Exosomal mRNA Cargo are biomarkers of tumor and immune cell populations in pediatric osteosarcoma.

Osteosarcoma is the commonest malignant bone tumor of children and adolescents and is characterized by a high risk of recurrence despite multimodal therapy, especially in metastatic disease. This suggests the presence of clinically undetected cancer cells that persist, leading to cancer recurrence. We sought to evaluate the utility of peripheral blood exosomes as a more sensitive yet minimally invasive blood test that could aid in evaluating treatment response and surveillance for potential disease recurrence. We extracted exosomes from the blood of pediatric osteosarcoma patients at diagnosis (n=7) and after neoadjuvant chemotherapy (n=5 subset), as well as from age-matched cancer-free controls (n=3). We also obtained matched tumor biopsy samples (n=7) from the cases. Exosome isolation was verified by CD9 immunoblot and characterized on electron microscopy. Profiles of 780 cancer-related transcripts were analysed in mRNA from exosomes of osteosarcoma patients at diagnosis and control patients, matched post-chemotherapy samples, and matched primary tumor samples. Peripheral blood exosomes of osteosarcoma patients at diagnosis were significantly smaller than those of controls and overexpressed extracellular matrix protein gene THBS1 and B cell markers MS4A1 and TCL1A. Immunohistochemical staining of corresponding tumor samples verified the expression of THBS1 on tumor cells and osteoid matrix, and its persistence in a treatment-refractory patient, as well as the B cell origin of the latter. These hold potential as liquid biopsy biomarkers of disease burden and host immune response in osteosarcoma. Our findings suggest that exosomes may provide novel and clinically-important insights into the pathophysiology of cancers such as osteosarcoma.

Deep-Learning-Enabled Computer-Aided Diagnosis in the Classification of Pancreatic Cystic Lesions on Confocal Laser Endomicroscopy.

Accurate classification of pancreatic cystic lesions (PCLs) is important to facilitate proper treatment and to improve patient outcomes. We utilized the convolutional neural network (CNN) of VGG19 to develop a computer-aided diagnosis (CAD) system in the classification of subtypes of PCLs in endoscopic ultrasound-guided needle-based confocal laser endomicroscopy (nCLE). From a retrospectively collected 22,424 nCLE video frames (50 videos) as the training/validation set and 11,047 nCLE video frames (18 videos) as the test set, we developed and compared the diagnostic performance of three CNNs with distinct methods of designating the region of interest. The diagnostic accuracy for subtypes of PCLs by CNNs with manual, maximal rectangular, and U-Net algorithm-designated ROIs was 100%, 38.9%, and 66.7% on a per-video basis and 88.99%, 73.94%, and 76.12% on a per-frame basis, respectively. Our per-frame analysis suggested differential levels of diagnostic accuracy among the five subtypes of PCLs, where non-mucinous PCLs (serous cystic neoplasm: 93.11%, cystic neuroendocrine tumor: 84.31%, and pseudocyst: 98%) had higher diagnostic accuracy than mucinous PCLs (intraductal papillary mucinous neoplasm: 84.43% and mucinous cystic neoplasm: 86.1%). Our CNN demonstrated superior specificity compared to the state-of-the-art for the classification of mucinous PCLs (IPMN and MCN), with high specificity (94.3% and 92.8%, respectively) but low sensitivity (46% and 45.2%, respectively). This suggests the complimentary role of CNN-enabled CAD systems, especially for clinically suspected mucinous PCLs.

Pro-metastatic and mesenchymal gene expression signatures characterize circulating tumor cells of neuroblastoma patients with bone marrow metastases and relapse.

Existing marker-based methods of minimal residual disease (MRD) determination in neuroblastoma do not effectively enrich for the circulating disease cell population. Given the relative size differential of neuroblastoma tumor cells over normal hematogenous cells, we hypothesized that cell size-based separation could enrich circulating tumor cells (CTCs) from blood samples and disseminated tumor cells (DTCs) from bone marrow aspirates (BMA) of neuroblastoma patients, and that their gene expression profiles could vary dynamically with various disease states over the course of treatment. Using a spiral microfluidic chip, peripheral blood of 17 neuroblastoma patients at 3 serial treatment timepoints (diagnosis, n=17; post-chemotherapy, n=11; and relapse, n=3), and bone marrow samples at diagnosis were enriched for large intact circulating cells. Profiling the resulting enriched samples with immunohistochemistry and mRNA expression of 1490 cancer-related genes via NanoString, 13 of 17 samples contained CTCs displaying cytologic atypia, TH and PHOX2B expression and/or upregulation of cancer-associated genes. Gene signatures reflecting pro-metastatic processes and the neuroblastoma mesenchymal super-enhancer state were consistently upregulated in 7 of 13 samples, 6 of which also had metastatic high-risk disease. Expression of 8 genes associated with PI3K and GCPR signaling were significantly upregulated in CTCs of patients with bone marrow metastases versus patients without. Correspondingly, in patients with marrow metastases, differentially-expressed gene signatures reflected upregulation of immune regulation in bone marrow DTCs versus paired CTCs samples. In patients who later developed disease relapse, 5 genes involved in immune cell regulation, JAK/STAT signaling and the neuroblastoma mesenchymal super-enhancer state (OLFML2B, STAT1, ARHGDIB, STAB1, TLR2) were upregulated in serial CTC samples over their disease course, despite urinary catecholamines and bone marrow aspirates not indicating the disease recurrences. In summary, using a label-free cell size-based separation method, we enriched and characterized intact circulating cells in peripheral blood indicative of neuroblastoma CTCs, as well as their DTC counterparts in the bone marrow. Expression profiles of pro-metastatic genes in CTCs correlated with the presence of bone marrow metastases at diagnosis, while longitudinal profiling identified persistently elevated expression of genes in CTCs that may serve as novel predictive markers of hematogenous MRD in neuroblastoma patients that subsequently relapse.

KIF1Bß increases ROS to mediate apoptosis and reinforces its protein expression through O 2- in a positive feedback mechanism in neuroblastoma.

Relapse-prone, poor prognosis neuroblastoma is frequently characterized by deletion of chr1p36 where tumor suppressor gene KIF1Bß resides. Interestingly, many 1p36-positive patients failed to express KIF1Bß protein. Since altered cellular redox status has been reported to be involved in cell death and protein modification, we investigated the relationship between reactive oxygen species (ROS) and KIF1Bß. Here, we showed that wild-type KIF1Bß protein expression positively correlates with superoxide (O2-) and total ROS levels in neuroblastoma cells, unlike apoptotic loss-of-function KIF1Bß mutants. Overexpression of KIF1Bß apoptotic domain variants increases total ROS and, specifically O2-, whereas knockdown of endogenous KIF1Bß decreases ROS and O2-. Interestingly, O2- increases KIF1Bß protein expression, independent of the proteasomal degradation pathway. Scavenging O2- or ROS decreases KIF1Bß protein expression and subsequent apoptosis. Moreover, treatment with investigational redox compound Gliotoxin increases O2-, KIF1Bß protein expression, apoptosis and colony formation inhibition. Overall, our findings suggest that ROS and O2- may be important downstream effectors of KIF1Bß-mediated apoptosis. Subsequently, O2- produced may increase KIF1Bß protein expression in a positive feedback mechanism. Therefore, ROS and, specifically O2-, may be critical regulators of KIF1Bß-mediated apoptosis and its protein expression in neuroblastoma.