The Great Immune Escape: Understanding the Divergent Immune Response in Breast Cancer Subtypes.

Breast cancer, the most common type of cancer affecting women, encompasses a collection of histologic (mainly ductal and lobular) and molecular subtypes exhibiting diverse clinical presentation, disease trajectories, treatment options, and outcomes. Immunotherapy has revolutionized treatment for some solid tumors but has shown limited promise for breast cancers. In this review, we summarize recent advances in our understanding of the complex interactions between tumor and immune cells in subtypes of breast cancer at the cellular and microenvironmental levels. We aim to provide a perspective on opportunities for future immunotherapy agents tailored to specific features of each subtype of breast cancer. SIGNIFICANCE: Although there are currently over 200 ongoing clinical trials testing immunotherapeutics, such as immune-checkpoint blockade agents, these are largely restricted to the triple-negative and HER2+ subtypes and primarily focus on T cells. With the rapid expansion of new in vitro, in vivo, and clinical data, it is critical to identify and highlight the challenges and opportunities unique for each breast cancer subtype to drive the next generation of treatments that harness the immune system.

Association Between Portal Vein Thrombosis and Pouchitis in Patients with Ulcerative Colitis.

BACKGROUND: Pouchitis is the most common long-term complication in patients requiring colectomy ileal pouch-anal anastomosis with medically refractory ulcerative colitis or colitis-associated neoplasia. A previous small case series suggests associated between portal vein thrombosis (PVT) and ischemic pouchitis. AIM: To evaluate the association between PVT and other demographic and clinical factors and pouchitis. METHODS: We used Explorys Inc., a population-based database, to search medical records between 1999 and 2020 with SNOMED-CT code criteria for "construction of pouch" and "ileal pouchitis." Patients with pouchitis were compared to those with previous pouch construction without pouchitis. Factors associated with pouchitis identified with univariable analysis were introduced into a multivariable model. A post hoc analysis further stratified demographical findings of the association between PVT and pouchitis. RESULTS: We identified 7900 patients with ileal pouchitis (7.5%) and 97,510 with pouch construction without pouchitis. In multivariate binary logistic regression, adjusted odds ratio (aOR) for the risk of pouchitis in patients with PVT was 10.78 (95% confidence interval [CI] 7.04-16.49, P < 0.001). Other significant factors associated with pouchitis included male gender (aOR 1.11, 95% CI 1.02-1.21, P = 0.018), deep vein thrombosis (aOR 1.46, 95% CI 1.23-1.72, P < 0.001), and the use of non-steroidal anti-inflammatory drugs (aOR 1.37, 95% CI 1.28-1.45, P < 0.001). Smoking was a protective factor (aOR 0.30, 95% CI 0.33-0.36, P < 0.001). Further sub-analysis showed a higher prevalence of younger patients with PVT and pouchitis. CONCLUSIONS: We report PVT as an independent risk factor associated with pouchitis. Our findings support that PVT is a potentially manageable perioperative complication, and intervention may reduce the risk of pouchitis.

MALT1 Is a Targetable Driver of Epithelial-to-Mesenchymal Transition in Claudin-Low, Triple-Negative Breast Cancer.

MALT1 is the effector protein of the CARMA/Bcl10/MALT1 (CBM) signalosome, a multiprotein complex that drives pro-inflammatory signaling pathways downstream of a diverse set of receptors. Although CBM activity is best known for its role in immune cells, emerging evidence suggests that it plays a key role in the pathogenesis of solid tumors, where it can be activated by selected G protein-coupled receptors (GPCR). Here, we demonstrated that overexpression of GPCRs implicated in breast cancer pathogenesis, specifically the receptors for Angiotensin II and thrombin (AT1R and PAR1), drove a strong epithelial-to-mesenchymal transition (EMT) program in breast cancer cells that is characteristic of claudin-low, triple-negative breast cancer (TNBC). In concert, MALT1 was activated in these cells and contributed to the dramatic EMT phenotypic changes through regulation of master EMT transcription factors including Snail and ZEB1. Importantly, blocking MALT1 signaling, through either siRNA-mediated depletion of MALT1 protein or pharmacologic inhibition of its activity, was effective at partially reversing the molecular and phenotypic indicators of EMT. Treatment of mice with mepazine, a pharmacologic MALT1 inhibitor, reduced growth of PAR1+, MDA-MB-231 xenografts and had an even more dramatic effect in reducing the burden of metastatic disease. These findings highlight MALT1 as an attractive therapeutic target for claudin-low TNBCs harboring overexpression of one or more selected GPCRs. IMPLICATIONS: This study nominates a GPCR/MALT1 signaling axis as a pathway that can be pharmaceutically targeted to abrogate EMT and metastatic progression in TNBC, an aggressive form of breast cancer that currently lacks targeted therapies.

Combination of polycarboxybetaine coating and factor XII inhibitor reduces clot formation while preserving normal tissue coagulation during extracorporeal life support.

Blood contact with high surface area medical devices, such as dialysis and extracorporeal life support (ECLS), induces rapid surface coagulation. Systemic anticoagulation, such as heparin, is thus necessary to slow clot formation, but some patients suffer from bleeding complications. Both problems might be reduced by 1) replacing heparin anticoagulation with artificial surface inhibition of the protein adsorption that initiates coagulation and 2) selective inhibition of the intrinsic branch of the coagulation cascade. This approach was evaluated by comparing clot formation and bleeding times during short-term ECLS using zwitterionic polycarboxybetaine (PCB) surface coatings combined with either a potent, selective, bicyclic peptide inhibitor of activated Factor XII (FXII900) or standard heparin anticoagulation. Rabbits underwent venovenous ECLS with small sham oxygenators for 60 min using three means of anticoagulation (n = 4 ea): (1) PCB coating + FXII900 infusion, (2) PCB coating + heparin infusion with an activated clotting time of 220-300s, and (3) heparin infusion alone. Sham oxygenator blood clot weights in the PCB + FXII900 and PCB + heparin groups were 4% and 25% of that in the heparin group (p < 10-6 and p < 10-5), respectively. At the same time, the bleeding time remained normal in the PCB + FXII900 group (2.4 ± 0.2 min) but increased to 4.8 ± 0.5 and 5.1 ± 0.7 min in the PCB + heparin and heparin alone groups (p < 10-4 and 0.01). Sham oxygenator blood flow resistance was significantly lower in the PCB + FXII900 and PCB + heparin groups than in the heparin only group (p < 10-6 and 10-5). These results were confirmed by gross and scanning electron microscopy (SEM) images and fibrinopeptide A (FPA) concentrations. Thus, the combined use of PCB coating and FXII900 markedly reduced sham oxygenator coagulation and tissue bleeding times versus the clinical standard of heparin anticoagulation and is a promising anticoagulation method for clinical ECLS.

Characterization of RNA-Binding Motif 3 (RBM3) Protein Levels and Nuclear Architecture Changes in Aggressive and Recurrent Prostate Cancer.

Background: The RNA-binding motif protein 3 (RBM3) has been shown to be up-regulated in several types of cancer, including prostate cancer (PCa), compared to normal tissues. Increased RBM3 nuclear expression has been linked to improved clinical outcomes. Aims: Given that RBM3 has been hypothesized to play a role in critical nuclear functions such as chromatin remodeling, DNA damage response, and other post-transcriptional processes, we sought to: (1) quantify RBM3 protein levels in archival PCa samples; (2) develop a nuclear morphometric model to determine if measures of RBM3 protein levels and nuclear features could be used to predict disease aggressiveness and biochemical recurrence. Methods & Results: This study utilized two tissue microarrays (TMAs) stained for RBM3 that included 80 total cases of PCa stratified by Gleason score. A software-mediated image processing algorithm identified RBM3-positive cancerous nuclei in the TMA samples and calculated twenty-two features quantifying RBM3 expression and nuclear architecture. Multivariate logistic regression (MLR) modeling was performed to determine if RBM3 levels and nuclear structural changes could predict PCa aggressiveness and biochemical recurrence (BCR). Leave-one-out cross validation (LOOCV) was used to provide insight on how the predictive capabilities of the feature set might behave with respect to an independent patient cohort to address issues such as model overfitting. RBM3 expression was found to be significantly downregulated in highly aggressive GS ≥ 8 PCa samples compared to other Gleason scores (P < 0.0001) and significantly down-regulated in recurrent PCa samples compared to non-recurrent samples (P = 0.0377). An eleven-feature nuclear morphometric MLR model accurately identified aggressive PCa, yielding a receiver operating characteristic area under the curve (ROC-AUC) of 0.90 (P < 0.0001) in the raw data set and 0.77 (95% CI: 0.83-0.97) for LOOCV testing. The same eleven-feature model was then used to predict recurrence, yielding a ROC-AUC of 0.92 (P = 0.0004) in the raw data set and 0.76 (95% CI: 0.64-0.87) for LOOCV testing. Conclusions: The RBM3 biomarker alone is a strong prognostic marker for the prediction of aggressive PCa and biochemical recurrence. Further, RBM3 appears to be down-regulated in aggressive and recurrent tumors.

Survival Trends for Resectable Pancreatic Cancer Using a Multidisciplinary Conference: the Impact of Post-operative Chemotherapy.

PURPOSE: Despite advances in various treatment modalities, surgical resection for pancreatic ductal adenocarcinoma (PDA) remains the only curative treatment. Data remains limited regarding survival rates for resectable PDA when managed by a multidisciplinary pancreas conference (MDPC). The aim of this study is to assess survival rates, identify significant predictors of mortality, and assess the benefits of adjuvant chemotherapy for resectable PDA following presentation at a MDPC. METHODS: All patients presented from April 2013 to August 2016 with resectable PDA were discussed at a MDPC at a tertiary care center and were followed prospectively until November 2017. Survival analysis was performed using Kaplan-Meier for age, tumor size, tumor differentiation, T-stage, lymph node status, and completion of adjuvant chemotherapy cycles. Independent predictors of survival were determined using multivariate Cox regression modeling. RESULTS: After MDPC consensus and exclusions, total of 64 patients underwent successful surgery. Amongst this cohort, 1-, 2-, and 3-year survival was 78.13%, 46.30%, and 27.27%, respectively. A total of 37 patients (58%) initiated and 16 patients (25%) finished chemotherapy following surgery. Log-rank analysis revealed that tumor size, age, surgical margins, lymph node status, and number of adjuvant chemotherapy cycles received significantly influenced post-operative survival. Tumor size (p < 0.001), lymph node status (p = 0.035), and number of adjuvant chemotherapy cycles (p = 0.041) remained significant after multivariate Cox regression model. CONCLUSIONS: Our results suggest that patients with PDA with tumor size > 50 mm and/or lymph node involvement have poor outcomes despite being surgically resectable. Successful completion of adjuvant chemotherapy has better survival outcomes as compared with incomplete or no adjuvant chemotherapy. The role of alternative management such as down-staging with neoadjuvant therapy should be considered.

High mobility group A1 (HMGA1) protein and gene expression correlate with ER-negativity and poor outcomes in breast cancer.

PURPOSE: The high mobility group A1 (HMGA1) chromatin remodeling protein is required for metastatic progression and cancer stem cell properties in preclinical breast cancer models, although its role in breast carcinogenesis has remained unclear. To investigate HMGA1 in primary breast cancer, we evaluated immunoreactivity score (IRS) in tumors from a large cohort of Asian women; HMGA1 gene expression was queried from two independent Western cohorts. METHODS: HMGA1 IRS was generated from breast tumors in Korean women as the product of staining intensity (weak = 1, moderate = 2, strong = 3) and percent positive cells (< 5% = 0, 5-30% = 1, 30-60% = 2, > 60% = 3), and stratified into three groups: low (< 3), intermediate (3-6), high (> 6). We assessed HMGA1 and estrogen receptor (ESR1) gene expression from two large databases (TCGA, METABRIC). Overall survival was ascertained from the METABRIC cohort. RESULTS: Among 540 primary tumors from Korean women (181 ER-negative, 359 ER-positive), HMGA1 IRS was < 3 in 89 (16.5%), 3-6 in 215 (39.8%), and > 6 in 236 (43.7%). High HMGA1 IRS was associated with estrogen receptor (ER)-negativity (χ2 = 12.07; P = 0.002) and advanced nuclear grade (χ2 = 12.83; P = 0.012). In two large Western cohorts, the HMGA1 gene was overexpressed in breast cancers compared to non-malignant breast tissue (P < 0.0001), including Asian, African American, and Caucasian subgroups. HMGA1 was highest in ER-negative tumors and there was a strong inverse correlation between HMGA1 and ESR1 gene expression (Pearson r = - 0.60, P < 0.0001). Most importantly, high HMGA1 predicted decreased overall survival (P < 0.0001) for all women with breast cancer and further stratified ER-positive tumors into those with inferior outcomes. CONCLUSIONS: Together, our results suggest that HMGA1 contributes to estrogen-independence, tumor progression, and poor outcomes. Moreover, further studies are warranted to determine whether HMGA1 could serve as a prognostic marker and therapeutic target for women with breast cancer.

Zwitterionic poly-carboxybetaine coating reduces artificial lung thrombosis in sheep and rabbits.

Current artificial lungs fail in 1-4 weeks due to surface-induced thrombosis. Biomaterial coatings may be applied to anticoagulate artificial surfaces, but none have shown marked long-term effectiveness. Poly-carboxybetaine (pCB) coatings have shown promising results in reducing protein and platelet-fouling in vitro. However, in vivo hemocompatibility remains to be investigated. Thus, three different pCB-grafting approaches to artificial lung surfaces were first investigated: 1) graft-to approach using 3,4-dihydroxyphenylalanine (DOPA) conjugated with pCB (DOPA-pCB); 2) graft-from approach using the Activators ReGenerated by Electron Transfer method of atom transfer radical polymerization (ARGET-ATRP); and 3) graft-to approach using pCB randomly copolymerized with hydrophobic moieties. One device coated with each of these methods and one uncoated device were attached in parallel within a veno-venous sheep extracorporeal circuit with no continuous anticoagulation (N = 5 circuits). The DOPA-pCB approach showed the least increase in blood flow resistance and the lowest incidence of device failure over 36-hours. Next, we further investigated the impact of tip-to-tip DOPA-pCB coating in a 4-hour rabbit study with veno-venous micro-artificial lung circuit at a higher activated clotting time of 220-300 s (N ≥ 5). Here, DOPA-pCB reduced fibrin formation (p = 0.06) and gross thrombus formation by 59% (p < 0.05). Therefore, DOPA-pCB is a promising material for improving the anticoagulation of artificial lungs. STATEMENT OF SIGNIFICANCE: Chronic lung diseases lead to 168,000 deaths each year in America, but only 2300 lung transplantations happen each year. Hollow fiber membrane oxygenators are clinically used as artificial lungs to provide respiratory support for patients, but their long-term viability is hindered by surface-induced clot formation that leads to premature device failure. Among different coatings investigated for blood-contacting applications, poly-carboxybetaine (pCB) coatings have shown remarkable reduction in protein adsorption in vitro. However, their efficacy in vivo remains unclear. This is the first work that investigates various pCB-coating methods on artificial lung surfaces and their biocompatibility in sheep and rabbit studies. This work highlights the promise of applying pCB coatings on artificial lungs to extend its durability and enable long-term respiratory support for lung disease patients.

72-Hour in vivo evaluation of nitric oxide generating artificial lung gas exchange fibers in sheep.

The large, densely packed artificial surface area of artificial lungs results in rapid clotting and device failure. Surface generated nitric oxide (NO) can be used to reduce platelet activation and coagulation on gas exchange fibers, while not inducing patient bleeding due to its short half-life in blood. To generate NO, artificial lungs can be manufactured with PDMS hollow fibers embedded with copper nanoparticles (Cu NP) and supplied with an infusion of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). The SNAP reacts with Cu NP to generate NO. This study investigates clot formation and gas exchange performance of artificial lungs with either NO-generating Cu-PDMS or standard polymethylpentene (PMP) fibers. One miniature artificial lung (MAL) made with 10 wt% Cu-PDMS hollow fibers and one PMP control MAL were attached to sheep in parallel in a veno-venous extracorporeal membrane oxygenation circuit (n = 8). Blood flow through each device was set at 300 mL/min, and each device received a SNAP infusion of 0.12 µmol/min. The ACT was between 110 and 180 s in all cases. Blood flow resistance was calculated as a measure of clot formation on the fiber bundle. Gas exchange experiments comparing the two groups were conducted every 24 h at blood flow rates of 300 and 600 mL/min. Devices were removed once the resistance reached 3x baseline (failure) or following 72 h. All devices were imaged using scanning electron microscopy (SEM) at the inlet, outlet, and middle of the fiber bundle. The Cu-PDMS NO generating MALs had a significantly smaller increase in resistance compared to the control devices. Resistance rose from 26 ±â€¯8 and 23 ±â€¯5 in the control and Cu-PDMS devices, respectively, to 35 ±â€¯8 mmHg/(mL/min) and 72 ±â€¯23 mmHg/(mL/min) at the end of each experiment. The resistance and SEM imaging of fiber surfaces demonstrate lower clot formation on Cu-PDMS fibers. Although not statistically significant, oxygen transfer for the Cu-PDMS MALs was 13.3% less than the control at 600 mL/min blood flow rate. Future in vivo studies with larger Cu-PDMS devices are needed to define gas exchange capabilities and anticoagulant activity over a long-term study at clinically relevant ACTs. STATEMENT OF SIGNIFICANCE: In artificial lungs, the large, densely-packed blood contacting surface area of the hollow fiber bundle is critical for gas exchange but also creates rapid, surface-generated clot requiring significant anticoagulation. Monitoring of anticoagulation, thrombosis, and resultant complications has kept permanent respiratory support from becoming a clinical reality. In this study, we use a hollow fiber material that generates nitric oxide (NO) to prevent platelet activation at the blood contacting surface. This material is tested in vivo in a miniature artificial lung and compared against the clinical standard. Results indicated significantly reduced clot formation. Surface-focused anticoagulation like this should reduce complication rates and allow for permanent respiratory support by extending the functional lifespan of artificial lungs and can further be applied to other medical devices.

Advances in the computational and molecular understanding of the prostate cancer cell nucleus.

Nuclear alterations are a hallmark of many types of cancers, including prostate cancer (PCa). Recent evidence shows that subvisual changes, ones that may not be visually perceptible to a pathologist, to the nucleus and its ultrastructural components can precede visual histopathological recognition of cancer. Alterations to nuclear features, such as nuclear size and shape, texture, and spatial architecture, reflect the complex molecular-level changes that occur during oncogenesis. Quantitative nuclear morphometry, a field that uses computational approaches to identify and quantify malignancy-induced nuclear changes, can enable a detailed and objective analysis of the PCa cell nucleus. Recent advances in machine learning-based approaches can now automatically mine data related to these changes to aid in the diagnosis, decision making, and prediction of PCa prognoses. In this review, we use PCa as a case study to connect the molecular-level mechanisms that underlie these nuclear changes to the machine learning computational approaches, bridging the gap between the clinical and computational understanding of PCa. First, we will discuss recent developments to our understanding of the molecular events that drive nuclear alterations in the context of PCa: the role of the nuclear matrix and lamina in size and shape changes, the role of 3-dimensional chromatin organization and epigenetic modifications in textural changes, and the role of the tumor microenvironment in altering nuclear spatial topology. We will then discuss the advances in the applications of machine learning algorithms to automatically segment nuclei in prostate histopathological images, extract nuclear features to aid in diagnostic decision making, and predict potential outcomes, such as biochemical recurrence and survival. Finally, we will discuss the challenges and opportunities associated with translation of the quantitative nuclear morphometry methodology into the clinical space. Ultimately, accurate identification and quantification of nuclear alterations can contribute to the field of nucleomics and has applications for computationally driven precision oncologic patient care.

PBOV1 as a potential biomarker for more advanced prostate cancer based on protein and digital histomorphometric analysis.

BACKGROUND: There are few tissue-based biomarkers that can accurately predict prostate cancer (PCa) progression and aggressiveness. We sought to evaluate the clinical utility of prostate and breast overexpressed 1 (PBOV1) as a potential PCa biomarker. METHODS: Patient tumor samples were designated by Grade Groups using the 2014 Gleason grading system. Primary radical prostatectomy tumors were obtained from 48 patients and evaluated for PBOV1 levels using Western blot analysis in matched cancer and benign cancer-adjacent regions. Immunohistochemical evaluation of PBOV1 was subsequently performed in 80 cancer and 80 benign cancer-adjacent patient samples across two tissue microarrays (TMAs) to verify protein levels in epithelial tissue and to assess correlation between PBOV1 proteins and nuclear architectural changes in PCa cells. Digital histomorphometric analysis was used to track 22 parameters that characterized nuclear changes in PBOV1-stained cells. Using a training and test set for validation, multivariate logistic regression (MLR) models were used to identify significant nuclear parameters that distinguish Grade Group 3 and above PCa from Grade Group 1 and 2 PCa regions. RESULTS: PBOV1 protein levels were increased in tumors from Grade Group 3 and above (GS 4 + 3 and ≥ 8) regions versus Grade Groups 1 and 2 (GS 3 + 3 and 3 + 4) regions (P = 0.005) as assessed by densitometry of immunoblots. Additionally, by immunoblotting, PBOV1 protein levels differed significantly between Grade Group 2 (GS 3 + 4) and Grade Group 3 (GS 4 + 3) PCa samples (P = 0.028). In the immunohistochemical analysis, measures of PBOV1 staining intensity strongly correlated with nuclear alterations in cancer cells. An MLR model retaining eight parameters describing PBOV1 staining intensity and nuclear architecture discriminated Grade Group 3 and above PCa from Grade Group 1 and 2 PCa and benign cancer-adjacent regions with a ROC-AUC of 0.90 and 0.80, respectively, in training and test sets. CONCLUSIONS: Our study demonstrates that the PBOV1 protein could be used to discriminate Grade Group 3 and above PCa. Additionally, the PBOV1 protein could be involved in modulating changes to the nuclear architecture of PCa cells. Confirmatory studies are warranted in an independent population for further validation.