Tumor microenvironmental determinants of high-risk DCIS progression.

Ductal carcinoma in situ (DCIS) constitutes an array of morphologically recognized intraductal neoplasms in the mammary ductal tree defined by an increased risk for subsequent invasive carcinomas at or near the site of biopsy detection. However, only 15-45% of untreated DCIS cases progress to invasive cancer, so understanding mechanisms that prevent progression is key to avoid overtreatment and provides a basis for alternative therapies and prevention. This study was designed to characterize the tumor microenvironment and molecular profile of high-risk DCIS that grew to a large size but remained as DCIS. All patients had DCIS lesions >5cm in size with at least one additional high-risk feature: young age (<45 years), high nuclear grade, hormone receptor negativity, HER2 positivity, the presence of comedonecrosis, or a palpable mass. The tumor immune microenvironment was characterized using multiplex immunofluorescence to identify immune cells and their spatial relationships within the ducts and stroma. Gene copy number analysis and whole exome DNA sequencing identified the mutational burden and driver mutations, and quantitative whole-transcriptome/gene expression analyses were performed. There was no association between the percent of the DCIS genome characterized by copy number variants (CNAs) and recurrence events (DCIS or invasive). Mutations, especially missense mutations, in the breast cancer driver genes PIK3CA and TP53 were common in this high-risk DCIS cohort (47% of evaluated lesions). Tumor infiltrating lymphocyte (TIL) density was higher in DCIS lesions with TP53 mutations (p=0.0079) compared to wildtype lesions, but not in lesions with PIK3CA mutations (p=0.44). Immune infiltrates were negatively associated with hormone receptor status and positively associated with HER2 expression. High levels of CD3+CD8- T cells were associated with good outcomes with respect to any subsequent recurrence (DCIS or invasive cancer), whereas high levels of CD3+Foxp3+ Treg cells were associated with poor outcomes. Spatial proximity analyses of immune cells and tumor cells demonstrated that close proximity of T cells with tumor cells was associated with good outcomes with respect to any recurrence as well as invasive recurrences. Interestingly, we found that myoepithelial continuity (distance between myoepithelial cells surrounding the involved ducts) was significantly lower in DCIS lesions compared to normal tissue (p=0.0002) or to atypical ductal hyperplasia (p=0.011). Gene set enrichment analysis identified several immune pathways associated with low myoepithelial continuity and a low myoepithelial continuity score was associated with better outcomes, suggesting that gaps in the myoepithelial layer may allow access/interactions between immune infiltrates and tumor cells. Our study demonstrates the immune microenvironment of DCIS, in particular the spatial proximity of tumor cells and T cells, and myoepithelial continuity are important determinants for progression of disease.

Maxillomandibular Advancement Efficacy in Obstructive Sleep Apnea Patients With Class 2 Versus 3 Dentofacial Deformity.

OBJECTIVE: To compare the efficacy of maxillomandibular advancement (MMA) for patients with obstructive sleep apnea (OSA) with class 2 versus 3 dentofacial deformities (DFDs). STUDY DESIGN: Retrospective chart review. SETTING: Tertiary sleep surgery center. METHODS: Patients with OSA and DFD class 2 versus 3 undergoing MMA at Stanford Sleep Surgery between 2014 and 2021 were matched by preoperative body mass index (BMI), age, and sex. Postoperative outcome was compared with polysomnography measures and patient-reported outcome measures (PROMs). RESULTS: Twenty-eight matched subjects, 14 in each deformity group were identified and assessed. The mean age (standard deviation) was 34.29 (10.21) and 33.86 (10.23) for classes 2 and 3, respectively. The apnea-hypopnea index (AHI) decreased from 43.42 (28.30) to 9.6 (5.29) (p < .001) and 37.17 (35.77) to 11.81 (15.74) (p = .042) in class 2 and 3 subjects, respectively. The oxygen desaturation index (ODI) changed from 30.48 (24.02) to 6.88 (3.39) (p = .024) and 11.43 (11.40) to 5.44 (7.96) (p = .85) in class 2 and 3 subjects, respectively. The Epworth sleepiness scale changed from 8.93 (5.28) to 3.91 (2.70) (p = .018) and 10.23 (4.38) to 4.22 (3.07) (p = .006) in class 2 and 3 subjects, respectively. CONCLUSION: Among age, sex, and BMI-matched subjects, MMA is equally effective in both dentofacial class 2 and 3 groups, both objectively and subjectively. Preoperatively, dentofacial class 2 patients with OSA presented with the more severe disease with higher AHI and ODI. Dentofacial class 3 patients with OSA may require additional attention to improve nasal function outcomes.

Identification of molecularly unique tumor-associated mesenchymal stromal cells in breast cancer patients.

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFß pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.

Xanthogranulomatous inflammation and spindle cell proliferation in response to silicone breast implant leakage.

This case study highlights the rare complications of silicone breast implants, as well as the diagnostic limitations of imaging. The patient initially presented with leakage of bilateral breast implants as discovered by a positron emission tomography (PET)-computerized tomography (CT) scan performed as part of a workup for small bowel Langerhans cell sarcoma metastases. The imaging results of the PET-CT scan revealed increased activity bilaterally with an enhancing, irregular, heterogeneously enhancing mass in the right breast. Given the clinical suspicion for breast implant-associated anaplastic large cell lymphoma, further investigation including surgical excision was undertaken. What initially was a concern for a serious complication of long-standing breast implants, fortuitously turned out to be a benign but exuberant xanthogranulomatous inflammatory reactive process. We hope that our report will add to the literature of this rare phenomenon and highlight it as a differential diagnosis of a mass in association with breast implants.

Novel post-acquisition image processing to attenuate red blood cell autofluorescence for quantitative image analysis.

Quantitative analysis of microscopy images from samples stained with fluorescent probes necessitates a very low fluorescence background signal. In tissues prepared by immersion in a chemical fixative, followed by conventional processing for paraffin embedding, red blood cell autofluorescence across several imaging channels can be a nuisance. Although many protocols have been proposed to suppress red blood cell autofluorescence prior to microscopy imaging, in many instances they may not prove totally effective. Moreover, in environments such as core facilities where control over tissue processing and staining may not be feasible, methods to address autofluorescence via post-image acquisition processing may be of some advantage. To this end, we have developed an image analysis algorithm using a commercially based software platform to remove contaminating red blood cell autofluorescence during quantitative evaluation of the fluorescence signal from an immunostaining protocol. The method is based upon the low autofluorescence signal of red blood cells exhibited in the blue channel (used to detect DAPI nuclear signal of all cells), which can be subtracted from the total channel signal by increasing the threshold for DAPI signal in the nuclear detection settings during nuclear segmentation. With the contributing signal from the red blood cells eliminated, the specific immunostained signal for the antigen of interest could be determined. We believe that this simple algorithm performed on post-acquisition microscopy images will be of use for quantitative fluorescence analyses whenever red blood cell autofluorescence is present, especially in amounts where creating regions of interest for evaluation is not possible.

The Shared Core Resource as a Partner in Innovative Scientific Research: Illustration from an Academic Microscopy Imaging Center.

Core facilities have a ubiquitous and increasingly valuable presence at research institutions. Although many shared cores were originally created to provide routine services and access to complex and expensive instrumentation for the research community, they are frequently called upon by investigators to design protocols and procedures to help answer complex research questions. For instance, shared microscopy resources are evolving from providing access to and training on complex imaging instruments to developing detailed innovative protocols and experimental strategies, including sample preparation techniques, staining, complex imaging parameters, and high-level image analyses. These approaches require close intellectual collaboration between core staff and research investigators to formulate and coordinate plans for protocol development suited to the research question. Herein, we provide an example of such coordinated collaboration between a shared microscopy facility and a team of scientists and clinician-investigators to approach a complex multiprobe immunostaining, imaging, and image analysis project investigating the tumor microenvironment from human breast cancer samples. Our hope is that this example may be used to convey to institute administrators the critical importance of the intellectual contributions of the scientific staff in core facilities to research endeavors.

Strain-Based Chemiresistive Polymer-Coated Graphene Vapor Sensors.

Suspended chemiresistive graphene sensors have been fabricated using well-established nanofabrication techniques to generate sensors that are highly sensitive to pyridine and with excellent discrimination between polar and nonpolar analytes. When coated with a polymer surface layer and suspended on 3-D patterned glass electrodes, a hybrid combination of polymer and graphene yields chemiresistive vapor sensors. Expansion and contraction of the polymer layer produces strain on the suspended graphene (Gr). Hence, when organic vapors permeate into the polymer layer, the high gauge factor of the graphene induces substantial electrical resistive changes as folds and creases are induced in the graphene. The hybrid suspended polymer/Gr sensor exhibits substantial responses to polar organic vapors, especially pyridine, while also exhibiting reversibility and increased discrimination between polar and nonpolar analytes compared to previous approaches. This sensor design also allows for potential tunability in the types of polymers used for the reactive surface layer, allowing for use in a variety of potential applications.

Genetic deletion of soluble epoxide hydrolase provides cardioprotective responses following myocardial infarction in aged mice.

BACKGROUND: Pathophysiological responses, including cardiovascular complications, often alter with age. Cardioprotective effects of epoxyeicosatrienoic acids (EETs) toward acute myocardial ischemia-reperfusion injury have been well documented. However, biological relevance of EET-evoked cardioprotection in the ageing myocardium remains unknown. EETs are metabolized to less active metabolites by the enzyme soluble epoxide hydrolase (sEH). This study uses permanent occlusion of the left anterior descending artery (LAD) in young and aged sEH null and WT mice to compare cardiac and mitochondrial function following ischemic injury. METHODS: Age-matched 16 month old (aged) and 3 month old (young) sEH null and littermate wild-type (WT) mice were subjected to permanent occlusion of the left anterior descending coronary artery. Echocardiography was used to assess cardiac structure and function prior-to and 7days post-myocardial infarction with tetrazolium chloride staining to determine infarct size. Mitochondrial ultrastructure was obtained using electron microscopy. Caspase-3, 20S proteasome, aconitase and mitochondrial ETC enzymatic activities were ascertained using established protocols. Mitochondrial respiration was assessed using a Clark electrode in permeabilized cardiac fibers to obtain respiratory control ratios. RESULTS: Markers of cell injury, mitochondrial efficiency and overall cardiac function were preserved in aged sEH null mice, although less robustly than in their young counterparts. While aged animals of both genotypes demonstrated a similar overall age-related decline, sEH deletion consistently demonstrated protection from myocardial ischemic injury regardless of age. CONCLUSION: Our data demonstrates the protection originating from sEH deletion in aged mice was markedly reduced compared to young animals, signifying unavoidable detrimental consequences of biological ageing on cardiac function.