Retinoblastoma co-repressor 1 (RB) and cyclin-dependent kinase inhibitor (CDKN) as a multi-gene panel for differentiating pulmonary from non-pulmonary origin in metastatic neuroendocrine carcinomas

BackgroundNeuroendocrine carcinomas (NECs) arise from neuroendocrine cells present throughout the body, and often present with metastases even with small and undetectable primary tumors. Additionally, neuroendocrine differentiation can be seen in carcinomas of non-neuroendocrine origin further complicating the landscape of metastatic NECs. Organ specific immunohistochemical markers such as TTF1, CDX2 and PAX8 are often lost in high grade tumors and may be non-contributory in localizing the primary site.Though NECs share a common cellular origin, they exhibit great variability in biologic behavior, prognosis and treatment based on the primary organ of origin.DesignTwenty one cases of metastatic NECs were retrieved from our archives and were classified based on location of the primary tumor derived from clinical and radiological findings. Next generation sequencing data was retrieved and analyzed for recurrent genetic abnormalities in these cases. Statistical analysis was performed using IBM SPSS25 software.ResultsRB1 mutations were exclusive to NECs metastasizing from lung primary and were detected in 5 of 12 (41.6 %) cases (p = 0.04). CDKN gene family (CDKN1B and 2 A) mutations were limited to metatstatic NECs of non-pulmonary origin and were detected in 4 of 9 (44.4 %) cases (p = 0.02).ConclusionThe location of the primary tumor in metastatic NECs appears to have significant prognostic and therapeutic implications. But due to the morphological homogeneity, higher grade of tumor, variable sensitivity of immunohistochemical markers, and small, often undetectable primary tumors, the localization of the primary tumor in cases of metastatic NECs is a challenge. In this study, RB1 and CDKN gene family mutations are identified as possible markers for differentiating pulmonary and non-pulmonary origin in metatstatic NECs.     

Association of serine/threonine kinase 11 mutations and response to programmed cell death 1 inhibitors in metastatic gastric cancer

Programmed cell death 1 (PD-1) inhibitors have shown therapeutic efficacy in metastatic gastric cancer (mGC). However, no predictive biomarkers have been established in mGC. Inactivating mutations in serine/threonine kinase 11 (STK11) are associated with poor response to PD-1 inhibitors in KRAS-mutant lung adenocarcinoma. Therefore, we hypothesized that STK11 inactivating mutations would be associated with inferior clinical response to PD-1 inhibitors in mGC. We analyzed 59 mGC patients who had been treated with PD-1 inhibitors and whose tumors had been analyzed by targeted high-throughput sequencing. STK11 mutations were identified in 30 (50.8%) patients, and were all missense mutations. Three patients (5.1%) had STK11 gene amplification and mutation, simultaneously. Patients with STK11 mutations had prolonged overall survival (median: 19.0 vs 11.6 months, p = 0.15), and progression-free survival (4.2 vs 1.9 months, p = 0.06) when treated with PD-1 inhibitors, but these differences were not statistically significant. Patients with STK11 inactivating mutations without STK11 gene amplification had significantly prolonged progression-free survival compared to patients with wild type STK11 or STK11 gene amplification (4.8 vs 1.0 months, p = 0.04). However, in multivariate Cox regression analysis with high microsatellite instability (MSI-H), the number of tumor mutations, PD Ligand-1 (PD-L1)+, Epstein-Barr virus positivity (EBV)+, and type of PD-1 inhibitor used (pembrolizumab vs nivolumab), only MSI-H and PD-L1+ were significantly associated with longer progression-free survival. In mGC, the presence of STK11 mutation was not predictive of the response to PD-1 inhibitors. Instead, patients with MSI-H or PD-L1+ tumors displayed superior clinical responses to PD-1 inhibitors.     

Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution.     

Maternal immunization modulates the primary immune response to 2-phenyl-oxazolone in BALB/c mice

The development of the antibody repertoire in newborn mice is greatly influenced by idiotypic network interactions. It has been demonstrated that anti-idiotypic antibodies either directly injected or transferred from the mother may alter the repertoire for life. For an elucidation of the underlying mechanisms we have analyzed the primary immune response to 2-phenyl-5-oxazolone (phOx) coupled to chicken serum albumin (CSA) in BALB/c mice after complete disappearance of maternal antibodies which originated from different stages of affinity maturation. Depending on the serum titers of the mothers after primary (1° mo), secondary (2° mo) or tertiary (3° mo) immunization, maternal anti-phOx IgG persisted in F1 mice for up to 9 months. In addition, F1 mice born to 2° mo developed – even without immunization – an anti-phOx IgM titer which reached levels similar to an antigen-induced primary response. An enhancement of the early primary anti-phOx as well as anti-CSA response was seen in F1 mice born from 1° mo, whereas the response was delayed when born to 2° mo and 3° mo. The antibody titers in the latter group of mice remained at a lower level for 3 months. In contrast, mice of the F2 generation which received a smaller amount of the same collection of maternal antibodies as F1 mice from 3° mo exhibited a quite different primary response: (i) They showed an earlier onset in their anti-CSA response. (ii) Whereas normally a plateau in antibody titer was reached by the 4th weak after immunization, in 55 % of the F2 mice a prolonged increase of the anti-phOx and anti-CSA antibody titers was observed. At 12 weeks after antigenic challenge, titers reached plateau levels of 6 × 10⁵ which were never before seen in a primary phOx or CSA response. Thus, depending on its own immunological experience, the maternal immune system induces a state of memory in the offspring which results in a faster and/or enhanced immune response in the F1 and F3 generations.     

Synaptic inhibition in the isolated respiratory network of neonatal rats

Gramicidin-perforated patch-clamp recording revealed phasic Cl^–-mediated hyperpolarizations in respiratory neurons of the brainstem–spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of γ-aminobutyric acid (GABA), i.e. GABA_A, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO₂/HCO₃^–-free solutions, but shifted from about ?65 mV to values more positive than ?20 mV upon dialysis of the cells with 144 instead of 4 mm Cl^–. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABA_B receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K⁺ channel-mediated hyperpolarization (reversal potential ?88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABA_A and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABA_B receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.