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Objectives Mesothelioma is a lethal disease that arises from the serosal lining of organ cavities. Several recurrent alterations have been observed in pleural and peritoneal ­mesotheliomas, including in BAP1, NF2, and CDKN2A. Although specific histopathologic parameters have been correlated with prognosis, it is not as well known whether genetic alterations correlate with histologic findings. Methods We reviewed 131 mesotheliomas that had undergone next-generation sequencing (NGS) at our institutions after pathologic diagnosis. There were 109 epithelioid mesotheliomas, 18 biphasic mesotheliomas, and 4 sarcomatoid mesotheliomas. All our biphasic and sarcomatoid cases arose in the pleura. Of the epithelioid mesotheliomas, 73 were from the pleura and 36 were from the peritoneum. On average, patients were 66 years of age (range, 26-90 years) and predominantly male (92 men, 39 women). Results The most common alterations identified were in BAP1, CDKN2A, NF2, and TP53. Twelve mesotheliomas did not show a pathogenic alteration on NGS. For epithelioid mesotheliomas in the pleura, the presence of an alteration in BAP1 correlated with low nuclear grade (P = .04), but no correlation was found in the peritoneum (P = .62). Similarly, there was no correlation between the amount of solid architecture in epithelioid mesotheliomas and any alterations in the pleura (P = .55) or peritoneum (P = .13). For biphasic mesotheliomas, cases with either no alteration detected or with an alteration in BAP1 were more likely to be epithelioid predominant (>50% of the tumor, P = .0001), and biphasic mesotheliomas with other alterations detected and no alteration in BAP1 were more likely to be sarcomatoid predominant (>50% of the tumor, P = .0001). Conclusions This study demonstrates a significant association between morphologic features associated with a better prognosis and an alteration in BAP1.

Table S1. Specifics of DCE-MRI Techniques Used; Table S2. Pathogenic mutations identified in study participants; Table S3. Patients' reasons of withdrawls from the study within 5 years

Purpose:To establish a cohort of high-risk women undergoing intensive surveillance for breast cancer.Experimental Design: We performed dynamic contrast-enhanced MRI every 6 months in conjunction with annual mammography (MG). Eligible participants had a cumulative lifetime breast cancer risk ≥20% and/or tested positive for a pathogenic mutation in a known breast cancer susceptibility gene.Results:Between 2004 and 2016, we prospectively enrolled 295 women, including 157 mutation carriers (75 BRCA1, 61 BRCA2); participants' mean age at entry was 43.3 years. Seventeen cancers were later diagnosed: 4 ductal carcinoma in situ (DCIS) and 13 early-stage invasive breast cancers. Fifteen cancers occurred in mutation carriers (11 BRCA1, 3 BRCA2, 1 CDH1). Median size of the invasive cancers was 0.61 cm. No patients had lymph node metastasis at time of diagnosis, and no interval invasive cancers occurred. The sensitivity of biannual MRI alone was 88.2% and annual MG plus biannual MRI was 94.1%. The cancer detection rate of biannual MRI alone was 0.7% per 100 screening episodes, which is similar to the cancer detection rate of 0.7% per 100 screening episodes for annual MG plus biannual MRI. The number of recalls and biopsies needed to detect one cancer by biannual MRI were 2.8 and 1.7 in BRCA1 carriers, 12.0 and 8.0 in BRCA2 carriers, and 11.7 and 5.0 in non-BRCA1/2 carriers, respectively.Conclusions:Biannual MRI performed well for early detection of invasive breast cancer in genomically stratified high-risk women. No benefit was associated with annual MG screening plus biannual MRI screening.See related commentary by Kuhl and Schrading, p. 1693

Figure S1. Study protocol design; Figure S2. MRI compliance during the first 5 years of study; Figure S3. Recall and biopsy rates by imaging modality

PDF file - 124K, Figure S2. Sub-network 'Calmodulin, c-Cbl, MET, PI3K reg class IA (p85), PDGF-R-beta' of Mitogenic signaling (Supplementary Table S1 No.1). Sub-network is made using 'Analyze network, Build Network' of GeneGo from the significant gene lists associated with H1993 treatment with LY2801653 (3 nM). STAT3 serves as both the divergence and convergence hub. Underexpressed genes (highlighted as blue targets) are significant genes from PamGene data. Eight out of 9 blue targets are represented. CD3Z was excluded from the network to simplify visualization because CD3Z is not related to NSCLC directly. The network objects to be placed in specific sectors of the network according to their sub-cellular localization. The localization sectors are Nucleus, Cytoplasm, Membrane, and Extracellular. Arrow between nodes describes positive (green), negative (red), and unspecified (black) interactions.

PDF file - 94K, Figure S3. Analysis of immunohistochemistry staining and evaluation. A. Scoring system is shown. A pathologist coded MET, p-MET, RON, and p-RON expression as the percentage of positive tumor cells (scale 0%-100%) with staining intensity from 0 to 3+. Positive IHC expression is defined as Scoring System. B. Phosphorylated MET was decreased modestly in LY2801653-treated mice in both A549 and H1993 studies. Moreover, in the treated arms of H1993, Phosphorylated RON was slightly decreased in LY2801653-treated mice.

PDF file - 62K, Figure S1. The protein tyrosine kinase activity profile obtained by using H1993 lysates treated with LY2801653 (10 nM) as tested on PamChip microarrays. For each of the 144 protein tyrosine kinase peptides on the array, treatment response was calculated as the log-transformed ratio of phosphorylation. 47 significant tyrosine kinase substrate modulation by LY2801653 (10 nM) treatment is shown (*: p-value

PDF file - 140K, Supplementary Table S1. Interactive gene networks of Mitogenic signaling of H1993 treatment with LY2801653 (3 nM). MetacoreTM (GeneGo) analysis using the tool 'Analyze network, Build Network'. Note: 'Size' represents the total number of genes evaluated in the Map Folder 'Mitogenic Signaling'. 'Target' denotes the number of genes out of the 12 significant genes that are common to that pathway.

Clinical manifestations of severe COVID-19 include coagulopathies that are exacerbated by the formation of neutrophil extracellular traps (NETs). Here, we report that pulmonary lymphatic vessels, which traffic neutrophils and other immune cells to the lung-draining lymph node (LDLN), can also be blocked by fibrin clots in severe COVID-19. Immunostained tissue sections from COVID-19 decedents revealed widespread lymphatic clotting not only in the lung but also in the LDLN, where the extent of clotting correlated with the presence of abnormal, regressed, or missing germinal centers (GCs). It strongly correlated with the presence of intralymphatic NETs. In mice, tumor necrosis factor α induced intralymphatic fibrin clots; this could be inhibited by DNase I, which degrades NETs. In vitro, TNF-α induced lymphatic endothelial cell upregulation of ICAM-1 and CXCL8, among other neutrophil-recruiting factors, as well as thrombomodulin downregulation; in decedents, lymphatic clotting in LDLNs. In a separate cohort of hospitalized patients, serum levels of Myeloperoxidase-DNA (MPO-DNA, a NET marker) inversely correlated with antiviral antibody titers, but D-dimer levels, indicative of blood thrombosis, did not correlate with either. Patients with high MPO-DNA but low D-dimer levels generated poor antiviral antibody titers. This study introduces lymphatic coagulation in lungs and LDLNs as a clinical manifestation of severe COVID-19 and suggests the involvement of NETosis of lymphatic-trafficking neutrophils. It further suggests that lymphatic clotting may correlate with impaired formation or maintenance of GCs necessary for robust antiviral antibody responses, although further studies are needed to determine whether and how lymphatic coagulation affects adaptive immune responses.

Autopsies of patients who have died from COVID‐19 have been crucial in delineating patterns of injury associated with SARS‐CoV‐2 infection. Despite their utility, comprehensive autopsy studies are somewhat lacking relative to the global burden of disease, and very few comprehensive studies contextualize the findings to other fatal viral infections. We developed a novel autopsy protocol in order to perform postmortem examinations on victims of COVID‐19 and herein describe detailed clinical information, gross findings, and histologic features observed in the first 16 complete COVID‐19 autopsies. We also critically evaluated the role of ancillary studies used to establish a diagnosis of COVID‐19 at autopsy, including immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy (EM). IHC and ISH targeting SARS‐CoV‐2 were comparable in terms of the location and number of infected cells in lung tissue; however, nonspecific staining of bacteria was seen occasionally with IHC. EM was unrevealing in blindly sampled tissues. We then compared the clinical and histologic features present in this series to six archival cases of fatal seasonal influenza and six archival cases of pandemic influenza from the fourth wave of the ‘Spanish Flu’ in the winter of 1920. In addition to routine histology, the inflammatory infiltrates in the lungs of COVID‐19 and seasonal influenza victims were compared using quantitative IHC. Our results demonstrate that the clinical and histologic features of COVID‐19 are similar to those seen in fatal cases of influenza, and the two diseases tend to overlap histologically. There was no significant difference in the composition of the inflammatory infiltrate in COVID‐19 and influenza at sites of acute lung injury at the time of autopsy. Our study underscores the relatively nonspecific clinical features and pathologic changes shared between severe cases of COVID‐19 and influenza, while also providing important caveats to ancillary methods of viral detection.

Malignant peritoneal mesothelioma historically carried a grim prognosis, but outcomes have improved substantially in recent decades. The prognostic significance of clinical, morphologic, and immunophenotypic features remains ill-defined. This multi-institutional cohort comprises 225 malignant peritoneal mesotheliomas, which were assessed for 21 clinical, morphologic, and immunohistochemical parameters. For epithelioid mesotheliomas, combining nuclear pleomorphism and mitotic index yielded a composite nuclear grade, using a previously standardized grading system. Correlation of clinical, morphologic, and immunohistochemical parameters with overall and disease-free survival was examined by univariate and multivariate analyses. On univariate analysis, longer overall survival was significantly associated with diagnosis after 2000 (P = 0.0001), age

Phyllodes tumors (PTs) pose a significant diagnostic challenge in breast pathology as histological criteria and cutoffs for grading are complex and arbitrary. Nevertheless, the clinical behavior of PTs varies widely and correlates, in part, with the histological grade. Methylation signatures have gained interest as diagnostic and prognostic tools in a variety of neoplasms. In particular, 5-hydroxymethylcytosine (5-hmC) has been found to be a useful biomarker. Herein, we assess 51 PTs of varying grades and 15 cellular fibroadenomas (cFA) for stromal expression of 5-hmC by immunohistochemistry. We demonstrate that nuclear 5-hmC decreases as PT grade increases, correlating with histologic predictors of adverse behavior, while it shows comparable levels in benign PTs and cFAs. We identify thresholds at which reduced 5-hmC levels help to distinguish borderline and malignant PT from their benign counterparts. Our results indicate that 5-hmC may serve as a valuable adjunct in classifying PTs in diagnostically difficult cases.

Maternal embryonic leucine-zipper kinase (MELK) regulates cell cycle progression and is highly expressed in many cancers. The molecular mechanism of MELK dysregulation has not been determined in aggressive forms of breast cancer, such as triple negative breast cancer (TNBC). To evaluate molecular markers of MELK aberrations in aggressive breast cancer, we assessed MELK gene amplification and expression in breast tumors. MELK mRNA expression is highly up-regulated in basal-like breast cancer (BLBC), the major molecular subtype of TNBC, compared to luminal or other subtypes of breast tumors. MELK copy number (CN) gains are significantly associated with BLBC, whereas no significant association of CpG site methylation or histone modifications with breast cancer subtypes was observed. Accordingly, the CN gains appear to contribute to an increase in MELK expression, with a significant correlation between mRNA expression and CN in breast tumors and cell lines. Furthermore, immunohistochemistry (IHC) assays revealed that both nuclear and cytoplasmic staining scores of MELK were significantly higher in invasive ductal carcinoma (IDC) tumors compared to ductal carcinoma in situ (DCIS) and normal breast tissues. Our data showed that upregulation of MELK in BLBC may be in part driven by CN gains, rather than epigenetic modifications, indicating a potential for overexpression and CN gains of MELK to be developed as a diagnostic and prognostic marker to identify patients who have more aggressive breast cancer.

The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.

Purpose: To establish a cohort of high-risk women undergoing intensive surveillance for breast cancer. Experimental Design: We performed dynamic contrast-enhanced MRI every 6 months in conjunction with annual mammography (MG). Eligible participants had a cumulative lifetime breast cancer risk ≥20% and/or tested positive for a pathogenic mutation in a known breast cancer susceptibility gene. Results: Between 2004 and 2016, we prospectively enrolled 295 women, including 157 mutation carriers (75 BRCA1, 61 BRCA2); participants' mean age at entry was 43.3 years. Seventeen cancers were later diagnosed: 4 ductal carcinoma in situ (DCIS) and 13 early-stage invasive breast cancers. Fifteen cancers occurred in mutation carriers (11 BRCA1, 3 BRCA2, 1 CDH1). Median size of the invasive cancers was 0.61 cm. No patients had lymph node metastasis at time of diagnosis, and no interval invasive cancers occurred. The sensitivity of biannual MRI alone was 88.2% and annual MG plus biannual MRI was 94.1%. The cancer detection rate of biannual MRI alone was 0.7% per 100 screening episodes, which is similar to the cancer detection rate of 0.7% per 100 screening episodes for annual MG plus biannual MRI. The number of recalls and biopsies needed to detect one cancer by biannual MRI were 2.8 and 1.7 in BRCA1 carriers, 12.0 and 8.0 in BRCA2 carriers, and 11.7 and 5.0 in non-BRCA1/2 carriers, respectively. Conclusions: Biannual MRI performed well for early detection of invasive breast cancer in genomically stratified high-risk women. No benefit was associated with annual MG screening plus biannual MRI screening. See related commentary by Kuhl and Schrading, p. 1693

Background Two-dimensional (2D) specimen radiography (SR) and tomosynthesis (DBT) for breast cancer yield data that lack high-depth resolution. A volumetric specimen imager (VSI) was developed to provide full-3D and thin-slice cross-sectional visualization at a 360° view angle. The purpose of this prospective trial was to compare VSI, 2D SR, and DBT interpretation of lumpectomy margin status with the final pathologic margin status of breast lumpectomy specimens. Methods The study enrolled 200 cases from two institutions. After standard imaging and interpretation was performed, the main lumpectomy specimen was imaged with the VSI device. Image interpretation was performed by three radiologists after surgery based on VSI, 2D SR, and DBT. A receiver operating characteristic (ROC) curve was created for each method. The area under the curve (AUC) was computed to characterize the performance of the imaging method interpreted by each user. Results From 200 lesions, 1200 margins were interpreted. The AUC values of VSI for the three radiologists were respectively 0.91, 0.90, and 0.94, showing relative improvement over the AUCs of 2D SR by 54%, 13%, and 40% and DBT by 32% and 11%, respectively. The VSI has sensitivity ranging from 91 to 94%, specificity ranging from 81 to 85%, a positive predictive value ranging from 25 to 30%, and a negative predicative value of 99%. Conclusions The ROC curves of the VSI were higher than those of the other specimen imaging methods. Full-3D specimen imaging can improve the correlation between the main lumpectomy specimen margin status and surgical pathology. The findings from this study suggest that using the VSI device for intraoperative margin assessment could further reduce the re-excision rates for women with malignant disease.

Objectives Numerous studies on malignant mesothelioma (MM) highlight the prognostic importance of histologic subtype, nuclear grade, and necrosis. This study compares these parameters in paired biopsy and resection specimens of pleural MM. Methods Histologic subtype, percentage of epithelioid morphology, nuclear grade, and the presence or absence of necrosis were compared in 429 paired biopsies and resection specimens of pleural MM from 19 institutions. Results Histologic subtype was concordant in 81% of cases (κ = 0.58). When compared with resection specimens, epithelioid morphology at biopsy had a positive predictive value (PPV) of 78.9% and a negative predictive value (NPV) of 93.5%; sarcomatoid morphology showed high PPV (92.9%) and NPV (99.3%), and biphasic morphology PPV was 89.7% and NPV was 79.7%. Agreement of the percentage of epithelioid morphology was fair (κ = 0.27). Nuclear grade and necrosis were concordant in 75% (κ = 0.59) and 81% (κ = 0.53) of cases, respectively. Nuclear grade showed moderate (κ = 0.53) and substantial (κ = 0.67) agreement from patients with and without neoadjuvant therapy, respectively, and necrosis showed moderate (κ = 0.47 and κ = 0.60) agreement, respectively, in the same subsets of paired specimens. Conclusions Paired biopsy-resection specimens from pleural MM show overall moderate agreement in pathologic parameters. These findings may help guide postbiopsy management and triage of patients with MM.

Acute respiratory distress syndrome (ARDS) occurred in ~12% of hospitalized COVID-19 patients in a recent New York City cohort. Pulmonary endothelial dysfunction, characterized by increased expression of inflammatory genes and increased monolayer permeability, is a major component of ARDS. Vascular leak results in parenchymal accumulation of leukocytes, protein, and extravascular water, leading to pulmonary edema, ischemia, and activation of coagulation associated with COVID-19. Endothelial inflammation further contributes to uncontrolled cytokine storm in ARDS. We have recently demonstrated that Krüppel-like factor 2 (KLF2), a transcription factor which promotes endothelial quiescence and monolayer integrity, is significantly reduced in experimental models of ARDS. Lung inflammation and high-tidal volume ventilation result in reduced KLF2, leading to pulmonary endothelial dysfunction and acute lung injury. Mechanistically, we found that KLF2 is a potent transcriptional activator of Rap guanine nucleotide exchange factor 3 (RAPGEF3) which orchestrates and maintains vascular integrity. Moreover, KLF2 regulates multiple genome-wide association study (GWAS)-implicated ARDS genes. Whether lung KLF2 is regulated by SARS-CoV-2 infection is unknown. Here we report that endothelial KLF2 is significantly reduced in human lung autopsies from COVID-19 patients, which supports that ARDS due to SARS-CoV-2 is a vascular phenotype possibly attributed to KLF2 down-regulation. We provide additional data demonstrating that KLF2 is down-regulated in SARS-CoV infection in mice.

Background: Fine needle aspiration (FNA) of renal masses can distinguish between benign and malignant neoplasms in 73–94% of cases. Previous studies suggested the correct subclassification of renal cell carcinomas (RCCs) by cytomorphology can be achieved in up to 80% of cases. However, as RCCs become increasingly subclassified by molecular signatures, correct subclassification based on cytology alone is increasingly difficult. Design: Two FNA passes (2 stained with Diff-Quik® and 2 with the Papanicolaou method) were performed on all fresh nephrectomy specimens for a 1-year period. There were 30 cases in this study, with 29 primary renal tumors and 1 case of metastatic lung adenocarcinoma. Each case was assigned a random number and came with 2 slides (1 from each staining method). Eight cytopathologists were asked to provide a diagnosis and the World Health Organization/International Society of Urological Pathology (WHO/ISUP) grading if applicable. Fleiss’ Kappa and Cohen’s Kappa equations were used to look at inter-rater variability. Results: When compared to the surgical pathology diagnosis, the average percent correct diagnosis for all cytopathologist was 35%. Chromophobe RCCs had the best average percent accuracy at 72% followed by clearcell RCC at 48%. Average accuracy for grading RCCs was 40%. Inter-rater variability among the cytopathologists for all RCC diagnoses was fair with a Fleiss’ Kappa coefficient of 0.28. For the WHO/ISUP grade, the weighted coefficient for each pathologist ranged from 0.11 to 0.45, ranging from fair to moderate, respectively. Conclusions: Renal tumors are difficult to classify on cytopathology alone. Core needle biopsy and ancillary studies are necessary if diagnosis will change management.

Anti-inflammatory therapies such as IL-6 inhibition have been proposed for COVID-19 in a vacuum of evidence-based treatment. However, abrogating the inflammatory response in infectious diseases may impair a desired host response and predispose to secondary infection. To determine whether IL-6 inhibition is associated with an increased occurrence of secondary infections in patients admitted to the intensive care unit (ICU). We retrospectively reviewed the medical record of patients during an 8-week span and compared the incidence of secondary infection in patients who did and did not receive tocilizumab. The study was approved by the IRB. Additionally, we included representative histopathologic post-mortem findings from several COVID-19 cases that underwent autopsy at our institution. The study was conducted in a cohorted COVID-19 ICU at a tertiary care university medical center. Patients 18 years of age or older admitted to the adult COVID-19 intensive care unit with COVID-19 were randomly selected. We reviewed the occurrence and nature of secondary infections and clinical outcomes in patients who did and did not receive tocilizumab. Measures were formulated prior to the study. For autopsy findings, we were interested in the lung pathology. 60 patients were selected of which 28 had received tocilizumab while 32 had not. Receiving tocilizumab was associated with a higher risk of secondary bacterial (64.3% vs. 31.3%, p=0.010) and fungal (7.1% vs. 0%, p=0.096) infections. 7 cases underwent autopsy. In 3 cases, tocilizumab had previously been given. All 3 patients demonstrated evidence of pneumonia on pathology. Of the 4 cases that had not been given tocilizumab, 2 showed evidence of aspiration pneumonia and 2 exhibited diffuse alveolar damage. Experimental therapies are currently being applied to COVID-19 outside of clinical trials. Anti-inflammatory therapies such as anti-IL-6 therapy have the potential to impair viral clearance, predispose to secondary infection, and cause harm. We seek to raise physician awareness of these issues and highlight the need to better understand the immune response in COVID-19.

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past magnetic resonance imaging (MRI) studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple-negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice. Virgin female C3(1)SV40Tag mice (n=8), weaned at 3-weeks old, were assigned to a LFD (n=4, 3.7-kcal/g; 17.2% kcal from vegetable oil) or a HAFD (n=4, 5.3-kcal/g; 60% kcal from lard) group. From ages 8–12 weeks, weekly fast spin echo MR images and time-of-flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood and mammary gland volumes for each time point were measured from manually traced regions-of-interest; tumors were classified as invasive by histopathology-blinded observers. Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from week 8–12 were 2-fold higher in HAFD-fed mice (0.42±0.14 week(−1)) than in LFD-fed mice (0.21±0.03 week(−1)), p

Background: As immunotherapy emerges as a possible treatment for triple negative breast cancer (TNBC), it is crucial to understand heterogeneity and the complexities of the tumor microenvironment. GeoMx, a new platform developed by NanoString, allows for gene expression analysis within localized segments of the tumor, and is therefore ideal for determining the spatial distribution of immune cell-types. To test the feasibility of applying spatial transcriptomic to breast tumors from diverse populations, we selected ten paraffin embedded blocks from a cohort of self-reported African American patients with TNBC in the Chicago Multi-Ethnic Breast Cancer Study (ChiMEC). Methods: Tissue sections were immunostained with four antibodies (PanCK, CD3E, CD20 and DAPI) and regions of interest (ROIs) were manually selected based on specific morphology, resulting in 10 to 39 ROIs per slide (with a total of 234 ROIs across the ten slides). Using the GeoMx Cancer Transcriptome Atlas assay, expression levels of 1825 genes were measured within each ROI. For this cell-type deconvolution analysis, we used the Spatial Deconvolution algorithm, developed specifically for GeoMx data. We first normalized read counts across all ROIs of a slide and removed the background expression levels. In order to account for cancer cell types in the deconvolution process, we selected ROIs with high tumor content to predict the gene expression profiles of these tumor cells. We then performed deconvolution using both cancer and immune expression profiles, and clustered ROIs into immune signature groups. Results: As expected, we saw a higher abundance of immune cells in the stroma compared with the tumor, which resulted in lower immune cell-type diversity within the tumor segments. Tumor segments were CD8+ T-cell and neutrophil enriched: 20.3% of immune cells in tumor segments were neutrophils, and 23.7% were CD8+ T-cells, whereas in the stroma, only 3.6% and 13.8% were neutrophils and CD8+ T-cells respectively. The proportion of naïve versus memory cytotoxic T-cells differed between the tumor and the stroma, with 84.1% of CD8+ T-cells in the stroma identified as memory versus only 23.9% for tumor segments. We were also able to compare two slides from the same patient and found similar immune patterns across the two sections of the tumor. Clustering of immune cell-type distribution revealed three major immune signatures within both sections of this tumor: macrophage-rich, B-cell rich, and CD8+ T-cell rich ROIs. Conclusion: Spatial transcriptomics allows for granular analysis of the tumor microenvironment. This level of detail is crucial to understanding the cancer-immune relationship, as our analysis revealed high heterogeneity of the immune landscape within tumors. Future work will compare the tumor microenvironment within and across racial groups. Citation Format: Jean-Baptiste Reynier, Jovian Yu, Anna Biernacka, Galina Khramtsova, Toshio Yoshimatsu, Qun Niu, Anna Woodard, Yonglan Zheng, Jeffrey Mueller, Mengjie Chen, Olufunmilayo I. Olopade. Cell-type deconvolution of African American breast tumors reveals spatial heterogeneity of the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2731.

BACKGROUND Programmed death-ligand 1 (PD-L1) expression testing is recommended by guidelines for patients with advanced non-small cell lung cancer (NSCLC). The primary objective of the current study was to determine the success rate of PD-L1 testing from cytology cell block samples obtained by bronchoscopic needle aspiration. The secondary objective was the assessment of the difference in specimen adequacy acquired via needles of different gauges. METHODS Patients with NSCLC who underwent bronchoscopic needle aspirations for which PD-L1 testing was requested between November 1, 2016, and February 6, 2017, were included in the current analysis. Patients underwent needle aspiration from intrathoracic adenopathy or a pulmonary lesion. Rapid on-site cytology evaluation was performed in all cases. PD-L1 immunohistochemistry was performed using the Abcam anti-PD-L1 antibody 28.8 clone on cell block specimen. RESULTS A total of 22 patients had PD-L1 testing requested on needle cytology samples obtained via bronchoscopy at the time of initial diagnosis (81.8%) and for progression of disease (18.2%). Twenty patients (90.9%) underwent successful PD-L1 testing. Sample acquisition was via endobronchial ultrasound-guided transbronchial needle aspiration in 72.7% of patients, endobronchial needle aspiration in 18.2% of patients, and peripheral nodule needle aspiration in 9.1% of patients. There was no statistical difference in PD-L1 test success rates between sample methods (P = .99) or needle sizes (P = 1.00). CONCLUSIONS Bronchoscopically obtained cytology needle–based samples are adequate for PD-L1 testing in patients with NSCLC. There was no difference noted between different needle sizes with regard to adequacy for PD-L1 testing. These findings are relevant for clinicians caring for patients with lung cancer because a vast majority of patients with advanced NSCLC are diagnosed by bronchoscopic needle-based techniques using a variety of commercially available needles. Cancer Cytopathol 2017. © 2017 American Cancer Society.

Background Imaging techniques can provide information about the tumor non-invasively and have been shown to provide information about the underlying genetic makeup. Correlating image-based phenotypes (radiomics) with genomic analyses is an emerging area of research commonly referred to as “radiogenomics” or “imaging-genomics”. The purpose of this study was to assess the potential for using an automated, quantitative radiomics platform on magnetic resonance (MR) breast imaging for inferring underlying activity of clinically relevant gene pathways derived from RNA sequencing of invasive breast cancers prior to therapy. Methods We performed quantitative radiomic analysis on 47 invasive breast cancers based on dynamic contrast enhanced 3 Tesla MR images acquired before surgery and obtained gene expression data by performing total RNA sequencing on corresponding fresh frozen tissue samples. We used gene set enrichment analysis to identify significant associations between the 186 gene pathways and the 38 image-based features that have previously been validated. Results All radiomic size features were positively associated with multiple replication and proliferation pathways and were negatively associated with the apoptosis pathway. Gene pathways related to immune system regulation and extracellular signaling had the highest number of significant radiomic feature associations, with an average of 18.9 and 16 features per pathway, respectively. Tumors with upregulation of immune signaling pathways such as T-cell receptor signaling and chemokine signaling as well as extracellular signaling pathways such as cell adhesion molecule and cytokine-cytokine interactions were smaller, more spherical, and had a more heterogeneous texture upon contrast enhancement. Tumors with higher expression levels of JAK/STAT and VEGF pathways had more intratumor heterogeneity in image enhancement texture. Other pathways with robust associations to image-based features include metabolic and catabolic pathways. Conclusions We provide further evidence that MR imaging of breast tumors can infer underlying gene expression by using RNA sequencing. Size and shape features were appropriately correlated with proliferative and apoptotic pathways. Given the high number of radiomic feature associations with immune pathways, our results raise the possibility of using MR imaging to distinguish tumors that are more immunologically active, although further studies are necessary to confirm this observation. Electronic supplementary material The online version of this article (10.1186/s40644-019-0233-5) contains supplementary material, which is available to authorized users.

Breast cancer is a major cause of morbidity and mortality in Western women. Tumor neoangiogenesis, the formation of new blood vessels from pre-existing ones, may be used as a prognostic marker for cancer progression. Clinical practice uses dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to detect cancers based on increased blood flow and capillary permeability. However, DCE-MRI requires repeated injections of contrast media. Therefore we explored the use of noninvasive time-of-flight (TOF) MR angiography for serial studies of mouse mammary glands to measure the number and size of arteries feeding mammary glands with and without cancer. Virgin female C3(1) SV40 TAg mice (n=9), aged 18-20 weeks, were imaged on a 9.4 Tesla small animal scanner. Multislice T2-weighted (T2W) images and TOF-MRI angiograms were acquired over inguinal mouse mammary glands. The data were analyzed to determine tumor burden in each mammary gland and the volume of arteries feeding each mammary gland. After in vivo MRI, inguinal mammary glands were excised and fixed in formalin for histology. TOF angiography detected arteries with a diameter as small as 0.1 mm feeding the mammary glands. A significant correlation (r=0.79; p< 0.0001) was found between tumor volume and the arterial blood volume measured in mammary glands. Mammary arterial blood volumes ranging from 0.08 mm3 to 3.81 mm3 were measured. Tumors and blood vessels found on in vivo T2W and TOF images, respectively, were confirmed with ex vivo histological images. These results demonstrate increased recruitment of arteries to mammary glands with cancer, likely associated with neoangiogenesis. Neoangiogenesis may be detected by TOF angiography without injection of contrast agents. This would be very useful in mouse models where repeat placement of I.V. lines is challenging. In addition, analogous methods could be tested in humans to evaluate the vasculature of suspicious lesions without using contrast agents.

This study reviews extensive genetic analysis in advanced non-small cell lung cancer (NSCLC) patients in order to: describe how targetable mutation genes interrelate with the genes identified as variants of unknown significance; assess the percentage of patients with a potentially targetable genetic alterations; evaluate the percentage of patients who had concurrent alterations, previously considered to be mutually exclusive; and characterize the molecular subset of KRAS. Thoracic Oncology Research Program Databases at the University of Chicago provided patient demographics, pathology, and results of genetic testing. 364 patients including 289 adenocarcinoma underwent genotype testing by various platforms such as FoundationOne, Caris Molecular Intelligence, and Response Genetics Inc. For the entire adenocarcinoma cohort, 25% of patients were African Americans; 90% of KRAS mutations were detected in smokers, including current and former smokers; 46% of EGFR and 61% of ALK alterations were detected in never smokers. 99.4% of patients, whose samples were analyzed by next-generation sequencing (NGS), had genetic alterations identified with an average of 10.8 alterations/tumor throughout different tumor subtypes. However, mutations were not mutually exclusive. NGS in this study identified potentially targetable genetic alterations in the majority of patients tested, detected concurrent alterations and provided information on variants of unknown significance at this time but potentially targetable in the future.

Long-noncoding RNAs (lncRNAs) have been shown to participate in oncogenesis across a variety of cancers and may represent novel therapeutic targets. However, little is known about the role of lncRNAs in basal-like breast cancer (BLBC), the aggressive form of breast cancer with no molecularly defined therapeutic target. To examine whether altered lncRNA expression contributes to the aggressive phenotype characteristic of BLBC, we performed a comparative analysis of BLBC versus non-BLBC using microarray profiling and RNA sequencing of primary breast cancer. We identified RP11-19E11.1 as a significantly up-regulated lncRNA in BLBC tumors and named it Basal-Like breast cancer Associated Transcript 1 (BLAT1). Analysis of pan-cancer datasets showed the highest expression of BLAT1 in BLBC tumors compared to all other cancers. Depletion of BLAT1 in breast cancer cells led to significantly increased apoptosis, partly because of accumulation of DNA damage. Mechanistically, BLAT1 expression is regulated at the epigenetic level via DNA methylation at CpG islands in the promoter. Concordantly, patients harboring tumors with BLAT1 hypomethylation showed decreased overall survival. Our results suggest that increased expression of BLAT1 via CpG site hypomethylation may contribute to the aggressive phenotype of BLBC, raising a possibility of new biomarkers for prognosis of aggressive BLBC tumors.

Advances in the surgical management of the axilla in patients treated with neoadjuvant chemotherapy, especially those with node positive disease at diagnosis, have led to changes in practice and more judicious use of axillary lymph node dissection that may minimize morbidity from surgery. However, there is still significant confusion about how to optimally manage the axilla, resulting in variation among practices. From the viewpoint of drug development, assessment of response to neoadjuvant chemotherapy remains paramount and appropriate assessment of residual disease—the primary endpoint of many drug therapy trials in the neoadjuvant setting—is critical. Therefore decreasing the variability, especially in a multicenter clinical trial setting, and establishing a minimum standard to ensure consistency in clinical trial data, without mandating axillary lymph node dissection, for all patients is necessary. The key elements which include proper staging and identification of nodal involvement at diagnosis, and appropriately targeted management of the axilla at the time of surgical resection are presented. The following protocols have been adopted as standard procedure by the I-SPY2 trial for management of axilla in patients with node positive disease, and present a framework for prospective clinical trials and practice.

The effects of consumption of different diets on the fatty acid composition in the mammary glands of SV40 T-antigen (Tag) transgenic mice, a well-established model of human triple-negative breast cancer, were investigated with magnetic resonance spectroscopy and spectroscopic imaging. Female C3(1) SV40 Tag transgenic mice (n = 12) were divided into three groups at 4 weeks of age: low fat diet (LFD), high animal fat diet (HAFD), and high fructose diet (HFruD). MRI scans of mammary glands were acquired with a 9.4 T scanner after 8 weeks on the diet. 1H spectra were acquired using point resolved spectroscopy (PRESS) from two 1 mm3 boxes on each side of inguinal mammary gland with no cancers, lymph nodes, or lymph ducts. High spectral and spatial resolution (HiSS) images were also acquired from nine 1-mm slices. A combination of Gaussian and Lorentzian functions was used to fit the spectra. The percentages of poly-unsaturated fatty acids (PUFA), mono-unsaturated fatty acids (MUFA), and saturated fatty acids (SFA) were calculated from each fitted spectrum. Water and fat peak height images (maps) were generated from HiSS data. The results showed that HAFD mice had significantly lower PUFA than both LFD (p < 0.001) and HFruD (p < 0.01) mice. The mammary lipid quantity calculated from 1H spectra was much larger in HAFD mice than in LFD (p = 0.03) but similar to HFruD mice (p = 0.10). The average fat signal intensity over the mammary glands calculated from HiSS fat maps was ~60% higher in HAFD mice than in LFD (p = 0.04) mice. The mean or median of calculated parameters for the HFruD mice were between those for LFD and HAFD mice. Therefore, PRESS spectroscopy and HiSS MRI demonstrated water and fat composition changes in mammary glands due to a Western diet, which was low in potassium, high in sodium, animal fat, and simple carbohydrates. Measurements of PUFA with MRI could be used to evaluate cancer risk, improve cancer detection and diagnosis, and guide preventative therapy.

MRI methods that accurately identify various stages of mouse mammary cancer could provide new knowledge that may have a direct impact on the management of breast cancer in patients. This research investigates whether we can accurately follow the progression from in situ to invasive cancer by the evaluation of in vivo and ex vivo MRI, and in comparison with histology as the gold standard for the diagnosis and staging of cancer. Six C3(1)SV40Tag virgin female mice, aged 12–16 weeks, were studied. At this age, these mice develop in situ cancer that resembles human ductal carcinoma in situ (DCIS). Fast spin-echo images of inguinal mammary glands were acquired at 9.4 T. After in vivo MRI, mice were sacrificed; inguinal mammary glands were excised and fixed in formalin for ex vivo MRI. Three-dimensional, volume-rendered, in vivo and ex vivo MR images were then correlated with histology. High-resolution ex vivo scans facilitated the comparison of in vivo scans with histology. The sizes of mammary cancers classified as in situ on the basis of histology ranged from 150 to 400 µm in largest diameter, and the average signal intensity relative to muscle was 1.40 ± 0.18 on T2-weighted images. Cancers classified as invasive on the basis of histology were >400 µm in largest diameter, and the average intensity relative to muscle on T2-weighted images was 2.34 ± 0.26. Using a cut-off of 400 µm in largest diameter to distinguish between in situ and invasive cancers, a T2-weighted signal intensity of at least 1.4 times that of muscle for in situ cancer, and at least 2.3 times that of muscle for invasive cancer, 96% of in situ and 100% of invasive cancers were correctly identified on in vivo MRI, using histology as the gold standard. Precise MRI–histology correlation demonstrates that MRI reliably detects early in situ cancer and differentiates in situ from invasive cancers in the SV40Tag mouse model of human breast cancer. Copyright © 2015 John Wiley & Sons, Ltd.

Computational fluid dynamics (CFD) modeling of the pulmonary vasculature has the potential to reveal continuum metrics associated with the hemodynamic stress acting on the vascular endothelium. It is widely accepted that the endothelium responds to flow-induced stress by releasing vasoactive substances that can dilate and constrict blood vessels locally. The objectives of this study are to examine the extent of patient specificity required to obtain a significant association of CFD output metrics and clinical measures in models of the pulmonary arterial circulation, and to evaluate the potential correlation of wall shear stress (WSS) with established metrics indicative of right ventricular (RV) afterload in pulmonary hypertension (PH). Right heart catheterization (RHC) hemodynamic data and contrast-enhanced computed tomography (CT) imaging were retrospectively acquired for 10 PH patients and processed to simulate blood flow in the pulmonary arteries. While conducting CFD modeling of the reconstructed patient-specific vasculatures, we experimented with three different outflow boundary conditions to investigate the potential for using computationally derived spatially averaged wall shear Stress (SAWSS) as a metric of RV afterload. SAWSS was correlated with both pulmonary vascular resistance (PVR) (R2 = 0.77, P < 0.05) and arterial compliance (C) (R2 = 0.63, P < 0.05), but the extent of the correlation was affected by the degree of patient specificity incorporated in the fluid flow boundary conditions. We found that decreasing the distal PVR alters the flow distribution and changes the local velocity profile in the distal vessels, thereby increasing the local WSS. Nevertheless, implementing generic outflow boundary conditions still resulted in statistically significant SAWSS correlations with respect to both metrics of RV afterload, suggesting that the CFD model could be executed without the need for complex outflow boundary conditions that require invasively obtained patient-specific data. A preliminary study investigating the relationship between outlet diameter and flow distribution in the pulmonary tree offers a potential computationally inexpensive alternative to pressure based outflow boundary conditions.

Pulmonary hypertension (PH) is a devastating disease affecting approximately 15-50 people per million, with a higher incidence in women. PH mortality is mostly attributed to right ventricle (RV) failure, which results from RV hypotrophy due to an overburdened hydraulic workload. The objective of this study is to correlate wall shear stress (WSS) with hemodynamic metrics that are generally accepted as clinical indicators of RV workload and are well correlated with disease outcome. Retrospective right heart catheterization data for 20 PH patients were analyzed to derive pulmonary vascular resistance (PVR), arterial compliance (C), and an index of wave reflections (Γ). Patient-specific contrast-enhanced computed tomography chest images were used to reconstruct the individual pulmonary arterial trees up to the seventh generation. Computational fluid dynamics analyses simulating blood flow at peak systole were conducted for each vascular model to calculate WSS distributions on the endothelial surface of the pulmonary arteries. WSS was found to be decreased proportionally with elevated PVR and reduced C. Spatially averaged WSS (SAWSS) was positively correlated with PVR (R (2) = 0.66), C (R (2) = 0.73), and Γ (R (2) = 0.5) and also showed promising preliminary correlations with RV geometric characteristics. Evaluating WSS at random cross sections in the proximal vasculature (main, right, and left pulmonary arteries), the type of data that can be acquired from phase-contrast magnetic resonance imaging, did not reveal the same correlations. In conclusion, we found that WSS has the potential to be a viable and clinically useful noninvasive metric of PH disease progression and RV health. Future work should be focused on evaluating whether SAWSS has prognostic value in the management of PH and whether it can be used as a rapid reactivity assessment tool, which would aid in selection of appropriate therapies.

High animal fat consumption is associated with an increase in triple-negative breast cancer (TNBC) risk. Based on previous MRI studies demonstrating the feasibility of detecting very early non-palpable mammary cancers in simian virus 40 large T antigen (SV40TAg) mice, we examined the effect of dietary fat fed from weaning to young adulthood in this model of TNBC. Virgin female C3(1)SV40TAg mice (n = 16) were weaned at 3–4 weeks of age and then fed either a low fat diet (LFD) (n = 8, 3.7 kcal/g; 17.2% kcal from vegetable oil) or a high animal fat diet (HAFD) (n = 8, 5.3 kcal/g; 60% kcal from lard). After 8 weeks on the diet (12 weeks of age), fast spin echo MR images of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed and inguinal mammary glands were excised and formalin fixed for ex vivo MRI. 3D volume-rendered MR images were then correlated with mammary gland histology to assess the glandular parenchyma and tumor burden. Using in vivo MRI, an average of 3.88 ± 1.03 tumors were detected per HAFD-fed mouse compared with an average of 1.25 ± 1.16 per LFD-fed mouse (p

When microinvasion cannot be ruled out on core needle biopsy (CNB) in the setting of ductal carcinoma in situ (DCIS), the surgeon must decide whether to perform a sentinel lymph node biopsy (SLNB) at the time of surgery. Up to 10 % of patients with T1mi have nodal disease, but the utility of SLNB in DCIS suspicious for microinvasion (Smic) is unclear. The University of Chicago pathology database was queried for a diagnosis of Smic or definite microinvasion (Mic) on CNB from 2000 to 2014. We analyzed histology, imaging, core needle size, and the use of myoepithelial immunohistochemistry (IHC) markers. We identified 103 women, 72 with Smic and 31 with Mic on CNB. After surgery, 32 % of Smic patients had infiltrating ductal carcinoma (IDC). Seventy-two percent of Smic patients underwent SLNB, with 67 % performed at the initial surgery. SLNB was positive in 6 % and 10 % of Smic and Mic patients, respectively (p = 0.66). Excluding N1mic, the incidence of macrometastatic nodal disease was 1.9 % for Smic patients and 3.3 % for Mic patients (p = 1.00). For Smic patients, IDC was associated with a larger lesion size and smaller CNB needle. In the setting of Smic, grade, necrosis, or presence of a mass did not increase the risk of IDC. In patients with Smic on CNB, the incidence of macrometastatic nodal disease after SLNB is rare. Surgeons may consider omitting SLNB until IDC is definitively confirmed, especially in patients with Smic apart from other high-risk features.