Single-Cell RNA Sequencing Reveals Alterations in Patient Immune Cells with Pulmonary Long COVID-19 Complications.

Since the emergence of the COVID-19 pandemic, the effects of SARS-CoV-2 have been extensively researched. While much is already known about the acute phase of the infection, increasing attention has turned to the prolonged symptoms experienced by a subset of individuals, commonly referred to as long COVID-19 patients. This study aims to delve deeper into the immune landscape of patients with prolonged symptoms by implementing single-cell mRNA analysis. A 71-year-old COVID-19 patient presenting with persistent viral pneumonia was recruited, and peripheral blood samples were taken at 3 and 2 years post-acute infection onset. Patients and control peripheral blood mononuclear cells (PBMCs) were isolated and single-cell sequenced. Immune cell population identification was carried out using the ScType script. Three months post-COVID-19 patients' PBMCs contained a significantly larger immature neutrophil population compared to 2-year and control samples. However, the neutrophil balance shifted towards a more mature profile after 18 months. In addition, a notable increase in the CD8+ NKT-like cells could be observed in the 3-month patient sample as compared to the later one and control. The subsequent change in these cell populations over time may be an indicator of an ongoing failure to clear the SARS-CoV-2 infection and, thus, lead to chronic COVID-19 complications.

Solitary fibrous tumor with IGF-II-induced non-islet cell tumor hypoglycemia: a case report and molecular characterization by next-generation sequencing.

Background: Non-islet cell tumor-induced hypoglycemia (NICTH) is a rare, life-threatening medical condition caused by excessive insulin-like growth factor II (IGF-II) secretion from tumors of most commonly mesenchymal origin. Using next-generation sequencing, we have characterized the genome and transcriptome of the resected IGF-II-secreting solitary fibrous tumor from a patient with severe hypoglycemia accompanied by hypoglycemia unawareness. Case presentation: A 69-year-old male patient presenting with abdominal discomfort was examined using computer tomography, revealing a large lesion at the lesser pelvis extending above the umbilicus. As no bone and lymph node metastases were detected, the patient was scheduled for laparotomy. Before surgery, the patient presented with symptoms of severe hypoglycemia. Suppressed C-peptide levels and subsequent hypokalemia indicated a possible case of NICTH. The patient was treated with methylprednisolone (8 mg) to assess hypoglycemia. After the surgery, mild hypoglycemia was present for the postoperative period, and no radiological recurrences were observed 3 and 12 months after discharge. Histopathological examination results were consistent with the diagnosis of malignant solitary fibrous tumor (SFT). Overexpression of IGF-II was confirmed by both immunohistochemistry and RNA sequencing. Further NGS analysis revealed an SFT characteristic alteration-NAB2-STAT6 fusion. Additionally, three deleterious missense variants were detected in oncogenes BIRC6, KIT, and POLQ, and one homozygous in-frame deletion in the RBM10 tumor suppressor gene. Conclusion: While the NAB2-STAT6 fusions are well characterized, the mutational landscape of SFTs remains understudied. This study reports the importance of NGS to characterize SFTs as we detected four coding variants in genes (BIRC6, KIT, POLQ, and RBM10) associated with tumorigenesis that could potentially contribute to the overall pathogenesis of SFT.

Transcriptome of GH-producing pituitary neuroendocrine tumours and models are significantly affected by somatostatin analogues.

Pituitary neuroendocrine tumours (PitNETs) are neoplasms of the pituitary that overproduce hormones or cause unspecific symptoms due to mass effect. Growth hormone overproducing GH-producing PitNETs cause acromegaly leading to connective tissue, metabolic or oncologic disorders. The medical treatment of acromegaly is somatostatin analogues (SSA) in specific cases combined with dopamine agonists (DA), but almost half of patients display partial or full SSA resistance and potential causes of this are unknown. In this study we investigated transcriptomic landscape of GH-producing PitNETs on several levels and functional models-tumour tissue of patients with and without SSA preoperative treatment, tumour derived pituispheres and GH3 cell line incubated with SSA to study effect of medication on gene expression. MGI sequencing platform was used to sequence total RNA from PitNET tissue, pituispheres, mesenchymal stromal stem-like cells (MSC), and GH3 cell cultures, and data were analysed with Salmon-DeSeq2 pipeline. We observed that the GH-producing PitNETs have distinct changes in growth hormone related pathways related to its functional status alongside inner cell signalling, ion transport, cell adhesion and extracellular matrix characteristic patterns. In pituispheres model, treatment regimens (octreotide and cabergoline) affect specific cell proliferation (MKI67) and core functionality pathways (RYR2, COL8A2, HLA-G, ARFGAP1, TGFBR2). In GH3 cells we observed that medication did not have transcriptomic effects similar to preoperative treatment in PitNET tissue or pituisphere model. This study highlights the importance of correct model system selection for cell transcriptomic profiling and data interpretation that could be achieved in future by incorporating NGS methods and detailed cell omics profiling in PitNET model research.

Liquid biopsy and preclinical tools for advancing diagnosis and treatment of patients with pancreatic neuroendocrine neoplasms.

Pancreatic neuroendocrine neoplasms (pNENs) are rare cancers with broad challenges for their management. The main clinical obstacles are the high rate of patients diagnosed at advanced stages, lack of prognostic markers for early detection of disease recurrence in resected patients, significant limitations in identifying those who will benefit from adjuvant therapy, and timely recognition of treatment response. Therefore, the discovery of new prognostic and predictive markers is necessary for patient stratification and clinical management. Liquid biopsy, which has revolutionized the field of clinical oncology, is extremely under-investigated in pNENs. This review highlights its potential and the recent advances in related technologies, as candidates for the delivery of the new tools that can help to refine pNEN diagnosis and to personalize treatment. In addition, the opportunities and limitations of available preclinical research models with regard to biomarker research are discussed in light of pNEN clinical needs.

Genome wide analysis of circulating miRNAs in growth hormone secreting pituitary neuroendocrine tumor patients' plasma.

Background: Circulating plasma miRNAs have been increasingly studied in the field of pituitary neuroendocrine tumor (PitNET) research. Our aim was to discover circulating plasma miRNAs species associated with growth hormone (GH) secreting PitNETs versus assess how the plasma levels of discovered miRNA candidates are impacted by SSA therapy and whether there is a difference in their levels between GH secreting PitNETs versus other PitNET types and healthy individuals. Design: We compared plasma miRNA content and levels before and after surgery focusing on GH secreting PitNET patients. Selected miRNA candidates from our data and literature were then tested in a longitudinal manner in somatostatin analogues (SSA) treatment group. Additionally, we validated selected targets in an independent GH secreting PitNET group. Methods: miRNA candidates were discovered using the whole miRNA sequencing approach and differential expression analysis. Selected miRNAs were then analyzed using real-time polymerase chain reaction (qPCR). Results: Whole miRNA sequencing discovered a total of 16 differentially expressed miRNAs (DEMs) in GH secreting PitNET patients' plasma 24 hours after surgery and 19 DEMs between GH secreting PitNET patients' plasma and non-functioning (NF) PitNET patients' plasma. Seven miRNAs were selected for further testing of which miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p showed a significant downregulation in plasma after 1 month of SSA treatment. mir-625-5p was found to be significantly downregulated in plasma of GH secreting PitNET patients vs. NF PitNET patients. miR-625-5p alongside miR-130b-3p were also found to be downregulated in GH PitNETs compared to healthy individuals. Conclusions: Our study suggests that expression of plasma miRNAs miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p in GH secreting PitNETs is affected by SSA treatment. Additionally, miR-625-5p can distinguish GH secreting PitNETs from other PitNET types and healthy controls warranting further research on these miRNAs for treatment efficacy.

Case Report: Micro-RNAs in Plasma From Bilateral Inferior Petrosal Sinus Sampling and Peripheral Blood From Corticotroph Pituitary Neuroendocrine Tumors.

Objective: Circulating miRNAs are found in bodily fluids including plasma and can serve as biomarkers for diseases. The aim of this study was to provide the first insight into the landscape of circulating miRNAs in close proximity to the adrenocorticotropic hormone (ACTH) secreting PitNET. To achieve this objective next-generation sequencing of miRNAs in plasma from bilateral inferior petrosal sinus sampling (BIPSS) - a gold standard in diagnosing ACTH-secreting PitNETs was carried out and selected miRNA candidates were further tested by RT-qPCR in independent patient cohorts. Methods: Sinistral (left) and dextral (right) BIPSS blood samples of the patient were collected in three time points: before the administration of corticotropin-releasing hormone, 5 and 15 minutes after stimulation. In differential expression analysis, sinistral plasma was compared with dextral. The selected miRNA candidates were tested in plasma by RT-qPCR in two patient groups: 1) in five ACTH secreting PitNET patients with plasma samples taken before and 24 hours after surgery, 2) in 12 ACTH secreting PitNET patients vs. 9 non-functioning PitNET patients. Results: BIPSS concluded that the highest amount of ACTH was released in the sinistral side at the 5th minute mark indicating a presence of a tumor. The highest amount of differentially expressed miRNAs was observed 5 minutes after stimulation (20 upregulated, 14 downregulated). At the 5th minute mark in sinistral plasma, two miRNAs were identified: hsa-miR-7-5p and hsa-miR-375-3p that were highly upregulated compared to other BIPSS samples and peripheral plasma samples. Further testing by qPCR revealed significant reduction of miR-7-5p in plasma 24 hours after surgery and upregulation in plasma of ACTH secreting PitNET patients compared to non-functioning PitNET patients (P =0.0013). Conclusions: By stimulating the ACTH secreting PitNET with CRH a rapid increase of two miRNAs (hsa-mir-7-5p, hsa-mir-375-3p) and ACTH can be observed in sinistral inferior petrosal (tumor side). A decrease of miR-7-5p in plasma after surgery and upregulation in plasma of ACTH secreting PitNET patients was discovered implying that further studies of this miRNA as diagnostic marker is needed.

Large Scale Molecular Studies of Pituitary Neuroendocrine Tumors: Novel Markers, Mechanisms and Translational Perspectives.

Pituitary neuroendocrine tumors (PitNETs) are non-metastatic neoplasms of the pituitary, which overproduce hormones leading to systemic disorders, or tumor mass effects causing headaches, vertigo or visual impairment. Recently, PitNETs have been investigated in large scale (exome and genome) molecular analyses (transcriptome microarrays and sequencing), to uncover novel markers. We performed a literature analysis on these studies to summarize the research data and extrapolate overlapping gene candidates, biomarkers, and molecular mechanisms. We observed a tendency in samples with driver mutations (GNAS, USP8) to have a smaller overall mutational rate, suggesting driver-promoted tumorigenesis, potentially changing transcriptome profiles in tumors. However, direct links from drivers to signaling pathways altered in PitNETs (Notch, Wnt, TGF-ß, and cell cycle regulators) require further investigation. Modern technologies have also identified circulating nucleic acids, and pinpointed these as novel PitNET markers, i.e., miR-143-3p, miR-16-5p, miR-145-5p, and let-7g-5p, therefore these molecules must be investigated in the future translational studies. Overall, large-scale molecular studies have provided key insight into the molecular mechanisms behind PitNET pathogenesis, highlighting previously reported molecular markers, bringing new candidates into the research field, and reapplying traditional perspectives to newly discovered molecular mechanisms.

Evaluation of the Possibility to Detect Circulating Tumor DNA From Pituitary Adenoma.

Objective: Circulating free DNA (cfDNA) in general and circulating tumor DNA (ctDNA) in particular is becoming an increasingly used form of liquid biopsy biomarkers. In this study, we are investigating the ability to detect ctDNA from the plasma of pituitary adenoma (PA) patients. Design: Tumor tissue samples were obtained from planed PA resections, before which blood plasma samples were taken. Somatic variants found in PA tissue samples were evaluated in related cfDNA, isolated from plasma samples. Methods: Sanger sequencing, as well as previously obtained whole-exome sequencing data, were used to evaluate somatic variants composition in tumor tissue samples. cfDNA was isolated from the same PA patients and competitive allele-specific TaqMan PCR and amplicon-based next-generation sequencing (NGS) approach were used for targeted detection of variants found in corresponding tumor tissue samples. Results: Using NGS-based analysis, we detected five out of 17 somatic variants in 40 to 60% of total reads, three variants in 0.50-5.00% of total read count, including GNAS c.601C>T, which was detected using ultra-deep NGS (1.78 million X) in 0.77% of amplicons reads. Nine variants were not detected. We also detected We were not able to detect variant found in PA tissue in cfDNA using cast-PCR, indicating that the portion of variant-containing ctDNA in total isolated cfDNA is too small to be detected with this method. Conclusions: For the first time, we demonstrate the possibility to detect somatic variants of PA in cfDNA isolated from patients' blood plasma. Whether the source of variant detected in cfDNA is PA should be further tested.