Characterization of immune exhaustion and suppression in the tumor microenvironment of splenic marginal zone lymphoma.

The role of the tumor microenvironment (TME) and intratumoral T cells in splenic marginal zone lymphoma (sMZL) is largely unknown. In the present study, we evaluated 36 sMZL spleen specimens by single cell analysis to gain a better understanding of the TME in sMZL. Using mass cytometry (CyTOF), we observed that the TME in sMZL is distinct from that of control non-malignant reactive spleen (rSP). We found that the number of TFH cells varied greatly in sMZL, ICOS+ TFH cells were more abundant in sMZL than rSP, and TFH cells positively correlated with increased numbers of memory B cells. Treg cell analysis revealed that TIGIT+ Treg cells are enriched in sMZL and correlate with suppression of TH17 and TH22 cells. Intratumoral CD8+ T cells were comprised of subsets of short-lived, exhausted and late-stage differentiated cells, thereby functionally impaired. We observed that T-cell exhaustion was present in sMZL and TIM-3 expression on PD-1low cells identified cells with severe immune dysfunction. Gene expression profiling by CITE-seq analysis validated this finding. Taken together, our data suggest that the TME as a whole, and T-cell population specifically, are heterogenous in sMZL and immune exhaustion is one of the major factors impairing T-cell function.

Estradiol and Dihydrotestosterone Levels in COVID-19 Patients.

OBJECTIVE: To determine differences in plasma sex hormone levels in male and female coronavirus disease 2019 (COVID-19) patients and healthy volunteers (HVs) because cell entry of severe acute respiratory syndrome coronavirus 2 occurs via the angiotensin-converting enzyme 2 receptor which is downregulated by 17ß-estradiol. PATIENTS AND METHODS: Citrated plasma samples were collected from 101 patients with COVID-19 upon presentation to the emergency department and from 40 HVs between November 1, 2020, and May 30, 2021. Plasma 17ß-estradiol and 5α-dihydrotestosterone (DHT) levels were measured using enzyme-linked immunosorbent assay (pg/mL). Data are presented as median and quartiles (IQR). Wilcoxon rank sum test with a P value less than .05 was considered significant. RESULTS: Patients with COVID-19 (median age, 49 years) included 51 males and 50 females (25 postmenopausal). Hospital admission was required for 58.8% of male patients (n = 30) and 48.0% of female patients (n = 24) (66.7% postmenopausal, n = 16) Healthy volunteers (median age, 41 years) included 20 males and 20 females (9 postmenopausal). Female patients with COVID-19 were found to have decreased 17ß-estradiol levels (18.5 [IQR, 10.5-32.3] pg/mL; 41.4 [IQR, 15.5-111.0] pg/mL, P=.025), and lower 17ß-estradiol to DHT ratios (0.073 [IQR, 0.052-0.159] pg/mL; 0.207 [IQR, 0.104-0.538] pg/mL, P=.015) than female HVs. Male patients with COVID-19 were found to have decreased DHT levels (302.8 [IQR, 249.9-470.8] pg/mL; 457.2 [IQR, 368.7-844.3] pg/mL, P=.005), compared with male HVs. Levels of DHT did not differ between female patients with COVID-19 and female HVs, whereas 17ß-estradiol levels did not differ between male patients with COVID-19 and male HVs. CONCLUSION: Sex hormone levels differ between patients with COVID-19 and HVs, with sex-specific patterns of hypogonadism in males and females. These alterations may be associated with disease development and severity.

Establishment and characterization of a novel Waldenstrom macroglobulinemia cell line, MWCL-1.

Waldenström macroglobulinemia (WM) is a rare, lymphoplasmacytic lymphoma characterized by hypersecretion of immunoglobulin M (IgM) protein and tumor infiltration into the bone marrow and lymphatic tissue. Our understanding of the mechanisms driving the development and progression of WM is currently by the shortage of representative cell models available for study. We describe here the establishment of a new WM cell line, MWCL-1. Comprehensive genetic analyses have unequivocally confirmed a clonal relationship between this novel cell line and the founding tumor. MWCL-1 cells exhibit an immunophenotype consistent with a diverse, tumor clone composed of both small B lymphocytes and larger lymphoplasmacytic cells and plasma cells: CD3⁻, CD19⁺, CD20⁺, CD27⁺, CD38⁺, CD49D⁺, CD138⁺, cIgM⁺, and κ⁺. Cytogenetic studies identified a monoallelic deletion of 17p13 (TP53) in both the cell line and the primary tumor. Direct DNA resequencing of the remaining copy of TP53 revealed a missense mutation at exon 5 (V143A, GTG>GCG). In accordance with primary WM tumors, MWCL-1 cells retain the ability to secrete high amounts of IgM protein in the absence of an external stimulus. The genetic, immunophenotypic, and biologic data presented here confirm the validity of the MWCL-1 cell line as a representative model of WM.

Effect of tissue shipping on plasma cell isolation, viability, and RNA integrity in the context of a centralized good laboratory practice-certified tissue banking facility.

The Multiple Myeloma Research Consortium has established a tissue bank for the deposition of bone marrow samples from patients with multiple myeloma to be mailed and processed under good laboratory practices. To date, over 1,000 samples have been collected. At this time, limited information is available on shipped bone marrow aspirates in regards to cell viability, yield, purity, and subsequent RNA yield and quality. To test these determinants, we did a pilot study on behalf of the Multiple Myeloma Research Consortium where samples were drawn at Mayo Clinic Rochester (MCR) pooled and split into two equal aliquots. One-half of each sample was processed following good laboratory practices compliant standard operating procedures, immediately after sample procurement, at MCR. The CD138+ cells were stored at -80 degrees C as a Trizol lysate. The other half of the aspirate was sent overnight to Mayo Clinic Scottsdale where they were processed using identical standard operating procedures. The RNA was extracted and analyzed in a single batch at MCR. At both locations, samples were assayed for the following quality determinants: Viability was assessed using a three-color flow cytometric method (CD45, CD38, and 7-AAD). Cell counts were done to determine plasma cell recovery and post-sort purity determined by means of a slide-based immunofluorescent assay. RNA recovery and integrity was assessed using the Agilent Bioanalyzer. Lastly, gene expression profiles were compared to determine the signature emanating from the shipment of samples. Despite minor differences, our results suggest that shipment of samples did not significantly affect these quality determinants in aggregate.

Functional gene expression analysis of clonal plasma cells identifies a unique molecular profile for light chain amyloidosis.

Immunoglobulin light chain amyloidosis (AL) is characterized by a clonal expansion of plasma cells within the bone marrow. Gene expression analysis was used to identify a unique molecular profile for AL using enriched plasma cells (CD138+) from the bone marrow of 24 patients with AL and 28 patients with multiple myeloma (MM) and 6 healthy controls. Class prediction analysis (PAM) revealed a subset of 12 genes, which included TNFRSF7 (CD27), SDF-1, and PSMA2, that distinguished between these 2 groups with an estimated and observed accuracy of classification of 92%. This model was validated with an independent dataset of 11 patients with AL and 12 patients with MM with 87% accuracy. Differential expression for the most discriminant genes in the 12-gene subset was validated using quantitative real-time polymerase chain reaction and protein expression analysis, which upheld the observations from the micro-array expression data. Functional analyses using a novel network mapping software revealed a number of potentially significant pathways that were dysregulated in patients with AL, with those regulating proliferation, apoptosis, cell signaling, chemotaxis, and migration being substantially represented. This study provides new insight into the molecular profile of clonal plasma cells and its functional relevance in the pathogenesis of light chain amyloidosis.

Analysis of somatic hypermutation and antigenic selection in the clonal B cell in immunoglobulin light chain amyloidosis (AL).

Light chain amyloidosis (AL) is a protein folding disorder with an underlying B cell neoplasia where the monoclonal immunoglobulin light chains (LCs) produced from insoluble amyloid fibrils. The deposition of these fibrillar aggregates in vital organs causes severe organ dysfunction over time and is associated with high mortality. We have identified the postgerminal center status of the B cell clone by evaluating the presence of somatic hypermutation in the variable region of the LC gene in 27 (13 of the lambda and 14 of the kappa subtype) AL patients. Seven of the 27 clones showed statistically significant evidence of antigenic selection, using a multinomial algorithm. The framework region mutations were selected for conservation of protein structure in 13 of the 27 patients. Additionally, mutational clusterspots were identified at specific positions in the nucleotide and deduced protein sequence that could potentially contribute to destabilizing interactions resulting in a propensity to form amyloid.

Immunoglobulin light chain variable (V) region genes influence clinical presentation and outcome in light chain-associated amyloidosis (AL).

Light chain-associated amyloidosis (AL) is a plasma cell dyscrasia in which the secreted monoclonal immunoglobulin (Ig) light chains form amyloid fibrils. There is considerable heterogeneity in clinical presentation, and prognosis of the disease relates to the severity of organ dysfunction induced by amyloid deposits. The mechanisms by which the amyloid fibrils are deposited as well as the predilection for specific organ sites have not been clearly elucidated. This study characterizes the repertoire of immunoglobulin light chain variable genes used by the clonal B cell in AL amyloid patients, and the association of light chain variable region (VL) genes with clinical presentation and outcome is assessed in 58 (32 lambda and 26 kappa) patients. A preferential use of VL germ-line genes was noted for both AL kappa and lambda patients. There was a significant correlation between the use of the Vlambda VI germ-line donor, 6a, and renal involvement as well as the Vlambda III gene, 3r, with soft-tissue AL. The use of a biased VL gene repertoire also correlated with clinical outcome, revealing important trends for predicting prognosis. The use of Vlambda II germ-line genes was associated with cardiac amyloidosis and affected survival adversely. The presence of multiple myeloma also correlated with a poor prognosis. The presence of renal disease, on the other hand, was associated with improved survival. Therefore, identification of the clonal VL gene in AL has important implications in determining clinical outcome.