma (STIC) is now recognized as the main precursor of ovarian high-grade serous carcinoma (HGSC). Other potential tubal lesions include p53 signatures and tubal intraepithelial lesions. We aimed to investigate the extent and pattern of aneuploidy in these epithelial lesions and HGSC to define the features that characterize stages of tumor initiation and progression. EXPERIMENTAL DESIGN: We applied RealSeqS to compare genome-wide aneuploidy patterns among the precursors, HGSC (cases, n = 85), and histologically unremarkable fallopian tube epithelium (HU-FTE; control, n = 65). On the basis of a discovery set (n = 67), we developed an aneuploidy-based algorithm, REAL-FAST (Repetitive Element AneupLoidy Sequencing Fallopian Tube Aneuploidy in STIC), to correlate the molecular data with pathology diagnoses. We validated the result in an independent validation set (n = 83) to determine its performance. We correlated the molecularly defined precursor subgroups with proliferative activity and histology. RESULTS: We found that nearly all p53 signatures lost the entire Chr17, offering a "two-hit" mechanism involving both TP53 and BRCA1 in BRCA1 germline mutation carriers. Proliferatively active STICs harbor gains of 19q12 (CCNE1), 19q13.2, 8q24 (MYC), or 8q arm, whereas proliferatively dormant STICs show 22q loss. REAL-FAST classified HU-FTE and STICs into 5 clusters and identified a STIC subgroup harboring unique aneuploidy that is associated with increased proliferation and discohesive growth. On the basis of a validation set, REAL-FAST showed 95.8% sensitivity and 97.1% specificity in detecting STIC/HGSC. CONCLUSIONS: Morphologically similar STICs are molecularly distinct. The REAL-FAST assay identifies a potentially "aggressive" STIC subgroup harboring unique DNA aneuploidy that is associated with increased cellular proliferation and discohesive growth. REAL-FAST offers a highly reproducible adjunct technique to assist the diagnosis of STIC lesions.