Post-radiotherapy stage III/IV non-small cell lung cancer radiomics research: a systematic review and comparison of CLEAR and RQS frameworks.

BACKGROUND: Lung cancer, the second most common cancer, presents persistently dismal prognoses. Radiomics, a promising field, aims to provide novel imaging biomarkers to improve outcomes. However, clinical translation faces reproducibility challenges, despite efforts to address them with quality scoring tools. OBJECTIVE: This study had two objectives: 1) identify radiomics biomarkers in post-radiotherapy stage III/IV nonsmall cell lung cancer (NSCLC) patients, 2) evaluate research quality using the CLEAR (CheckList_for_EvaluAtion_of_Radiomics_research), RQS (Radiomics_Quality_Score) frameworks, and formulate an amalgamated CLEAR-RQS tool to enhance scientific rigor. MATERIALS AND METHODS: A systematic literature review (Jun-Aug 2023, MEDLINE/PubMed/SCOPUS) was conducted concerning stage III/IV NSCLC, radiotherapy, and radiomic features (RF). Extracted data included study design particulars, such as sample size, radiotherapy/CT technique, selected RFs, and endpoints. CLEAR and RQS were merged into a CLEAR-RQS checklist. Three readers appraised articles utilizing CLEAR, RQS, and CLEAR-RQS metrics. RESULTS: Out of 871 articles, 11 met the inclusion/exclusion criteria. The Median cohort size was 91 (range: 10-337) with 9 studies being single-center. No common RF were identified. The merged CLEAR-RQS checklist comprised 61 items. Most unreported items were within CLEAR's "methods" and "open-source," and within RQS's "phantom-calibration," "registry-enrolled prospective-trial-design," and "cost-effective-analysis" sections. No study scored above 50% on RQS. Median CLEAR scores were 55.74% (32.33/58 points), and for RQS, 17.59% (6.3/36 points). CLEAR-RQS article ranking fell between CLEAR and RQS and aligned with CLEAR. CONCLUSION: Radiomics research in post-radiotherapy stage III/IV NSCLC exhibits variability and frequently low-quality reporting. The formulated CLEAR-RQS checklist may facilitate education and holds promise for enhancing radiomics research quality. CLINICAL RELEVANCE STATEMENT: Current radiomics research in the field of stage III/IV postradiotherapy NSCLC is heterogenous, lacking reproducibility, with no identified imaging biomarker. Radiomics research quality assessment tools may enhance scientific rigor and thereby facilitate radiomics translation into clinical practice. KEY POINTS: There is heterogenous and low radiomics research quality in postradiotherapy stage III/IV nonsmall cell lung cancer. Barriers to reproducibility are small cohort size, nonvalidated studies, missing technical parameters, and lack of data, code, and model sharing. CLEAR (CheckList_for_EvaluAtion_of_Radiomics_research), RQS (Radiomics_Quality_Score), and the amalgamated CLEAR-RQS tool are useful frameworks for assessing radiomics research quality and may provide a valuable resource for educational purposes in the field of radiomics.

Are parental condition transfer effects more widespread than is currently appreciated?

It has long been recognized that the environment experienced by parents can influence the traits of offspring (i.e. 'parental effects'). Much research has explored whether mothers respond to predictable shifts in environmental signals by modifying offspring phenotypes to best match future conditions. Many organisms experience conditions that theory predicts should favor the evolution of such 'anticipatory parental effects', but such predictions have received limited empirical support. 'Condition transfer effects' are an alternative to anticipatory effects that occur when the environment experienced by parents during development influences offspring fitness. Condition transfer effects occur when parents that experience high-quality conditions produce offspring that exhibit higher fitness irrespective of the environmental conditions in the offspring generation. Condition transfer effects are not driven by external signals but are instead a byproduct of past environmental quality. They are also likely adaptive but have received far less attention than anticipatory effects. Here, we review the generality of condition transfer effects and show that they are much more widespread than is currently appreciated. Condition transfer effects are observed across taxa and are commonly associated with experimental manipulations of resource conditions experienced by parents. Our Review calls for increased research into condition transfer effects when considering the role of parental effects in ecology and evolution.

Expanding Complex Morpholines Using Systematic Chemical Diversity.

The morpholine heterocycle is a structural unit found in many bioactive compounds and FDA-approved drugs, but the generation of more complex C-functionalized morpholine derivatives remains considerably underexplored. Using systematic chemical diversity (SCD), a concept that guides the expansion of saturated drug-like scaffolds through regiochemical and stereochemical variation, we describe the synthesis of a collection of methyl-substituted morpholine acetic acid esters starting from enantiomerically pure amino acids and amino alcohols. In total, 24 diverse substituted morpholines were produced that vary systematically in regiochemistry and stereochemistry (relative and absolute). These diverse C-substituted morpholines can be directly applied in fragment screening or incorporated as building blocks in medicinal chemistry and library synthesis.

Investigation of the molecular switching process between spin crossover states of triazole complexes as basis for optical sensing applications.

With the advent of the first laser sources and suitable detectors, optical sensor applications immediately also came into focus. During the last decades, a huge variety of optical sensor concepts were developed, yet the forecast for the future application potential appears even larger. In this context, the development of new sensor probes at different scales down to the atomic or molecular level open new avenues for research and development. We investigated an iron based triazole molecular spin-crossover complex changing its absorption characteristics significantly by varying environmental parameters such as humidity, temperature, magnetic or electric field, respectively, with respect to its suitability for a new class of versatile molecular sensor probes. Hereby, besides the investigation of synthesized pure bulk material using different analyzing methods, we also studied amorphous micro particles which were applied in or onto optical waveguide structures. We found that significant changes of the reflection spectra can also be obtained after combining the particles with different types of optical waveguides.The obtained results demonstrate the suitability of the material complex for a broad field of future sensor applications.

Identification of potential microbial risk factors associated with fecal indicator exceedances at recreational beaches.

BACKGROUND: Fecal bacterial densities are proxy indicators of beach water quality, and beach posting decisions are made based on Beach Action Value (BAV) exceedances for a beach. However, these traditional beach monitoring methods do not reflect the full extent of microbial water quality changes associated with BAV exceedances at recreational beaches (including harmful cyanobacteria). This proof of concept study evaluates the potential of metagenomics for comprehensively assessing bacterial community changes associated with BAV exceedances compared to non-exceedances for two urban beaches and their adjacent river water sources. RESULTS: Compared to non-exceedance samples, BAV exceedance samples exhibited higher alpha diversity (diversity within the sample) that could be further differentiated into separate clusters (Beta-diversity). For Beach A, Cyanobacterial sequences (resolved as Microcystis and Pseudanabaena at genus level) were significantly more abundant in BAV non-exceedance samples. qPCR validation supported the Cyanobacterial abundance results from metagenomic analysis and also identified saxitoxin genes in 50% of the non-exceedance samples. Microcystis sp and saxitoxin gene sequences were more abundant on non-exceedance beach days (when fecal indicator data indicated the beach should be open for water recreational purposes). For BAV exceedance days, Fibrobacteres, Pseudomonas, Acinetobacter, and Clostridium sequences were significantly more abundant (and positively correlated with fecal indicator densities) for Beach A. For Beach B, Spirochaetes (resolved as Leptospira on genus level) Burkholderia and Vibrio sequences were significantly more abundant in BAV exceedance samples. Similar bacterial diversity and abundance trends were observed for river water sources compared to their associated beaches. Antibiotic Resistance Genes (ARGs) were also consistently detected at both beaches. However, we did not observe a significant difference or correlation in ARGs abundance between BAV exceedance and non-exceedance samples. CONCLUSION: This study provides a more comprehensive analysis of bacterial community changes associated with BAV exceedances for recreational freshwater beaches. While there were increases in bacterial diversity and some taxa of potential human health concern associated with increased fecal indicator densities and BAV exceedances (e.g. Pseudomonas), metagenomics analyses also identified other taxa of potential human health concern (e.g. Microcystis) associated with lower fecal indicator densities and BAV non-exceedances days. This study can help develop more targeted beach monitoring strategies and beach-specific risk management approaches.

Supraclavicular brown adipocytes originate from Tbx1+ myoprogenitors.

Brown adipose tissue (BAT) dissipates energy as heat, contributing to temperature control, energy expenditure, and systemic homeostasis. In adult humans, BAT mainly exists in supraclavicular areas and its prevalence is associated with cardiometabolic health. However, the developmental origin of supraclavicular BAT remains unknown. Here, using genetic cell marking in mice, we demonstrate that supraclavicular brown adipocytes do not develop from the Pax3+/Myf5+ epaxial dermomyotome that gives rise to interscapular BAT (iBAT). Instead, the Tbx1+ lineage that specifies the pharyngeal mesoderm marks the majority of supraclavicular brown adipocytes. Tbx1Cre-mediated ablation of peroxisome proliferator-activated receptor gamma (PPARγ) or PR/SET Domain 16 (PRDM16), components of the transcriptional complex for brown fat determination, leads to supraclavicular BAT paucity or dysfunction, thus rendering mice more sensitive to cold exposure. Moreover, human deep neck BAT expresses higher levels of the TBX1 gene than subcutaneous neck white adipocytes. Taken together, our observations reveal location-specific developmental origins of BAT depots and call attention to Tbx1+ lineage cells when investigating human relevant supraclavicular BAT.

Retrospective Analysis of Radiation-Induced Complications of Uveal Melanoma Patients Treated With Brachytherapy in the Era of Anti-VEGF.

PURPOSE: To associate clinical factors and radiation doses delivered by iodine-125 plaque brachytherapy to visual outcomes and development of radiation-induced ocular complications in patients with uveal melanoma in the era of anti-vascular endothelial growth factor (anti-VEGF) injections. DESIGN: Retrospective cohort study. METHODS: A retrospective chart review was performed for 225 patients treated with iodine-125 brachytherapy for uveal melanoma. The effects of radiation doses (focal doses, average dose to the entire eye, and integral dose) on visual outcomes and development of radiation complications (radiation retinopathy, radiation optic neuropathy, vitreous hemorrhage, and neovascular glaucoma) were analyzed using multivariate Cox regression snalysis. RESULTS: Median follow-up was 33.6 months (range, 12-105.6 months). Radiation retinopathy was associated with younger age, tumor distance to optic nerve <6 mm, and maximum radiation dose to fovea. Radiation optic neuropathy was associated with White race, tumor distance to optic nerve <6 mm, and integral radiation dose. Vitreous hemorrhage was associated with White race and integral radiation dose. Incidence of neovascular glaucoma was low in our study, with 2 patients (0.9%) developing the complication. Of the 123 patients who developed radiation retinopathy, 82 patients (66.7% of radiation retinopathy patients, 37.3% of total patients) received anti-VEGF injections. CONCLUSIONS: Our study found multiple associations between radiation doses and complications as well as visual outcomes on multivariate analysis. Given that the majority of our patients who developed radiation retinopathy received anti-VEGF injections, our study helps to illustrate the course and progression of radiation-induced complications in the new era of anti-VEGF.

A TrkB and TrkC partial agonist restores deficits in synaptic function and promotes activity-dependent synaptic and microglial transcriptomic changes in a late-stage Alzheimer's mouse model.

Introduction: TrkB and TrkC receptor signaling promotes synaptic plasticity and interacts with pathways affected by amyloid-ß (Aß)-toxicity. Upregulating TrkB/C signaling could reduce Alzheimer's disease (AD)-related degenerative signaling, memory loss, and synaptic dysfunction. Methods: PTX-BD10-2 (BD10-2), a small molecule TrkB/C receptor partial agonist, was orally administered to aged London/Swedish-APP mutant mice (APP L/S ) and wild-type controls (WT). Effects on memory and hippocampal long-term potentiation (LTP) were assessed using electrophysiology, behavioral studies, immunoblotting, immunofluorescence staining, and RNA-sequencing. Results: Memory and LTP deficits in APP L/S mice were attenuated by treatment with BD10-2. BD10-2 prevented aberrant AKT, CaMKII, and GLUA1 phosphorylation, and enhanced activity-dependent recruitment of synaptic proteins. BD10-2 also had potentially favorable effects on LTP-dependent complement pathway and synaptic gene transcription. Conclusions: BD10-2 prevented APP L/S /Aß-associated memory and LTP deficits, reduced abnormalities in synapse-related signaling and activity-dependent transcription of synaptic genes, and bolstered transcriptional changes associated with microglial immune response.

Dynamic DNA methylation turnover in gene bodies is associated with enhanced gene expression plasticity in plants.

BACKGROUND: In several eukaryotes, DNA methylation occurs within the coding regions of many genes, termed gene body methylation (GbM). Whereas the role of DNA methylation on the silencing of transposons and repetitive DNA is well understood, gene body methylation is not associated with transcriptional repression, and its biological importance remains unclear. RESULTS: We report a newly discovered type of GbM in plants, which is under constitutive addition and removal by dynamic methylation modifiers in all cells, including the germline. Methylation at Dynamic GbM genes is removed by the DRDD demethylation pathway and added by an unknown source of de novo methylation, most likely the maintenance methyltransferase MET1. We show that the Dynamic GbM state is present at homologous genes across divergent lineages spanning over 100 million years, indicating evolutionary conservation. We demonstrate that Dynamic GbM is tightly associated with the presence of a promoter or regulatory chromatin state within the gene body, in contrast to other gene body methylated genes. We find Dynamic GbM is associated with enhanced gene expression plasticity across development and diverse physiological conditions, whereas stably methylated GbM genes exhibit reduced plasticity. Dynamic GbM genes exhibit reduced dynamic range in drdd mutants, indicating a causal link between DNA demethylation and enhanced gene expression plasticity. CONCLUSIONS: We propose a new model for GbM in regulating gene expression plasticity, including a novel type of GbM in which increased gene expression plasticity is associated with the activity of DNA methylation writers and erasers and the enrichment of a regulatory chromatin state.

Pre-analytical effects on whole transcriptome and targeted RNA sequencing analysis in cytology: The effects of prolonged time in storage of effusion specimens prior to preservation.

OBJECTIVES: To investigate the pre-analytics of the molecular testing of cytology specimens, we studied the effects of time in refrigerator storage (4°C) of malignant effusions on RNA sequencing (RNAseq) results. METHODS: Ten effusion specimens were stored in a refrigerator (4°C) for different durations (day 0, 1, 4, and 7). All specimens were prepared as cytospins fixed in either Carnoy's solution or 95% ethanol (EtOH) and in an RNA preservative for a fresh frozen (FF) high-quality reference. Whole transcriptome (wt) and targeted (t)RNAseq of two multigene expression signatures were performed. We then compared transcript expression levels (including mutant allele fraction) according to pre-analytical variables using a concordance correlation coefficient (CCC) and a mixed effect model. RESULTS: Sequencing results were mostly stable over increasing time in storage. Cytospins fixed in Carnoy's solution were more concordant with FF samples than cytospins fixed in 95% EtOH at all timepoints. This finding was consistent for both wtRNAseq (averages: day 0 CCC = 0.98 vs 0.91; day 7 CCC = 0.88 vs 0.78) and tRNAseq methods (averages: day 0 CCC = 0.98 vs 0.81; day 7 CCC = 0.98 vs 0.90). Cytospins fixed in Carnoy's solution did not show significant changes in expression over timepoints or between expression signatures, whereas 95% EtOH did. CONCLUSION: RNAseq can be accurately performed on effusion specimens after prolonged refrigerator storage. RNA extracted from scraped cytospin slides fixed in Carnoy's solution was marginally superior to 95% EtOH fixation, but either method had comparable analytic performance to high-quality FF RNA samples.

Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity.

Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.

Heterocyclic Merging of Stereochemically Diverse Chiral Piperazines and Morpholines with Indazoles.

We report a heterocyclic merging approach to construct novel indazolo-piperazines and indazolo-morpholines. Starting from chiral diamines and amino alcohols, novel regiochemically (1,3 and 1,4) and stereochemically diverse (relative and absolute) cohorts of indazolo-piperazines and indazolo-morpholines were obtained within six or seven steps. The key transformations involved are a Smiles rearrangement to generate the indazole core structure and a late-stage Michael addition to build the piperazine and morpholine heterocycles. We further explored additional vector diversity by incorporating substitutions on the indazole aromatic ring, generating a total of 20 unique, enantiomerically pure heterocyclic scaffolds.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Incorporation and Deposition of Spin Crossover Materials into and onto Electrospun Nanofibers.

We synthesized iron(II)-triazole spin crossover compounds of the type [Fe(atrz)3]X2 and incorporated and deposited them on electrospun polymer nanofibers. For this, we used two separate electrospinning methods with the goal of obtaining polymer complex composites with intact switching properties. In view of possible applications, we chose iron(II)-triazole-complexes that are known to exhibit spin crossover close to ambient temperature. Therefore, we used the complexes [Fe(atrz)3]Cl2 and [Fe(atrz)3](2ns)2 (2ns = 2-Naphthalenesulfonate) and deposited those on fibers of polymethylmethacrylate (PMMA) and incorporated them into core-shell-like PMMA fiber structures. These core-shell structures showed to be inert to outer environmental influences, such as droplets of water, which we purposely cast on the fiber structure, and it did not rinse away the used complex. We analyzed both the complexes and the composites with IR-, UV/Vis, Mössbauer spectroscopy, SQUID magnetometry, as well as SEM and EDX imaging. The analysis via UV/Vis spectroscopy, Mössbauer spectroscopy, and temperature-dependent magnetic measurements with the SQUID magnetometer showed that the spin crossover properties were maintained and were not changed after the electrospinning processes.

Para-conduit diaphragmatic hernia following esophagectomy-the new price of minimally invasive surgery?

Esophageal Cancer is the seventh commonest cancer worldwide with poor overall survival. Significant morbidity related to open esophagectomy has driven practice toward hybrid, totally minimally invasive and robotic procedures. With the increase in minimally invasive approaches, it has been suggested that there might be an increased incidence of subsequent para-conduit diaphragmatic hernia. To assess the incidence, modifiable risk factors and association with operative approach of this emerging complication, we evaluated outcomes following esophagectomy from two Australian Centers. Prospectively collected databases were examined to identify patients who developed versus did not develop a para-conduit hernia. Patient characteristics, disease factors, treatment factors, operative and post-operative factors were compared for these two groups. A total of 24 of 297 patients who underwent esophagectomy were diagnosed with a symptomatic para-conduit diaphragmatic hernia (8.1%). The significant risk factor for hernia was a minimally invasive abdominal approach (70.8% vs. 35.5%; P = 0.004, odds ratio = 12.876, 95% CI 2.214-74.89). Minimally invasive thoracic approaches were not associated with increased risk. Minimally invasive abdominal approaches to esophagectomy doubled the risk of developing a para-conduit diaphragmatic hernia. Effective operative solutions to address this complication are required.

Prognostic impact of biologically equivalent dose in stereotactic body radiotherapy for renal cancer.

Purpose /Objectives Materials/Methods: The National Cancer Database (NCDB) was queried (2004-2017) for patients with RCC who did not have surgical resection but received definitive SBRT. Kaplan-Meier analysis with log-rank test was used to evaluate overall survival (OS). Univariable (UVA) and multivariable (MVA) analysis were conducted using cox proportional hazard models to determine prognostic factors for OS. Results: A total of 344 patients with median age 77 (IQR 70-85) were included in this study. Median BED3 was 180 Gy (IQR 126.03-233.97). Median OS was 90 months in the highest quartile compared to 36-52 months in the lower three quartiles (p < 0.01). On UVA, the highest BED3 quartile was a positive prognostic factor (HR 0.67, p < 0.01 CI 0.51-0.91) while age, tumor size, T-stage, metastasis, renal pelvis location, and transitional cell histology were negative factors. On MVA, the highest BED3 quartile was remained significant (HR 0.69, p = 0.02; CI 0.49-0.95) as a positive factor, while age, metastasis were negative factors. Conclusion: Higher BED may be associated with improved OS. Prospective investigation is needed to clearly define optimal BED for SBRT used to treat RCC.

Untangling the intertwined: metallic to semiconducting phase transition of colloidal MoS2 nanoplatelets and nanosheets.

2D semiconducting transition metal dichalcogenides (TMDCs) are highly promising materials for future spin- and valleytronic applications and exhibit an ultrafast response to external (optical) stimuli which is essential for optoelectronics. Colloidal nanochemistry on the other hand is an emerging alternative for the synthesis of 2D TMDC nanosheet (NS) ensembles, allowing for the control of the reaction via tunable precursor and ligand chemistry. Up to now, wet-chemical colloidal syntheses yielded intertwined/agglomerated NSs with a large lateral size. Here, we show a synthesis method for 2D mono- and bilayer MoS2 nanoplatelets with a particularly small lateral size (NPLs, 7.4 nm ± 2.2 nm) and MoS2 NSs (22 nm ± 9 nm) as a reference by adjusting the molybdenum precursor concentration in the reaction. We find that in colloidal 2D MoS2 syntheses initially a mixture of the stable semiconducting and the metastable metallic crystal phase is formed. 2D MoS2 NPLs and NSs then both undergo a full transformation to the semiconducting crystal phase by the end of the reaction, which we quantify by X-ray photoelectron spectroscopy. Phase pure semiconducting MoS2 NPLs with a lateral size approaching the MoS2 exciton Bohr radius exhibit strong additional lateral confinement, leading to a drastically shortened decay of the A and B exciton which is characterized by ultrafast transient absorption spectroscopy. Our findings represent an important step for utilizing colloidal TMDCs, for example small MoS2 NPLs represent an excellent starting point for the growth of heterostructures for future colloidal photonics.

Molecular testing opportunities on cytology effusion specimens: the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing.

INTRODUCTION: RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections. MATERIALS AND METHODS: We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC). RESULTS: The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods. CONCLUSIONS: Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.

Role of 21-Gene Recurrence Score in Predicting Prognostic Benefit of Radiation Therapy After Breast-Conserving Surgery for T1N0 Breast Cancer.

PURPOSE: The 21-gene RT-PCR recurrence score (RS) is performed in patients with hormone receptor-positive (ER+, PR+), human epidermal growth factor receptor 2 (HER2)-negative, N0 breast cancer to determine which patients will likely benefit from chemotherapy after breast-conserving surgery (BCS). The purpose of this study was to evaluate whether the RS can predict for patients likely to benefit from radiation therapy (RT) after BCS. METHODS AND MATERIALS: The National Cancer Database was queried (2004-2017) for female patients with pT1N0 ER+ PR+ HER2-negative breast cancer treated with BCS who had an available RS. Patients were stratified based on their RS (low risk [LR], 1-10; intermediate risk [IR], 11-25; high risk [HR], 26-100). For each RS cohort, propensity score matching was conducted to create 1:1 matched cohorts of patients who received RT and patients who did not. Kaplan-Meier analysis evaluated overall survival (OS). Univariable and multivariable (MVA) Cox proportional hazard analysis identified clinical and treatment factors prognostic for OS. RESULTS: A total of 79,040 patients met the selection criteria: 18,823 in the LR cohort, 52,341 in the IR cohort, and 7876 in the HR cohort. A total of 92% of patients received RT: 91% in the LR cohort, 93% in the IR cohort, and 92% in the HR cohort. After propensity score matching, the 5-year OS in the LR cohort was 95% for those who received RT and 93% for those who did not (P = .184). In the IR cohort, the 5-year OS was 95% for those who received RT and 93% for those who did not (P = .001). In the HR cohort, the 5-year OS was 95% for those who received RT and 84% for those who did not (P < .001). MVA demonstrated that RT was a positive prognostic factor for OS in both the IR cohort (P = .001) and HR cohort (P < .001). On MVA in the LR cohort, RT (P = .186) was not predictive of improved OS. CONCLUSIONS: An OS benefit was observed with the use of RT in patients with IR or HR RS but not in patients with LR RS. Future prospective evaluation is warranted.

Systematic profiling of conditional degron tag technologies for target validation studies.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.