Previous work has shown that circulating miR-371a-3p has higher sensitivity and specificity than current biomarkers for malignant germ cell tumors (MGCTs). Herein, the performance of two methods commonly used to measure miR-371a-3p levels was compared: quantitative RT-PCR (RT-qPCR) and droplet digital PCR (ddPCR). Samples from the University of Texas Southwestern Medical Center (Dallas, TX) and the University of Cambridge (Cambridge, UK) were evaluated using both RT-qPCR and ddPCR, as per current protocols (RT-qPCR) or standard manufacturer's recommendations (ddPCR). Data were available for 70 individuals: 36 patients with MGCT and 34 control participants (nonmalignant GCT, non-GCT cancer, and noncancer). The performance of the two assays was compared using receiver operating characteristic curves and the area under the curve. Raw miR-371a-3p Cq values (RT-qPCR) were generally lower (ie, miR-371a-3p was more abundant) and the number of positive droplets (ddPCR) higher for patients with MGCT compared with control participants. The area under the curve (95% CI) was 0.94 (0.90-0.99) and 0.82 (0.70-0.93) when using RT-qPCR and ddPCR, respectively. Thus, RT-qPCR had better classification performance compared with ddPCR in the present cohort, supporting the continued use of RT-qPCR in standard clinical practice. Further investigation is required to optimize ddPCR before it should be considered for clinical adoption.

Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5-year survival (∼13%). Thus, new, effective, and ideally, less toxic therapies are desperately needed. Epidemiologic studies have found that patients with PDAC prescribed H1-antihistamines have improved survival. Expression of the histamine H1 receptor (HRH1), a G protein-coupled receptor which is blocked by approved H1-antihistamines, is increased by ∼20-fold in PDAC tumors compared with normal pancreas. Here, we used bioinformatic and molecular biological techniques to identify the cellular localization of HRH1 in the PDAC tumor microenvironment, assess functional responses to HRH1 activation, and define its potential biological roles in PDAC. We found that HRH1 is primarily expressed in cancer cells of PDAC tumors in humans and KPC mice (mice engineered to develop PDAC) and signals via G protein q/11 to increase intracellular Ca2+. HRH1 activation increases migration and invasion by PDAC cancer cells. Orally administered fexofenadine, an H1-antihistamine, was bioavailable in the tumors of KPC mice and yielded smaller pancreatic tumor tissue weights and lower expression of immunomodulatory (interleukin 6 and PD-1) and fibrotic (Col1A1) genes than in vehicle-control KPC mice. Thus, PDAC cancer cells express HRH1, which is functional in vitro and in vivo, suggesting that the repurposing of approved H1-antihistamines may be an efficacious and safe therapeutic approach for patients with PDAC. SIGNIFICANCE STATEMENT: Pancreatic ductal adenocarcinoma (PDAC) has a ∼13% 5-year survival rate, highlighting the need for new therapies. The HRH1 (histamine) receptor, associated with poorer survival, is upregulated in PDAC tumors. This study found that HRH1 is functional in PDAC cells, increasing intracellular Ca2+ via Gq/11 and promoting tumorigenic responses. KPC mice treated with an H1-antihistamine have reduced pancreas weight and lower proinflammatory and fibrotic markers in PDAC tumors. Thus, HRH1 may be a potential target for repurposing approved H1-antihistamines to treat PDAC.

MicroRNAs, short non-protein coding RNAs, are overexpressed in GCTs. Circulating levels of germ cell tumor (GCT)- associated miRNAs, such as miR-371a-3p, can be utilized as efficient and cost-effective alternatives in diagnosing and managing patients presenting with GCTs. This quality of miRNAs has demonstrated favorable performance characteristics as a reliable blood-based biomarker with high diagnostic accuracy compared to current serum tumor markers (STMs) including α-fetoprotein (AFP), beta human chorionic gonadotropin (β-hCG), and lactate dehydrogenase (LDH). The conventional STMs exhibit limited specificity and sensitivity. Potential clinical implications of miRNAs include impact on de-escalating or intensifying treatment, detecting recurrence at earlier stages, and lessening the necessity of cross-sectional imaging or invasive tissue biopsy for non-teratomatous GCTs. Here we also highlight the outstanding issues that must be addressed prior to clinical implementation. Standards for measuring circulating miRNAs and determining ideal cutoff values are essential for integration into current clinical guidelines.

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics.