Differential response of placental cells to high D-glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras-related protein RAB-7A.

Placental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5-mM D-glucose (i.e. control) or 25-mM D-glucose (high glucose). Cell-conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K-g, pellet 10K-g, and pellet 100K-g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRMHR) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n = 6) and those with GDM (n = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.

Exploration of novel cationic amino acid-enriched short peptides: design, SPPS, biological evaluation and in silico study.

Antimicrobial resistance (AMR) represents a critical challenge worldwide, necessitating the pursuit of novel approaches to counteract bacterial and fungal pathogens. In this context, we explored the potential of cationic amino acid-enriched short peptides, synthesized via solid-phase methods, as innovative antimicrobial candidates. Our comprehensive evaluation assessed the antibacterial and antifungal efficacy of these peptides against a panel of significant pathogens, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, and Aspergillus niger. Utilizing molecular docking techniques, we delved into the molecular interactions underpinning the peptides' action against these microorganisms. The results revealed a spectrum of inhibitory activities, with certain peptide sequences displaying pronounced effectiveness across various pathogens. These findings underscore the peptides' potential as promising antimicrobial agents, with molecular docking offering valuable insights into their mechanisms of action. This study enriches antimicrobial peptide (AMP) research by identifying promising candidates for further refinement and development toward therapeutic application, highlighting their significance in addressing the urgent issue of AMR.

Dataset for reporting of the invasive carcinoma of the breast: recommendations from the International Collaboration on Cancer Reporting (ICCR).

BACKGROUND AND OBJECTIVES: Current national or regional guidelines for the pathology reporting on invasive breast cancer differ in certain aspects, resulting in divergent reporting practice and a lack of comparability of data. Here we report on a new international dataset for the pathology reporting of resection specimens with invasive cancer of the breast. The dataset was produced under the auspices of the International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organizations. METHODS AND RESULTS: The established ICCR process for dataset development was followed. An international expert panel consisting of breast pathologists, a surgeon, and an oncologist prepared a draft set of core and noncore data items based on a critical review and discussion of current evidence. Commentary was provided for each data item to explain the rationale for selecting it as a core or noncore element, its clinical relevance, and to highlight potential areas of disagreement or lack of evidence, in which case a consensus position was formulated. Following international public consultation, the document was finalized and ratified, and the dataset, which includes a synoptic reporting guide, was published on the ICCR website. CONCLUSIONS: This first international dataset for invasive cancer of the breast is intended to promote high-quality, standardized pathology reporting. Its widespread adoption will improve consistency of reporting, facilitate multidisciplinary communication, and enhance comparability of data, all of which will help to improve the management of invasive breast cancer patients.

Magnetic resonance imaging of tumor response to stroma-modifying pegvorhyaluronidase alpha (PEGPH20) therapy in early-phase clinical trials.

Pre-clinical and clinical studies have shown that PEGPH20 depletes intratumoral hyaluronic acid (HA), which is linked to high interstitial fluid pressures and poor distribution of chemotherapies. 29 patients with metastatic advanced solid tumors received quantitative magnetic resonance imaging (qMRI) in 3 prospective clinical trials of PEGPH20: HALO-109-101 (NCT00834704), HALO-109-102 (NCT01170897), and HALO-109-201 (NCT01453153). Apparent Diffusion Coefficient of water (ADC), T1, ktrans, vp, ve, and iAUC maps were computed from qMRI acquired at baseline and ≥ 1 time point post-PEGPH20. Tumor ADC and T1 decreased, while iAUC, ktrans, vp, and ve increased, on day 1 post-PEGPH20 relative to baseline values. This is consistent with HA depletion leading to a decrease in tumor extracellular water content and an increase in perfusion, permeability, extracellular matrix space, and vascularity. Baseline parameter values predictive of pharmacodynamic responses were: ADC > 1.46 × 10-3 mm2/s (Balanced Accuracy (BA) = 72%, p < 0.01), T1 > 0.54 s (BA = 82%, p < 0.01), iAUC < 9.2 mM-s (BA = 76%, p < 0.05), ktrans < 0.07 min-1 (BA = 72%, p = 0.2), ve < 0.17 (BA = 68%, p < 0.01), and vp < 0.02 (BA = 60%, p < 0.01). A low ve at baseline was moderately predictive of response in any parameter (BA = 65.6%, p < 0.01 averaged across patients). These qMRI biomarkers are potentially useful for guiding patient pre-selection and post-treatment follow-up in future clinical studies of PEGPH20 and other tumor stroma-modifying anti-cancer therapies.

B7-H3 Expression in Breast Cancer and Brain Metastasis.

Brain metastasis is a significant challenge for some breast cancer patients, marked by its aggressive nature, limited treatment options, and poor clinical outcomes. Immunotherapies have emerged as a promising avenue for brain metastasis treatment. B7-H3 (CD276) is an immune checkpoint molecule involved in T cell suppression, which is associated with poor survival in cancer patients. Given the increasing number of clinical trials using B7-H3 targeting CAR T cell therapies, we examined B7-H3 expression across breast cancer subtypes and in breast cancer brain metastases to assess its potential as an interventional target. B7-H3 expression was investigated using immunohistochemistry on tissue microarrays of three clinical cohorts: (i) unselected primary breast cancers (n = 347); (ii) brain metastatic breast cancers (n = 61) and breast cancer brain metastases (n = 80, including a subset of 53 patient-matched breast and brain metastasis cases); and (iii) mixed brain metastases from a range of primary tumours (n = 137). In primary breast cancers, B7-H3 expression significantly correlated with higher tumour grades and aggressive breast cancer subtypes, as well as poorer 5-year survival outcomes. Subcellular localisation of B7-H3 impacted breast cancer-specific survival, with cytoplasmic staining also correlating with a poorer outcome. Its expression was frequently detected in brain metastases from breast cancers, with up to 90% expressing B7-H3. However, not all brain metastases showed high levels of expression, with those from colorectal and renal tumours showing a low frequency of B7-H3 expression (0/14 and 2/16, respectively). The prevalence of B7-H3 expression in breast cancers and breast cancer brain metastases indicates potential opportunities for B7-H3 targeted therapies in breast cancer management.

A dedicated structured data set for reporting of invasive carcinoma of the breast in the setting of neoadjuvant therapy: recommendations from the International Collaboration on Cancer Reporting (ICCR).

AIMS: The International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organisations, is an initiative aimed at providing a unified international approach to reporting cancer. ICCR recently published new data sets for the reporting of invasive breast carcinoma, surgically removed lymph nodes for breast tumours and ductal carcinoma in situ, variants of lobular carcinoma in situ and low-grade lesions. The data set in this paper addresses the neoadjuvant setting. The aim is to promote high-quality, standardised reporting of tumour response and residual disease after neoadjuvant treatment that can be used for subsequent management decisions for each patient. METHODS: The ICCR convened expert panels of breast pathologists with a representative surgeon and oncologist to critically review and discuss current evidence. Feedback from the international public consultation was critical in the development of this data set. RESULTS: The expert panel concluded that a dedicated data set was required for reporting of breast specimens post-neoadjuvant therapy with inclusion of data elements specific to the neoadjuvant setting as core or non-core elements. This data set proposes a practical approach for handling and reporting breast resection specimens following neoadjuvant therapy. The comments for each data element clarify terminology, discuss available evidence and highlight areas with limited evidence that need further study. This data set overlaps with, and should be used in conjunction with, the data sets for the reporting of invasive breast carcinoma and surgically removed lymph nodes from patients with breast tumours, as appropriate. Key issues specific to the neoadjuvant setting are included in this paper. The entire data set is freely available on the ICCR website. CONCLUSIONS: High-quality, standardised reporting of tumour response and residual disease after neoadjuvant treatment are critical for subsequent management decisions for each patient.

Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) Samples from Advanced Non-Small Cell Lung Cancer for Whole Genome, Whole Exome and Comprehensive Panel Sequencing.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often the only source of tumor tissue from patients with advanced, inoperable lung cancer. EBUS-TBNA aspirates are used for the diagnosis, staging, and genomic testing to inform therapy options. Here we extracted DNA and RNA from 220 EBUS-TBNA aspirates to evaluate their suitability for whole genome (WGS), whole exome (WES), and comprehensive panel sequencing. For a subset of 40 cases, the same nucleic acid extraction was sequenced using WGS, WES, and the TruSight Oncology 500 assay. Genomic features were compared between sequencing platforms and compared with those reported by clinical testing. A total of 204 aspirates (92.7%) had sufficient DNA (100 ng) for comprehensive panel sequencing, and 109 aspirates (49.5%) had sufficient material for WGS. Comprehensive sequencing platforms detected all seven clinically reported tier 1 actionable mutations, an additional three (7%) tier 1 mutations, six (15%) tier 2-3 mutations, and biomarkers of potential immunotherapy benefit (tumor mutation burden and microsatellite instability). As expected, WGS was more suited for the detection and discovery of emerging novel biomarkers of treatment response. WGS could be performed in half of all EBUS-TBNA aspirates, which points to the enormous potential of EBUS-TBNA as source material for large, well-curated discovery-based studies for novel and more effective predictors of treatment response. Comprehensive panel sequencing is possible in the vast majority of fresh EBUS-TBNA aspirates and enhances the detection of actionable mutations over current clinical testing.

Discrepancies in tumor mutation burden reporting from sequential endobronchial ultrasound transbronchial needle aspiration samples within single lymph node stations - brief report.

Introduction: Tumour Mutation Burden (TMB) is a potential biomarker for immune cancer therapies. Here we investigated parameters that might affect TMB using duplicate cytology smears obtained from endobronchial ultrasound transbronchial needle aspiration (EBUS TBNA)-sampled malignant lymph nodes. Methods: Individual Diff-Quik cytology smears were prepared for each needle pass. DNA extracted from each smear underwent sequencing using large gene panel (TruSight Oncology 500 (TSO500 - Illumina)). TMB was estimated using the TSO500 Local App v. 2.0 (Illumina). Results: Twenty patients had two or more Diff-Quik smears (total 45 smears) which passed sequencing quality control. Average smear TMB was 8.7 ± 5.0 mutations per megabase (Mb). Sixteen of the 20 patients had paired samples with minimal differences in TMB score (average difference 1.3 ± 0.85). Paired samples from 13 patients had concordant TMB (scores below or above a threshold of 10 mutations/Mb). Markedly discrepant TMB was observed in four cases, with an average difference of 11.3 ± 2.7 mutations/Mb. Factors affecting TMB calling included sample tumour content, the amount of DNA used in sequencing, and bone fide heterogeneity of node tumour between paired samples. Conclusion: TMB assessment is feasible from EBUS-TBNA smears from a single needle pass. Repeated samples of a lymph node station have minimal variation in TMB in most cases. However, this novel data shows how tumour content and minor change in site of node sampling can impact TMB. Further study is needed on whether all node aspirates should be combined in 1 sample, or whether testing independent nodes using smears is needed.

Targeted Hyperbranched Nanoparticles for Delivery of Doxorubicin in Breast Cancer Brain Metastasis.

Breast cancer brain metastases (BM) are associated with a dismal prognosis and very limited treatment options. Standard chemotherapy is challenging in BM patients because the high dosage required for an effective outcome causes unacceptable systemic toxicities, a consequence of poor brain penetration, and a short physiological half-life. Nanomedicines have the potential to circumvent off-target toxicities and factors limiting the efficacy of conventional chemotherapy. The HER3 receptor is commonly expressed in breast cancer BM. Here, we investigate the use of hyperbranched polymers (HBP) functionalized with a HER3 bispecific-antibody fragment for cancer cell-specific targeting and pH-responsive release of doxorubicin (DOX) to selectively deliver and treat BM. We demonstrated that DOX-release from the HBP carrier was controlled, gradual, and greater in endosomal acidic conditions (pH 5.5) relative to physiologic pH (pH 7.4). We showed that the HER3-targeted HBP with DOX payload was HER3-specific and induced cytotoxicity in BT474 breast cancer cells (IC50: 17.6 µg/mL). Therapeutic testing in a BM mouse model showed that HER3-targeted HBP with DOX payload impacted tumor proliferation, reduced tumor size, and prolonged overall survival. HER3-targeted HBP level detected in ex vivo brain samples was 14-fold more than untargeted-HBP. The HBP treatments were well tolerated, with less cardiac and oocyte toxicity compared to free DOX. Taken together, our HER3-targeted HBP nanomedicine has the potential to deliver chemotherapy to BM while reducing chemotherapy-associated toxicities.

Neutralizing Antibody Response to Genotypically Diverse Measles Viruses in Clinically Suspected Measles Cases.

The neutralizing antibody (Nt-Ab) response to vaccine and wild-type measles viruses (MeV) was studied in suspected measles cases reported during the years 2012-2016. The neutralization activity against MeV A, D4 and D8 genotypes was studied on sera (Panel A; n = 68 (measles-immunized) and Panel B; n = 50 (unvaccinated)) that were either laboratory confirmed or not confirmed by the presence of IgM antibodies. Additionally, the Nt-Ab response in Panel A was measured against the MeV vaccine and four wild-type viruses. Neutralization results were compared using homology modeling and molecular dynamics simulation (MDS) of MeV-hemagglutinin (H) and fusion (F) proteins. Overall, the Nt-Ab titres for MeV-A were found to be significantly lower than MeV-D4 and MeV-D8 viruses for Panel A. No major difference was noted in Nt-Ab titres between MeV-D8 viruses (Jamnagar and New Delhi), whereas MeV-D4 (Sindhudurg and Bagalkot (BGK) viruses) showed significant differences between Nt-Ab titres for Panel B. Interestingly, the substitutions observed in epitopes of H-protein, L249P and G316A are observed to be unique to MeV-BGK. MDS of H-protein revealed significant fluctuations in neutralizing epitopes due to L249P substitution. The majority of the clinically suspected cases showed Nt-Abs to MeV wild-types. Higher IgG antibody avidity and Nt-Ab titres were noted in IgM-negatives than in IgM-positives cases, indicating reinfection or breakthrough. MDS revealed reduced neutralization due to decreased conformational flexibility in the H-epitope.

Predicting breast cancer-specific survival in metaplastic breast cancer patients using machine learning algorithms.

Metaplastic breast cancer (MpBC) is a rare and aggressive subtype of breast cancer, with data emerging on prognostic factors and survival prediction. This study aimed to develop machine learning models to predict breast cancer-specific survival (BCSS) in MpBC patients, utilizing a dataset of 160 patients with clinical, pathological, and biological variables. An in-depth variable selection process was carried out using gain ratio and correlation-based methods, resulting in 10 variables for model estimation. Five models (decision tree with bagging; logistic regression; multilayer perceptron; naïve Bayes; and, random forest algorithms) were evaluated using 10-fold cross-validation. Despite the constraints posed by the absence of therapeutic information, the random forest model exhibited the highest performance in predicting BCSS, with an ROC area of 0.808. This study emphasizes the potential of machine learning algorithms in predicting prognosis for complex and heterogeneous cancer subtypes using clinical datasets, and their potential to contribute to patient management. Further research that incorporates additional variables, such as treatment response, and more advanced machine learning techniques will likely enhance the predictive power of MpBC prognostic models.

Tumor Response to Stroma-Modifying Therapy: Magnetic Resonance Imaging Findings in Early-Phase Clinical Trials of Pegvorhyaluronidase alpha (PEGPH20).

Pre-clinical and clinical studies have shown that PEGPH20 depletes intratumoral hyaluronic acid (HA), which is linked to high interstitial fluid pressures and poor distribution of chemotherapies. 29 patients with metastatic advanced solid tumors received quantitative magnetic resonance imaging (qMRI) in 3 prospective clinical trials of PEGPH20, HALO-109-101 (NCT00834704), HALO-109-102 (NCT01170897), and HALO-109-201 (NCT01453153). Apparent Diffusion Coefficient of water (ADC), T1, ktrans, vp, ve, and iAUC maps were computed from qMRI acquired at baseline and ≥ 1 time point post-PEGPH20. Tumor ADC and T1 decreased, while iAUC, ktrans, vp, and ve increased, on day 1 post-PEGPH20 relative to baseline values. This is consistent with HA depletion leading to a decrease in tumor water content and an increase in perfusion, permeability, extracellular matrix space, and vascularity. Baseline parameter values that were predictive of pharmacodynamic responses were: ADC > 1.46×10-3 mm2/s (Balanced Accuracy (BA) = 72%, p < 0.01), T1 > 0.54s (BA = 82%, p < 0.01), iAUC < 9.2 mM-s (BA = 76%, p < 0.05), ktrans<0.07min-1 (BA = 72%, p = 0.2), ve<0.17 (BA = 68%, p < 0.01), and vp<0.02 (BA = 60%, p < 0.01). Further, ve<0.39 at baseline was moderately predictive of response in any parameter (BA = 65.6%, p < 0.01 averaged across patients). These qMRI biomarkers are potentially useful for guiding patient pre-selection and post-treatment follow-up in future clinical studies of PEGPH20 and other tumor stroma-modifying anti-cancer therapies.

A novel series of dipeptide derivatives containing indole-3-carboxylic acid conjugates as potential antimicrobial agents: the design, solid phase peptide synthesis, in vitro biological evaluation, and molecular docking study.

A new library of peptide-heterocycle hybrids consisting of an indole-3-carboxylic acid constituent conjugated with short dipeptide motifs was designed and synthesized by using the solid phase peptide synthesis methodology. All the synthesized compounds were characterized by spectroscopic techniques. Additionally, the synthesized compounds were subjected to in vitro antimicrobial activities. Two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive (Streptococcus pyogenes and Staphylococcus aureus) were used for the evaluation of the antibacterial activity of the targeted dipeptide derivatives. Good antibacterial activity was observed for the screened analogues by comparing their activities with that of ciprofloxacin, the standard drug. Also, two fungi (Aspergillus niger and Candida albicans) were employed for the evaluation of the antifungal activity of the synthesized compounds. When compared to the standard drug Fluconazole, it was observed that the screened analogues exhibited good antifungal activity. In continuation, all the synthesized derivatives were subjected to integrated molecular docking studies and molecular dynamics simulations to investigate binding affinities, intermolecular interaction networks, and conformational flexibilities with deoxyribonucleic acid (DNA) gyrase and lanosterol-14-alpha demethylase. The molecular docking studies revealed that indole-3-carboxylic acid conjugates exhibited encouraging binding interaction networks and binding affinity with DNA gyrase and lanosterol-14 alpha demethylase to show antibacterial and antifungal activity, respectively. Such synthesis, biological activity, molecular dynamics simulations, and molecular docking studies of short peptides with an indole conjugate unlock the door for the near future advancement of novel medicines containing peptide-heterocycle hybrids with the ability to be effective as antimicrobial agents.

Whole Genome Sequencing in Advanced Lung Cancer can be Performed Using Diff-Quik Cytology Smears Derived from Endobronchial Ultrasound, Transbronchial Needle Aspiration (EBUS TBNA).

INTRODUCTION: Maximising alternative sample types for genomics in advanced lung cancer is important because bronchoscopic samples may sometimes be insufficient for this purpose. Further, the clinical applications of comprehensive molecular analysis such as whole genome sequencing (WGS) are rapidly developing. Diff-Quik cytology smears from EBUS TBNA is an alternative source of DNA, but its feasibility for WGS has not been previously demonstrated. METHODS: Diff-Quik smears were collected along with research cell pellets. RESULTS: Tumour content of smears were compared to research cell pellets from 42 patients, which showed good correlation (Spearman correlation 0.85, P < 0.0001). A subset of eight smears underwent WGS, which presented similar mutation profiles to WGS of the matched cell pellet. DNA yield was predicted using a regression equation of the smears cytology features, which correctly predicted DNA yield > 1500 ng in 7 out of 8 smears. CONCLUSIONS: WGS of commonly collected Diff-Quik slides is feasible and their DNA yield can be predicted.

Obesity-associated changes in molecular biology of primary breast cancer.

Obesity is associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet its impact on BC biology remains understudied in humans. This study investigates how the biology of untreated primary BC differs according to patients' body mass index (BMI) using data from >2,000 patients. We identify several genomic alterations that are differentially prevalent in overweight or obese patients compared to lean patients. We report evidence supporting an ageing accelerating effect of obesity at the genetic level. We show that BMI-associated differences in bulk transcriptomic profile are subtle, while single cell profiling allows detection of more pronounced changes in different cell compartments. These analyses further reveal an elevated and unresolved inflammation of the BC tumor microenvironment associated with obesity, with distinct characteristics contingent on the estrogen receptor status. Collectively, our analyses imply that obesity is associated with an inflammaging-like phenotype. We conclude that patient adiposity may play a significant role in the heterogeneity of BC and should be considered for BC treatment tailoring.

Improved concordance of challenging human epidermal growth factor receptor 2 dual in-situ hybridisation cases with the use of a digital image analysis algorithm in breast cancer.

AIMS: Accurate assessment of human epidermal growth factor receptor 2 (HER2) expression by HER2 immunohistochemistry and in-situ hybridisation (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines define 5 groups based on HER2 expression and copy number. Manual pathologist quantification by light microscopy of equivocal and less common HER2 ISH groups (groups 2-4) can be challenging, and there are no data on interobserver variability in reporting of these cases. We sought to determine whether a digital algorithm could improve interobserver variability in the assessment of difficult HER2 ISH cases. METHODS AND RESULTS: HER2 ISH was evaluated in a cohort enriched for less common HER2 patterns using standard light microscopy versus analysis of whole slide images using the Roche uPath HER2 dual ISH image analysis algorithm. Standard microscopy demonstrated significant interobserver variability with a Fleiss's kappa value of 0.471 (fair-moderate agreement) improving to 0.666 (moderate-good) with the use of the algorithm. For HER2 group designation (groups 1-5), there was poor-moderate reliability between pathologists by microscopy [intraclass correlation coefficient (ICC) = 0.526], improving to moderate-good agreement (ICC = 0.763) with the use of the algorithm. In subgroup analysis, the algorithm improved concordance particularly in groups 2, 4 and 5. Time to enumerate cases was also significantly reduced. CONCLUSION: This work demonstrates the potential of a digital image analysis algorithm to improve the concordance of pathologist HER2 amplification status reporting in less common HER2 groups. This has the potential to improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers.

Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

Redefining normal breast cell populations using long noncoding RNAs.

Single-cell RNAseq has allowed unprecedented insight into gene expression across different cell populations in normal tissue and disease states. However, almost all studies rely on annotated gene sets to capture gene expression levels and sequencing reads that do not align to known genes are discarded. Here, we discover thousands of long noncoding RNAs (lncRNAs) expressed in human mammary epithelial cells and analyze their expression in individual cells of the normal breast. We show that lncRNA expression alone can discriminate between luminal and basal cell types and define subpopulations of both compartments. Clustering cells based on lncRNA expression identified additional basal subpopulations, compared to clustering based on annotated gene expression, suggesting that lncRNAs can provide an additional layer of information to better distinguish breast cell subpopulations. In contrast, these breast-specific lncRNAs poorly distinguish brain cell populations, highlighting the need to annotate tissue-specific lncRNAs prior to expression analyses. We also identified a panel of 100 breast lncRNAs that could discern breast cancer subtypes better than protein-coding markers. Overall, our results suggest that lncRNAs are an unexplored resource for new biomarker and therapeutic target discovery in the normal breast and breast cancer subtypes.

Intra-operative interstitial brachytherapy in a novel infra-zygomatic approach for partially resectable head and neck cancers around the base of skull: A technical note.

Purpose: To report an intra-operative catheter insertion technique into the base of skull tumor bed following surgical resection for maxillary tumors. Material and methods: A 42-year-old male patient diagnosed with carcinoma of the maxilla was treated with neoadjuvant chemotherapy followed by chemo-radiation using external beam technique combined with brachytherapy boost to post-operative bed. Brachytherapy was delivered via intra-operative catheter placement at the base of skull to residual disease, which was surgically unresectable. Initially, catheters were placed cranio-caudally. This was later changed into an infra-zygomatic approach to improve planning and dose coverage. High-risk clinical tumor volume (CTV) was generated with a 3 mm margin to residual gross tumor. Planning was done using Varian Eclipse brachytherapy planning system, and an optimal plan was generated. Conclusions: An innovative, beneficial, and safe brachytherapy approach is necessary in a difficult and critical area, such as the base of skull. Our novel method of implant insertion through infra-zygomatic approach resulted in a safe and successful procedure.

Immuno-Colorimetric Neutralization Test: A Surrogate for Widely Used Plaque Reduction Neutralization Tests in Public Health Virology.

Since their first documentation in 1952, plaque reduction neutralization tests (PRNTs) have become the choice of test for the measurement of neutralizing antibodies against a particular virus. However, PRNTs can be performed only against viruses that cause cytopathic effects (CPE). PRNTs also require skilled personnel and can be time-consuming depending on the time required for the virus to cause CPE. Hence, their application limits large-scale studies or epidemiological and laboratory investigations. Since 1978, many surrogate PRNTs or immunocolorimetric assay (ICA)-based focus reduction neutralization tests (FRNT) have been developed. In this article, ICAs and their utility in FRNTs for the characterization of neutralizing antibodies, homologous or heterologous cross-neutralization, and laboratory diagnosis of viruses of public health importance have been discussed. Additionally, possible advancements and automations have been described that may help in the development and validation of novel surrogate tests for emerging viruses.