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Introduction: Ductal carcinoma in situ (DCIS) of the breast is a premalignant lesion representing a spectrum of biology from indolent to aggressive. A minority of women present with clinically high-risk features associated with poor outcome. Yet even in patients with biologically aggressive DCIS, the risk of breast cancer mortality is only 3.3% compared to 30-40% in patients with biologically aggressive invasive cancer of the same size.1 We hypothesize that the tumor immune microenvironment could play a proactive role in preventing invasion in high grade clinically high-risk DCIS and that HER2 status, including specific HER2 isoform expression and post-translational modification of HER2, could impact progression. Methods: DCIS: Elaboration of Factors from Enlarged lesions that Nevertheless remain Stage 0 Entities (DEFENSE) is a study of high-risk DCIS, defined as having at least two of the following characteristics: large (>5cm), high grade, hormone receptor-negative status and/or HER2+ status. Slides obtained from FFPE tissue blocks were stained with fluorescence-based multiplex immunohistochemistry (mIHC) panels and imaged to characterize immune infiltrate within the ducts and the stromal compartments. mIHC was also used to detect extracellular and intracellular domains of HER2 with imaging analysis performed to identify HER2 isoforms of HER2+ specimens. Isoforms were characterized as 1) full-length/extracellular domain (ECD) intact, 2) pure/complete loss of ECD (p95 isoform), 3) subclonal populations of both full-length ECD and p95 and 4) gradient/representing partial loss of ECD per cell (reflecting post-translational cleavage). Clinical characteristics were correlated with molecular profile, tumor immune infiltrates, and HER2 isoform. Finally, expression profiling with a 44k array was conducted by Agendia, and MammaPrint and BluePrint results were generated. Results: Of 92 total patients, median age is 46 years. The average DCIS lesion size is 8.2cm, and 33% are hormone receptor negative (HR-). Based upon initial analysis, mIHC demonstrates significant heterogeneity in immune infiltrate populations of pathologically identical DCIS specimens and within regions of the same specimen. High-grade HR- disease has highly reactive stroma, characterized by dense CD3+, CD34+, and CD68+ infiltrate within the stromal compartment. HER2 testing of the first 51 cases demonstrates a high rate of positivity of 67% (34/51). Of those tested for HER2 isoform expression (n=21), none had homogeneous intact full-length HER2. Six (29%) demonstrate the pure p95 isoform, 3 (14%) demonstrate the subclonal isoform, and 12 (57%) demonstrate a gradient HER2 isoform phenotype. Preliminary data from expression profiling shows that the HER2+ cases are also HER2 intrinsic sub-type by BluePrint. Across all of the high-risk DCIS cases, all were scored as MammaPrint high risk, either Luminal B or HER2-type, with only 1 basal and no Luminal A. Additional analyses are ongoing, including completion of testing for the whole data set as well whole exome DNA sequencing and SMART-3SEQ RNA sequencing. Conclusions: Clinically high-risk and pathologically homogenous DCIS lesions demonstrate significant immune infiltrate heterogeneity. Nearly 70% of these large clinically high-risk DCIS lesions are HER2+ with HER2 isoforms most commonly representing either partial or complete loss of the HER2 ECD. This is significantly higher than what is reported for invasive HER2+ breast cancer. A comparison to size and molecularly matched invasive cancers, including from the I-SPY 2 trial, is underway in an effort to elucidate how molecularly aggressive lesions remain in situ despite their large size. References:1 Narod SA et al (2015). Breast Cancer Mortality After a Diagnosis of Ductal Carcinoma in Situ. JAMAOncol. 1(7): 888-96. Citation Format: Alexa Glencer, Alexander Borowsky, Hidetoshi Mori, Michael Campbell, Olivier Harismendy, Janet Stein, Prachi Ghule, Mark Evans, Robert West, Gillian Hirst, Nicole Schindler, Phoebe Miller, Kyra Lee, Donald Weaver, Laura Esserman. The tumor immune microenvironment and HER2 landscape of high-risk ductal carcinoma in situ: The DEFENSE study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-01.

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.

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.

Seasonal windows of opportunity are intervals within a year that provide improved prospects for growth, survival, or reproduction. However, few studies have sufficient temporal resolution to examine how multiple factors combine to constrain the seasonal timing and extent of developmental opportunities. Here, we document seasonal changes in milkweed (

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 TGFβ 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.

Patients undergoing lower extremity bypasses (LEB) are at a high risk of developing post-discharge complications requiring readmission. Health systems have developed several strategies to mitigate this risk. One such measure is developing comprehensive Transitional Care Program (TCP), which includes phone calls to patients after being discharged from the hospital. Our study aimed to assess short-term readmission, mortality, and amputation rates of patients who participated in TCP by completing at least one post-discharge follow-up phone call after undergoing LEB for revascularization of peripheral arterial disease (PAD).A retrospective review was completed for patients who underwent LEB surgery between January 2010 and January 2020 to treat PAD at Penn State Hershey Medical Center. Immediate discharge follow-up was done via telephone calls using a standardized script. Patients were then divided into two groups, those who did not have a successful follow-up call (Group I) and those who had at least one successful follow-up call within seven days after discharge (Group II). Univariate analysis was used to compare preoperative demographics, intraoperative variables, and postoperative outcomes. The probability of readmission and risk factors contributing to it were computed using multiple stepwise forward regression analyses. Epidemiological analysis was done to evaluate the risk of readmission in the group receiving post-discharge follow-up calls.A total of 457 patients underwent LEB from 2010 to 2020 and qualified for inclusion in the study. Among these patients, 126 (27.6%) did not have a successful post-discharge follow-up call (Group I), whereas, 331 (72.4%) patients did complete a successful call (Group II). The mean age of patients was 66.7 years. There were no significant differences in preoperative baseline patient characteristics or intraoperative factors. Patients who completed a successful call had lower readmission rates within thirty days of the operation (8.8 vs. 17.5%, P = 0.008), and this was sustained in multivariate analysis (adjusted odds ratio AOR: 0.18, [confidence interval CI: 0.05-0.66], P = 0.009). However, no differences were observed for thirty-day mortality (Group-I: 3.2% versus Group-II: 1.2%, P = 0.152) or amputation (Group-I: 9.6% versus Group II 5.9%, P = 0.162). Among those who had a successful call, patients with a history of smoking (AOR: 4.05 [CI: 1.21, 17.12] P = 0.025), diabetes mellitus (AOR: 3.42 [CI: 1.35, 8.7] P = 0.01) and myocardial infarction (AOR: 7.15 [CI: 1.76, 20.1] P = 0.006) had a much higher chances of readmission. Risk analysis using epidemiological methods showed that by receiving a call, the risk of readmission could be dropped to half (RR: 0.50 [CI: 0.30, 0.84]), with an attributable risk reduction of -8.7% (CI: -15.9%, -1.4%).This single-institution retrospective study demonstrates the importance of immediate discharge follow-up phone calls in patients who undergo open lower extremity revascularization to reduce thirty-day readmissions. Our analysis showed patients who received immediate follow-up phone calls were less likely to be readmitted to the hospital. The development of reliable and efficient systems to enhance immediate discharge follow-up in vascular surgery patients is pivotal to improving quality of care, preventing readmissions, and reducing healthcare costs.

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 7 days 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.