Inositol Polyphosphate 4-Phosphatase Type II Is a Tumor Suppressor in Multiple Myeloma

Inositol polyphosphate-4-phosphatase type II (INPP4B) has been identified as a tumor suppressor, while little is known about its expression and function in multiple myeloma (MM). In this study, we evaluated the expression of INPP4B in 28 cases of newly diagnosed MM patients and 42 cases of extramedullary plasmacytoma (EMP) patients compared with normal plasma cells and found that low INPP4B expression was correlated with poor outcomes in MM patients. Moreover, expression of INPP4B in seven MM cell lines was all lower than that in normal plasma cells. In addition, loss of function of INPP4B promoted cell proliferation in MM cells; however, gain of function suppressed MM cells proliferation and arrested the cell cycle at G0/G1 phage. Meanwhile, knockdown of INPP4B enhanced resistance, but overexpression promoted sensitivity to bortezomib treatment in MM cells. Mechanistically, we found that INPP4B exerted its role via inhibiting the phosphorylation of Akt at lysine 473 but not threonine 308, which attenuated the activation of the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway. Therefore, we identified an inhibitory effect of INPP4B in MM, and our findings suggested that loss of INPP4B expression is a risk factor of aggressive MM.

Humoral immune reconstitution after anti-BCMA CAR-T cell therapy in relapse/refractory multiple myeloma

Systematic and dynamic humoral immune reconstitution is little known for relapse/refractory (R/R) multiple myeloma (MM) patients who received anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR)-T cell therapy. We investigated the kinetics of B cell, normal plasma cell and immunoglobulin recovery in 40 patients who achieved ongoing response after anti-BCMA CAR-T cell therapy. All patients developed B-cell aplasia and the median duration of B-cell aplasia was 70 days (23-270). B cell count reached nadir on a median of day 7 and returned to baseline level on a median of day 97. BCMA positive cells in bone marrow turned undetectable on a median time of day 28 (13-159) in 94.87% (37/39) patients. Normal plasma cells in bone marrow were first re-detectable on a median of day 212. All patients developed a significant decrease in serum IgG, IgA, and IgM on a median of day 60. Recovery of serum IgG, IgM and IgA was observed in 53.33% (8/15) patients (non- IgG MM), 73.08% (19/26) patients (non- IgM MM) and 23.81% (5/21) patients (non- IgA MM) at 1-year, respectively. Median times to IgG, IgM and IgA recovery were on day 386, 254 and not reached during follow-up, respectively. Virus-specific IgG levels decreased with loss of protection. Twenty-three of 40 (57.5%) patients developed a total of 44 infection events. No infection-related deaths. These results reveal a 7-month aplasia of bone marrow normal plasma cells and a longer hypogammaglobulinemia, suggesting a profound and lasting humoral immune deficiency after anti-BCMA CAR-T cell therapy, especially for IgA.

Lipemia in the Plasma Sample Affects Fentanyl Measurements by Means of HPLC-MS2 after Liquid-Liquid Extraction

Examination of fentanyl levels is frequently performed in certain scientific evaluations and forensic toxicology. It often involves the collection of very variable blood samples, including lipemic plasma or serum. To date, many works have reported the methods for fentanyl detection, but none of them have provided information about the impact on the assay performance caused by an excessive amount of lipids. This aspect may be, however, very important for highly lipophilic drugs like fentanyl. To address this issue, we developed the liquid chromatography method with mass spectrometry detection and utilized it to investigate the impact of lipids presence in rabbit plasma on the analytical method performance and validation. The validation procedure, conducted for normal plasma and lipemic plasma separately, resulted in good selectivity, sensitivity and linearity. The limits of detection and quantification were comparable between the two matrices, being slightly lower in normal plasma (0.005 and 0.015 µg/L) than in lipemic plasma (0.008 and 0.020 µg/L). Liquid–liquid extraction provided a low matrix effect regardless of the lipid levels in the samples (<10%), but pronounced differences were found in the recovery and accuracy. In the normal plasma, this parameter was stable and high (around 100%), but in the lipemic matrix, much more variable and less efficient results were obtained. Nevertheless, this difference had no impact on repeatability and reproducibility. In the present work, we provided reliable, convenient and sensitive method for fentanyl detection in the normal and lipemic rabbit plasma. However, construction of two separate validation curves was necessary to provide adequate results since the liquid-liquid extraction was utilized. Therefore, special attention should be paid during fentanyl quantification that involves lipemic plasma samples purified by this technique.

Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases.ImportanceRespiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.

Identification of potential plasma biomarkers in early-stage nasopharyngeal carcinoma-derived exosomes based on RNA sequencing

Background Early diagnosis of nasopharyngeal carcinoma (NPC) is vital to improve the prognosis of these patients. However, early diagnosis of NPC is typically challenging. Therefore, we explored the pathogenetic roles and associated mechanisms of exosomes in plasma of patients with early-stage NPC. Methods Exosomes in plasma were extracted by ultra-high-speed centrifugation. Western blot and transmission electron microscopy (TEM) were used to verify the purity of exosomes. The sequencing data (6 plasma samples from healthy volunteers vs. 6 NPC plasma samples) were analyzed by principal component analysis (PCA), DESeq2, gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and TargetScan. The differentially expressed miRNAs (DEmiRNAs) were obtained from the dataset (GSE118720) downloaded from the Gene Expression Omnibus (GEO) repository. Additionally, the datasets downloaded from the GEO database (GSE12452, GSE13597, GSE53819, GSE64634) were used to predict the target genes and functions of hsa-miR-1301-3p. qPCR was applied to verify the differences in the expressions of hsa-miR-1301-3p between 10 normal plasma and 10 NPC plasma samples. Results Western blot, TEM, and Nanoparticle Tracking Analysis showed adequate purity of the extracted exosomes. RNA-seq analysis revealed 21 upregulated miRNAs, and 10 downregulated miRNAs in plasma exosomes of early-stage NPC patients. GO analysis showed that the target genes of DEmiRNAs were mainly enriched in DNA synthesis and transcription regulation. KEGG analysis revealed that DEmiRNAs were mainly enriched in PI3K-Akt and MAPK signaling pathways. Moreover, the expression of hsa-mir-1301-3p was verified to be significantly upregulated in enlarged samples of plasma exosomes. Conclusions We identified several DEmiRNAs extracted from tumor-derived exosomes between normal plasma and early-stage NPC plasma. Bioinformatics analyses indicated that these DEmiRNAs may be related to NPC development. Our study may provide novel insights into underlying biomarkers and mechanisms of plasma exosomes in early-stage NPC.

Circulating tumor DNA methylation as markers for early detection of pancreatic ductal adenocarcinoma (PDAC).

395 Background: PDAC is a cancer of high mortality and low survival. Its early detection is critical due to symptoms often occur only at advanced stages. However there is no reliable screening tool to identify high-risk patients. ctDNA methylation has recently emerged as a promising new target to differentiate PDAC plasma from normal plasma for its early detection. Methods: Reduced representation bisulfite sequencing libraries were made in 46 PDAC tissues, 30 para-PDAC tissues and 20 PDAC plasmas to screen PDAC-specific markers, which was done by quantifying and comparing methylation levels of genomic regions and individual CpG sites between those groups. Markers were validated in plasma samples from 84 PDAC patients and 64 normal controls to propose a blood classifier. The best-performing markers were developed into a targeted sequencing panel, which was tested on a larger collection of plasma samples from patients of a variety of pancreatic diseases to build and validate a PDAC-predicting model. Results: We profiled genome-wide methylation patterns of tissues samples to identify 171 PDAC-specific markers. We reiterated training and cross-validating PDAC classification models using SVM method, and achieved an average sensitivity of 86% and specificity of 88%. To prove the feasibility of a non-invasive detection in plasma, a targeted methylation assay using those markers was tested on PDAC and normal plasmas, and yielded an average sensitivity of 68.4% and a specificity of 85.8%. We refined the panel by selecting the most discriminatory markers and built the version II of the panel, which was named PANcreatic Cancer Detection Assay, or PANDA, for a more efficient target capture, which was validated in an independent cohort of plasma samples that included 94 PDAC cases, 25 chronic pancreatitis (CP) cases and 80 normal samples from multiple centers. The PANDA achieved an AUC of 0.906 when classifying PDAC from normal, and an AUC of 0.882 when separating PDAC from CPs, both of which are more accurate than CA19-9, the conventional blood marker for PDAC. We further integrated test subjects’ age and their CA19-9 level as features into the PANDA model, which further elevated their AUC to 0.882 and 0.933 when classifying PDAC plasma from either CP plasma or normal plasma, respectively. Conclusions: We have developed PANDA, an NGS based target assay covering PDAC-specific DNA methylation targets by screening and validation on PDAC tissues and plasmas. Combined with age and CA19-9 blood level, PANDA has shown encouraging results to classify PDAC plasma from non-malignant diseases, demonstrating its potential to be optimized into non-invasive diagnostics for blood-based early PDAC screening.