A novel quantitative real-time PCR diagnostic assay for fecal and nasal swab detection of an otariid lungworm, Parafilaroides decorus.

Parafilaroides decorus, also known as sea lion lungworm, is a metastrongyloid nematode that infects otariid hosts, such as the charismatic California sea lion, Zalophus californianus. P. decorus causes bronchointerstitial pneumonia, respiratory distress, reduced ability to swim, dive and hunt and as a result, increased mortality particularly in young animals. Respiratory disease is a leading cause of stranding and admission to rehabilitation centers on the Pacific coast. Low-coverage genomic sequencing of four P. decorus individuals analyzed through Galaxy's RepeatExplorer identified a novel repeat DNA family we employed to design a sensitive quantitative PCR (qPCR) assay for diagnosing infections from fecal or sputum samples. The assay detects as little as 10 fg of P. decorus DNA and a linear regression model developed using a standard curve can be used to estimate the concentration of P. decorus DNA in a sample, ± 0.015 ng. This knowledge can be leveraged to estimate the level of parasite burden, which can be used to design improved treatments for animals in rehabilitation. Improved treatment of infections will aid in more animals being successfully released back into the wild.

Genetic Alterations Detected in Cell-Free DNA Are Associated With Enzalutamide and Abiraterone Resistance in Castration-Resistant Prostate Cancer.

PURPOSE: Androgen receptor (AR) gene alterations, including ligand-binding domain mutations and copy number (CN) gain, have yet to be fully established as predictive markers of resistance to enzalutamide and abiraterone in men with metastatic castration-resistant prostate cancer (mCRPC). The goal of this study was to validate AR gene alterations detected in cell-free DNA (cfDNA) as markers of enzalutamide and abiraterone resistance in patients with mCRPC. METHODS: Patients with mCRPC (N = 62) were prospectively enrolled between 2014 and 2018. Blood was collected before therapies-enzalutamide (n = 25), abiraterone (n = 35), or enzalutamide and abiraterone (n = 2)-and at disease progression. We used deep next-generation sequencing to analyze cfDNA for sequence variants and CN status in AR and 45 additional cancer-associated genes. Primary end points were prostate-specific antigen response, progression-free survival (PFS), and overall survival (OS). RESULTS: Elevated tumor-specific cfDNA (circulating tumor DNA) was associated with a worse prostate-specific antigen response (hazard ratio [HR], 3.17; 95% CI, 1.11 to 9.05; P = .031), PFS (HR, 1.76; 95% CI, 1.03 to 3.01; P = .039), and OS (HR, 2.92; 95% CI, 1.40 to 6.11; P = .004). AR ligand-binding domain missense mutations (HR, 2.51; 95% CI, 1.15 to 5.72; P = .020) were associated with a shorter PFS in multivariable models. AR CN gain was associated with a shorter PFS; however, significance was lost in multivariable modeling. Genetic alterations in tumor protein p53 (HR, 2.70; 95% CI, 1.27 to 5.72; P = .009) and phosphoinositide 3-kinase pathway defects (HR, 2.62; 95% CI, 1.12 to 6.10; P = .026) were associated with a worse OS in multivariable models. CONCLUSION: These findings support the conclusion that high circulating tumor DNA burden is associated with worse outcomes to enzalutamide and abiraterone in men with mCRPC. Tumor protein p53 loss and phosphoinositide 3-kinase pathway defects were associated with worse OS in men with mCRPC. AR status associations with outcomes were not robust, and additional validation is needed.

Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression.

Tumor progression to metastasis is not cancer cell autonomous, but rather involves the interplay of multiple cell types within the tumor microenvironment. Here we identify asporin (ASPN) as a novel, secreted mesenchymal stromal cell (MSC) factor in the tumor microenvironment that regulates metastatic development. MSCs expressed high levels of ASPN, which decreased following lineage differentiation. ASPN loss impaired MSC self-renewal and promoted terminal cell differentiation. Mechanistically, secreted ASPN bound to BMP-4 and restricted BMP-4-induced MSC differentiation prior to lineage commitment. ASPN expression was distinctly conserved between MSC and cancer-associated fibroblasts (CAF). ASPN expression in the tumor microenvironment broadly impacted multiple cell types. Prostate tumor allografts in ASPN-null mice had a reduced number of tumor-associated MSCs, fewer cancer stem cells, decreased tumor vasculature, and an increased percentage of infiltrating CD8+ T cells. ASPN-null mice also demonstrated a significant reduction in lung metastases compared with wild-type mice. These data establish a role for ASPN as a critical MSC factor that extensively affects the tumor microenvironment and induces metastatic progression. SIGNIFICANCE: These findings show that asporin regulates key properties of mesenchymal stromal cells, including self-renewal and multipotency, and asporin expression by reactive stromal cells alters the tumor microenvironment and promotes metastatic progression.

Detection fidelity of AR mutations in plasma derived cell-free DNA.

Somatic genetic alterations including copy number and point mutations in the androgen receptor (AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%. The lower bound threshold for accurate mutation detection by deep sequencing of cfDNA has not been comprehensively determined and may have locus specific variability. Herein, we used NGS for AR mutation discovery in plasma-derived cfDNA from patients with mCRPC and then used droplet digital polymerase chain reaction (ddPCR) for validation. Our findings show the AR (tTC>cTC) F877L hotspot was prone to false positive mutations during NGS. The rate of error at AR (tTC>cTC) F877L during amplification prior to ddPCR was variable among high fidelity polymerases. These results highlight the importance of validating low-abundant mutations detected by NGS and optimizing and controlling for amplification conditions prior to ddPCR.

Characterization of right ventricular remodeling and failure in a chronic pulmonary hypertension model.

In pulmonary hypertension (PH), right ventricular (RV) dysfunction and failure is the main determinant of a poor prognosis. We aimed to characterize RV structural and functional differences during adaptive RV remodeling and progression to RV failure in a large animal model of chronic PH. Postcapillary PH was created surgically in swine (n = 21). After an 8- to 14-wk follow-up, two groups were identified based on the development of overt heart failure (HF): PH-NF (nonfailing, n = 12) and PH-HF (n = 8). In both groups, invasive hemodynamics, pressure-volume relationships, and echocardiography confirmed a significant increase in pulmonary pressures and vascular resistance consistent with PH. Histological analysis also demonstrated distal pulmonary arterial (PA) remodeling in both groups. Diastolic dysfunction, defined by a steeper RV end-diastolic pressure-volume relationship and longitudinal strain, was found in the absence of HF as an early marker of RV remodeling. RV contractility was increased in both groups, and RV-PA coupling was preserved in PH-NF animals but impaired in the PH-HF group. RV hypertrophy was present in PH-HF, although there was evidence of increased RV fibrosis in both PH groups. In the PH-HF group, RV sarcoplasmic reticulum Ca(2+)-ATPase2a expression was decreased, and endoplasmic reticulum stress was increased. Aldosterone levels were also elevated in PH-HF. Thus, in the swine pulmonary vein banding model of chronic postcapillary PH, RV remodeling occurs at the structural, histological, and molecular level. Diastolic dysfunction and fibrosis are present in adaptive RV remodeling, whereas the onset of RV failure is associated with RV-PA uncoupling, defective calcium handling, and hyperaldosteronism.