CD56 Dim Research Articles

Development of an 18-color flow cytometry panel for B-lymphoblastic leukemia minimal residual disease detection

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is characterized by a neoplastic proliferation of precursor B cells that have blast cytomorphology and immature immunophenotype. Following treatment of B-ALL, flow cytometry is utilized to detect minimal residual disease (MRD), which aids in monitoring therapeutic response and informing prognosis. In our institution, the BD FACSCanto cytometer is used in conjunction with two standardized 8-antibody EuroFlow panels to detect MRD in B-ALL patients. The objective of this study was to validate a new 18-color B-ALL MRD panel for bone marrow aspirate specimens to be run on a BD LSRFortessa cytometer, with the intention of improving sensitivity and consolidating the testing into one antibody panel. The proposed panel consisted of 22 antibodies, some of which were stacked within the same fluorescence channel, in addition to a viability stain. Twelve bone marrow aspirate specimens collected in 2023 (five positive and seven negative B-ALL MRD specimens) were stained with the new antibody panel and analyzed on the Fortessa cytometer. CD45 dim, CD19-positive blasts were analyzed using BD FACSDiva Software. For the five specimens previously reported as B-ALL MRD-positive using the EuroFlow panels, we were able to detect phenotypically identical B-ALL cells with the 18-color panel. A paired t-test done on the five MRD-positive specimens showed no significant difference in the percentage of residual B-ALL cells (out of total viable cells) measured by the 18-color panel when compared to the EuroFlow panels (EuroFlow/18-color: 0.0096%/0.0097%, 0.024%/0.012%, 0.94%/0.99%, 0.85%/0.85%, 0.022%/0.017%; p=0.58). All seven specimens that were previously reported as B-ALL MRD-negative using the EuroFlow panels were also negative for residual B-ALL cells when stained with the 18-color panel and analyzed on the Fortessa cytometer. The antibody cost per test of the EuroFlow panels ($20-$25, ~$44 for both panels) and new 18-color panel ($63) were calculated based on the unit cost of each antibody aliquot and the required volume of reagent per test. In summary, the new 18-color B-ALL MRD panel run on the BD LSRFortessa cytometer appears to provide similar results as the EuroFlow panels run on the BD FACSCanto cytometer. Although compared to the two EuroFlow panels, the single 18-color panel is more expensive per test in terms of antibodies, it may be preferable when there is scant tissue (such as cerebrospinal fluid) as there may not be enough material to run both EuroFlow panels.

A discussion of pediatric therapy-related acute myeloid leukemia with a rare t(8;16) translocation

Abstract Introduction/Objective Secondary acute myeloid leukemia is a type of blood cancer that arises after a previous myeloid neoplasm or after exposure to chemotherapy. Certain genetic mutations and chromosomal abnormalities can occur as a result of said cytotoxic drug-related effects. Here, we discuss a case of secondary acute myeloid leukemia with a rare translocation following hemophagocytic lymphohistiocytosis Etoposide treatment in a 13-year-old female. After less than a year into her initial treatment, the patient presented to the emergency department with severe bone pain. A full hematologic workup was performed, including bone marrow biopsy, flow cytometry analysis, and subsequent genetic studies. A diagnosis of therapy-related acute myeloid leukemia was made. Genetic studies revealed a rare t(8;16) translocation which presents with a unique morphology and prognosis that will be discussed here. Methods/Case Report A 13-year-old female presented to the emergency department for evaluation of fever, myalgia, and swelling in her clavicle. The patient’s history was notable for hemophagocytic lymphohistiocytosis with subsequent Etoposide chemotherapy treatment which she received less than a year prior to her visit. CBC was notable for low white blood cell count, low hemoglobin, increased neutrophilic bands, and decreased lymphocytes. Given her past medical history and worsening pain without a clear reason, hematology was consulted. Results (if a Case Study enter NA) A blast count of 61.8% was reported via flow cytometry with the cells seen in the dim CD45+, moderate to bright side-scatter region, co-expressing CD4, CD14 (dim, heterogeneous), CD15, CD16 (dim), CD64, HLA-DR, and MPO. The bone marrow biopsy was described as hypercellular with the predominant cell type as blasts at 66.4%. Numerous hemophagocytic blasts were noted as well. Acute leukemia next generation sequencing panel revealed a t(8;16)(p11;p13.3) KATA6A::CREBBP fusion without other abnormalities in the evaluated genes. This workup as a whole rendered the diagnosis of acute myeloid leukemia with KAT6A::CREBBP. Conclusion Diagnosing acute myeloid leukemia with KATA6A::CREBBP is important for prognostic factors. Knowing the patient’s history regarding her prior chemotherapy with Etoposide is also imperative for distinguishing a de novo presentation of this rare subtype, versus a therapy-related myeloid neoplasm. The patient was started on chemotherapy for her acute myeloid leukemia shortly thereafter the diagnosis.

A Rare Case of Diffuse Large B Cell Lymphoma Co-occurring with Small Lymphocytic Leukemia: De Novo Process or Richter’s Syndrome?

Abstract Introduction/Objective Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of lymphoma and can arise either de novo or through transformation from indolent lymphomas, such as Chronic Lymphocytic Leukemia (CLL), known as Richter syndrome (RS). DLBCL derived from RS tends to exhibit more aggressive behavior compared to de novo DLBCL. Methods/Case Report We present a case of a 57-year-old female with no significant medical history, who initially presented with antibiotic and prednisone-resistant sinusitis, progressing to bilateral neck pain and eye congestion. Computed tomography (CT) imaging revealed a 3 cm mass in the right nasal cavity extending to the right maxillary sinus and medial right orbit. Histopathological examination showed diffuse sheets of intermediate to large cells with irregular nuclear contours, dispersed chromatin, variably conspicuous nucleoli, and moderate cytoplasm. Additionally, scattered foci of monotonous, atypical lymphoid cells with lower grade morphology, including small size and condensed chromatin, were also present. Immunohistochemical analysis revealed differential staining patterns between the two cell populations. The larger atypical cells expressed CD20, PAX5, CD10, BCL-6, MUM1, BCL-2, MYC, CD19 (dim), and exhibited a high Ki-67 proliferation index (>80%). In contrast, the smaller atypical cells were positive for CD5, CD19, CD20, PAX5, BCL-2, and CD23 (subset), with a lower Ki-67 proliferation index (<5%). Flow cytometry analysis of tissue samples revealed two distinct clones: Surface Kappa-restricted CD19 dim B cells (38.9%) with CD5- CD10 dim CD20+ and surface Lambda-restricted CD19+ B cells (58.7%) with CD5+ CD10- CD20+. FISH analysis revealed no gene rearrangements. The final diagnosis was Diffuse large B-cell lymphoma, germinal center B- cell phenotype, with aggressive features and focal involvement by small lymphocytic lymphoma/chronic lymphocytic leukemia. Results (if a Case Study enter NA) NA Conclusion The relationship between the large B-cell lymphoma and SLL/CLL remains uncertain; however, given the different immunophenotype and light chain expression observed by flow cytometry, they are likely to represent separate processes.

Flow cytometric immunophenotypic features of acute myeloid leukemia with mast cell differentiation.

Acute myeloid leukemia (AML) with mast cell (MC) differentiation was recently described as an aggressive subgroup of AML cases. The objectives of this study were to assess the flow cytometric immunophenotypic features of AML-MC cases. We characterized the immunophenotypic features of 21 AML-MC cases by flow cytometry and compared them to 20 reactive/regenerating bone marrow specimens. The number of MCs detected by flow cytometry in AML-MC cases ranged from 0.4% to 21.1%, with a median of 3.5%, significantly higher than that of normal/reactive bone marrow (BM) (median, 0.01%; range, 0.000%-0.396%; P < .0001). Immunophenotypically, MCs in AML-MC cases demonstrated immaturity, differing from MCs in normal/reactive BMs, including dimmer CD45 (100% vs 0%), lower side scatter (100% vs 0%), more frequent CD34 (81% vs 20%), and CD123 (100% vs 10%) positivity, and more frequent uniform/increased CD38 expression (95% vs 20%) (all P ≤ .0001). CD2 (0/5) and CD25 (2/6, 1 uniform and 1 partial) were assessed in a subset of cases. The myeloblasts in AML-MC were typically CD34+CD117+HLA-DR+ with unusually frequent expression of CD56 (57%, all partial) and CD25 (63%, mostly partial), increased CD117 (62%), and decreased CD38 (86%). The MC percentage determined by flow cytometry correlated well with MCs detected by tryptase immunohistochemistry (r = 0.76, P < .001). The MCs in AML-MC cases are characterized by dim CD45, low side scatter, CD34 and CD123 positivity, and uniform and increased CD38 expression. Flow cytometry is an excellent tool for identifying AML-MC cases.

Exploring the role of pyroptosis and immune infiltration in sepsis based on bioinformatic analysis

PurposeSepsis is a disease that is typically treated in intensive care units with high mortality and morbidity. Pyroptosis is a newly identified type of programmed cell death and is characterized by inflammatory cytokine secretion. However, the role of pyroptosis in sepsis remains unclear. MethodsGSE28750 and GSE134347 datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed pyroptosis genes (DEPGs) were identified between sepsis and healthy controls. Machine learning was used to further narrow the gene range. Receiver operating curves (ROC) were generated to estimate the diagnostic efficacy. Immune infiltration levels were estimated via single-sample gene set enrichment analysis (ssGSEA). A network database was used to predict the upstream transcription factors and miRNAs of DEPGs. Finally, the expression of the genes was validated by qRT-PCR between sepsis patients and healthy controls. ResultsWe found that the pyroptosis pathway was enriched and activated in sepsis. 8 DEPGs were identified. A heatmap showed that the genes, NLRC4, NAIP, IL-18, AIM2 and ELANE, were abundant in the sepsis samples, and the genes, NLRP1, CHMP7 and TP53, were abundant in the healthy control samples. The ssGSEA results showed that the abundances of activated dendritic cells, MDSC, macrophage, plasmacytoid dendritic cells, regulatory T-cells, and Th17-cells were significantly higher, while the activated B-cell, activated CD8 T-cell, CD56 dim tural killer cell, immature B-cell, monocyte, and T follicular helper cell abundances were lower in sepsis samples compared to healthy controls. The qRT-PCR results showed that the expression levels of NAIP, IL-18, TP53, CHMP7, NLRC4, ELANE and NLRP1 were consistant with the bioinformatic analyses, while the expression level of AIM2 has no significant difference. ConclusionOur study identified seven potential pyroptosis-related genes, NAIP, IL-18, TP53, CHMP7, NLRC4, ELANE and NLRP1. This study revealed that pyroptosis may promote sepsis development by activating the immune response.

Abstract 112: The NSG-Flt3KO; Tg(hFLT3LG); hIL15 mouse strain improves engraftment of innate immune cells post-CD34+ humanization

Abstract Human immune system mouse models play critical roles in numerous preclinical drug development pipelines, including immunology, infectious diseases, and immuno-oncology. These models are constantly evolving, with addition of human cytokines to sustain engraftment of human immune cells. To date, most humanized models have primarily focused on T lymphocyte cell biology, leaving room for improvement in developing models that effectively represent NK cells and the myeloid lineage. Previously, we demonstrated that the introduction of human IL-15 (NSG-IL-15) or FLT3L (NSG-FLT3L) to NOD-scid IL2rγnull (NSG) mice enhanced engraftment of innate immune cells, including human CD56+ NK cells, CD141+ and CD1c+ DC subsets, CD123+ pDCs, and CD14+ monocytes. In this study, we describe a new NSG mouse variant (NSG-Flt3KO; Tg(hFLT3LG); hIL15) that expresses physiological levels of both human IL-15 and FLT3L and detail the immune cell diversity following HSC engraftment. NSG-Flt3KO; Tg(hFLT3LG); hIL15 mice, along with control strains NSG, NSG-IL15, and NSG-FLT3L, were engrafted with human umbilical cord blood derived CD34+ hematopoietic stem cells from three different donors, and blood samples were collected until Week 20 post-transplantation. Blood samples were analyzed via flow cytometry to assess the engraftment of innate and adaptive immune cells. Spleen and bone marrow samples were collected at Week 12 post-transplantation and analyzed for lymphoid and myeloid immune cell populations via flow cytometry. To compare the differences among the strains in terms of immune cell sub-populations across weeks, a Two-Way ANOVA (mixed-model) was conducted, with p&amp;lt;0.05 considered statistically significant. Overall, the results demonstrate that NSG-Flt3KO; Tg(hFLT3LG); hIL15 mice have enhanced CD56+/dim CD16+ NK cells compared to NSG-FTL3L strain (20.5±12.8% vs 7.0±3.5% at Week 12), but comparable to NSG-IL15. Furthermore, myeloid sub-lineage cells in NSG-IL15-FLT3L mice, such as monocytes, macrophages (M1), granulocytes, dendritic cells, and neutrophils, were greater than in NSG or NSG-IL15 strains, but comparable to NSG-FLT3L mice. Overall levels of hCD45+ and T cell sub-populations (CD3+ or CD4+ or CD8+) were comparable across all strains tested. In sum, our NSG-Flt3KO; Tg(hFLT3LG); hIL15 engraftment data suggest an improved model that supports drug development targeting human myeloid and NK cell populations. Citation Format: Srinivas Reddy Boreddy, Shuang Hu, Brian Soper. The NSG-Flt3KO; Tg(hFLT3LG); hIL15 mouse strain improves engraftment of innate immune cells post-CD34+ humanization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 112.

Relapsing-remitting multiple sclerosis patients exhibit differential natural killer functional subpopulations.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) and has been known as T-cell mediated. However, the contribution of multiple cell types, notably natural killer (NK) cells, has also been reported. To quantify circulating total NK cells and its subpopulations, CD56 dim and bright, and to characterize the functional phenotype and IFN-γ and TNF-α production in relapsing-remitting patients treated with IFN-β and in apparently healthy controls. CD56bright NK cells were found to be the least represented subpopulation. In relapse patients, the frequencies of IFN-γ-producing NK cells and their subpopulations were significantly decreased. In remission patients, CD56dim NK cells expressed high levels of HLA-DR and CD54. These results suggest that remission RRMS patients, although in an inactive stage of MS, present circulating NK cells with an activation phenotype, supporting the idea that NK cells may be relevant mediators in the MS pathophysiology.

Ovarian myeloid sarcoma with concurrent acute myeloid leukemia- A case report

Myeloid sarcoma is a rare extramedullary tumor of immature myeloid cells that can be composed of cells showing granulocytic or monocytic differentiation. It is frequently detected as masses in subcutaneous soft tissue, bone, skin, GIT, reproductive organs, CNS, heart, lungs, kidney and breast. We report a pediatric case of myeloid sarcoma presenting with bilateral ovarian masses.A 12 years old female presented with complaints of fever, lethargy, decreased appetite and easy fatiguability. Patient further developed swelling and pain in large joints along with slurring of speech and difficulty in swallowing. Examination revealed polyserositis along with hepatosplenomegaly. ANA IFA profile was positive. CECT abdomen showed heterogeneous lesions in bilateral adnexae along with a few enlarged matted lymph nodes in left para-aortic region (?Neoplastic/ ?Histiocytosis/ ?EM hematopoiesis). GBP was suggestive of leucoerythroblastic reaction. BM study with flowcytometry was suggestive of Acute myeloid leukemia with monocytic differentiation. Biopsy from both adnexae showed features consistent with poorly differentiated malignancy, favour haematolymphoid neoplasm. Immunophenotyping by IHC showed the cells to be positive for CD45 (dim), MPO &amp; CD117, thus establishing diagnosis of Myeloid sarcoma, both ovaries.Myeloid sarcoma is uncommon ovarian neoplasm and the clinical presentation may be with or without acute leukemia / MPN. Immunohistochemistry and ancillary studies are necessary to recognize this entity.

PRDM1 Promotes Primary Human Circulating CD56 dim NK-Cell Differentiation

Background: Natural killer (NK) cells are innate lymphocytes that mount immune responses against viral infection and malignant cells. Circulating NK-cells can be generally divided into two groups, CD56 bright and CD56 dim, with the latter constituting ~90% of NK-cells in peripheral blood. While CD56 bright NK-cells are considered less mature and capable of producing more cytokines, CD56 dim population represents more differentiated cells with greater cytotoxicity. Transcription factor (TF) regulatory circuits were proposed for CD56 bright and CD56 dim NK-cells, indicating that TCF1/LEF1 and BACH2 are important in the former population and that Blimp1 and MAF are master TFs in the latter group. Blimp1 (encoded by PRDM1) has been previously found to regulate terminal differentiation of B-cells and CD8 + T-cells. In NK-cells, deletion of PRDM1 has been shown to promote cell growth and survival in vitro, and its loss of function was commonly detected in NK-cell lymphomas. Here, we employed a multi-omics approach to investigate the role of PRDM1 in the differentiation of human primary NK-cells. Methods: We used CRISPR-Cas9 system to knock out PRDM1 in primary human NK-cells isolated from PBMCs of healthy donors and performed whole transcriptome sequencing. ChIP-seq was employed to investigate the global binding spectrum of PRDM1 in primary human NK-cells cultured with K562-mbIL21-Cl9 feeder cells or with IL-2 alone. We also performed integrative genomics analyses to examine the transcriptomic regulation of NK-cell differentiation by PRDM1. Results: Gene set enrichment analyses (GSEA) demonstrated that PRDM1 KO resulted in enrichment of genes highly expressed in CD56 bright cells and depletion of genes upregulated in CD56 dim NK-cells. Specifically, we found that PRDM1 deficient NK-cells expressed CD56 bright NK-cell master TFs at higher levels, such as TCF7, RUNX2, and MYC. In contrast, TFs that play an important role in more differentiated CD56 dim NK cells were downregulated in PRDM1-KO cells, including IKZF3, MAF, and TBX21. Notably, BCL11B, a TF that has been reported to promote canonical and adaptive NK-cell differentiation, was significantly downregulated along with its target gene ZBTB16 when PRDM1 was deleted. Moreover, genes encoding cytotoxic molecules such as PRF1 and GZMB were also repressed in PRDM1-KO NK-cells. Together, these results indicate that PRDM1-KO NK-cells closely resembled the less mature CD56 bright NK-cells than the CD56 dim terminally differentiated counterpart. ChIP-seq analysis of PRDM1 revealed that many of the differentially expressed genes (DEGs) between PRDM1 KO and WT cells were directly bound by PRDM1. When integrated with publicly available ATAC-seq datasets, we found that some of these PRDM1 bound sites showed differential chromatin accessibility between CD56 bright and CD56 dim NK-cells. To explore the transcriptional program regulated by PRDM1, we utilized a regulated system to re-express PRDM1 in KHYG1, an NK-cell lymphoma cell line that is sensitive to PRDM1 overexpression. RNA-seq analysis showed that the DEGs between KHYG1 with short-term re-expression of PRDM1 and control cells overlapped with DEGs between PRDM1 KO vs WT NK-cells. These genes included BCL11B, MAF, PRF1, and GZMB. Therefore, PRDM1 directly regulates genes that are crucial in NK-cell differentiation. Furthermore, we detected upregulation of memory T-cell or progenitor exhausted T-cell signature genes in PRDM1 KO NK-cells compared with WT, including TCF7, SELL, CCR7, and IL7R. Consistent with this, GSEA analysis demonstrated enrichment of TCF1 + progenitor exhausted T cell or memory T cell upregulated genes in PRDM1 deleted NK-cells, suggesting that PRDM1 KO NK-cells may share some features with memory T-cells or progenitor exhausted T cells. Importantly, MYB, which was found to be essential in progenitor exhausted T cells, was also a direct target of PRDM1 and was upregulated in PRDM1 KO NK-cells. GSEA also showed that PRDM1 depletion in NK-cells led to upregulation of genes highly expressed in mast cell, Th1 cell, B cell, or CD8 + T cell compared to NK cells, which may indicate a role of PRDM1 in maintaining lineage commitment. Conclusions: Our findings collectively show that PRDM1 may be a master TF that regulates human NK-cell differentiation and functions through direct transcriptional regulation of key target genes.

CD123 As a Therapeutic Target in Accelerated and Blast Phase Myeloproliferative Neoplasms (MPNs)

Background: CD123 expression in MPNs has been reported in case series and CD123 has been a therapeutic target in one phase I study of patients with chronic myelomonocytic leukemia or myelofibrosis in chronic phase. CD123 has also been described as a marker of leukemic stem cells and incorporated in chimeric antigen receptor T-cell products for treatment of acute myeloid leukemia in clinical trials. Herein, we describe the clinical phenotype of patients with accelerated (AP) or blast phase (BP) MPNs paired with CD123 co-expression on CD45(dim) myeloid blasts. Methods: This is a retrospective, single-institution study of patients with AP and BP MPNs. Subjects were identified by search for MPN diagnoses in the Ohio State University Comprehensive Cancer Center Leukemia Tissue Bank (OSUCCC-LTB). Relevant clinical data was collected by retrospective chart review with approval of Institutional Review Board. CD123 expression was measured by immunophenotyping of cryopreserved peripheral blood (PB) or marrow (BM) samples. Blasts were identified by CD45(dim) gating of lymphocytes. Lymphocytes were also gated on CD7, CD13, CD33, CD34, and CD117. CD123 expression was stratified by quartile and correlation with disease phase, symptoms, splenomegaly, transfusion-dependence, and molecular mutations measured by Chi-square test. Results: Forty-one PB or BM samples from 24 subjects were identified from the OSUCCC-LTB. Nine subjects had paired PB and BM specimens and 6 subjects had samples obtained at multiple timepoints in the course of their disease. Median age at time of sample collection was 65.8 years (range 35.2-79.7) and median time from initial diagnosis to sampling was 13.1 months (range 0-96.5). Constitutional symptoms were present at the time of collection in 24 specimens (58.5%) and splenomegaly present in 20 specimens (51.3%). At sample collection, 19.5% had never received treatment, 19.5% had been previously treated, and 61% were currently on treatment. Most patients on treatment (n=16, 64%) were receiving JAK inhibitor at sample collection. Nineteen samples were obtained in chronic phase (46.3%), nine in AP (22.0%), and thirteen in BP (31.7%). Median WBC count was 4.37K/uL (range 0.3-174.6), median peripheral blasts were 3.2% (range 0-76%), median hemoglobin was 8.8 g/dL (range 5.6-13.2), and median platelets were 42K/uL (range 5-564). Sixteen samples (39.0%) were from transfusion-dependent patients, of which, 68.8% were both red blood cell (RBC) and platelet transfusion-dependent, 25.0% were RBC transfusion-dependent only, and 6.2% were platelet transfusion-dependent only. Median lactate dehydrogenase at time of sampling was 300 U/L (range 116-2,142). Median bone marrow blasts at time of sampling were 3% (range 0-82%). Molecular sequencing and karyotype +/- 30 days of sample collection were available for 18 (43.9%) and 27 (65.9%) of specimens, respectively. The most common mutations identified were JAK2 (50%), ASXL1 (27.8%), SRSF2 (22.2%), CALR (16.7%), and TP53 (16.7%). Normal karyotype was reported in 37% of the samples (one was consistent with donor chimerism post-allogeneic SCT), 33.3% had complex karyotype, 11.1% had del 5q or 7q/-7, and 18.5% had del 13q or 20q. CD123 expression on the CD45(dim) gated blasts ranged from 0.3-95.6% with a median value of 11.02%. CD123 expression is presented in figure 1 box plot. Figure 2a shows correlation between CD123 expression and AP/BP compared to chronic phase disease (p &amp;lt; 0.01). Figure 2b shows CD123 expression correlation with ASXL1 compared to unmutated ASXL1 (p =0.023). There were no statistically significant associations between CD123 expression and any other tested variables. Conclusions: These single-institution data suggest that high CD123 expression on blasts is associated with leukemic transformation and high-risk disease. This is demonstrated by correlation of higher expression in AP and BP MPN as well as correlation with ASXL1 mutation. CD123 expression in MPNs has been previously reported and explored as a therapeutic target in a single phase I trial, though these data suggest that specifically targeting high risk disease, defined by blasts &amp;gt;10% or the presence of ASXL1 mutation, may be more effective with greater target availability. This is an especially important subpopulation of patients with MPNs in need of more effective and less toxic therapy than current standards of care that typically include cytotoxic chemotherapy.

Single-Cell Analysis of Immune Recognition in Chronic Myeloid Leukemia Patients Following Tyrosine Kinase Inhibitor Discontinuation

Background: Modern treatment goal for patients with chronic myeloid leukemia (CML) is to maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. The control of residual leukemia cells may occur with immunological mechanisms as several studies have shown that a higher proportion of CD56 dim NK cells before TKI discontinuation is associated with a better probability of TFR. In addition, CD8+ T cell abundances and phenotypes have been associated with responses to TKIs. However, the more detailed phenotypes and mechanisms and how to translate these findings to increased TFR rates have remained unknown. Methods: We analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced (scRNA+TCRαβ-seq) samples from patients with CML ( n=13, N=25), other cancers (AML n=11, CLL n=13, solid tumors n=4), and healthy ( n=7). Additionally, we profiled 90 CML and 786 healthy TCRβ-seq samples, and sorted T cells specific to leukemia-associated antigen PR1 and matched these back to the scRNA+TCRαβ-seq and TCRβ-seq data. To validate and refine our findings, we performed co-culture of primary and expanded NK cells with CML cell lines (K562 and LAMA84) with a multiplexed scRNA-seq readout. Results: In the pan-cancer scRNA+TCRαβ-seq comparison of &amp;gt;250,000 cells, CML was separated from other cancers and healthy by the high number and active phenotype NK cells. The other phenotype more pronounced in CML was CD8+ T EM/T EMRA phenotype. Most NK cells in CML had an active CD56 dim phenotype with high expression of GZMA/B, PRF1, CCL3/4 and IFNG. In the off-TKI samples from patients with TFR, these NK cells upregulated NK cell activation genes such as TNFRSF4/OX40, TNFRSF9/4-1BB, and TNFRSF18/GITR. The most important predicted interactions between NK cells and CD34+ leukemic cells were inhibitory LGALS9- TIM3 and PVR- TIGIT interactions. Following TKI discontinuation, the inhibitory markers HAVCR2/TIM3 and TIGIT decreased in patients with TFR, which was not seen in patients with relapse following TFR. Accordingly, in our co-culture data of NK cells with CML cell lines, we noted activation of NK cells to a similar phenotype as noted in ex vivo off-TKI samples. In co-culture with CML target cells, NK cells gained an activation by upregulation of genes like TNFRSF4/OX40, TNFRSF9/4-1BB, TNFRSF18/GITR and the inhibitory markers HAVCR2/TIM3 and TIGIT. In co-culture with NK cells, CML target cells upregulated the IFN-γ response pathway including elevated HLA-expression and accordingly, upregulation of LGALS9 and PVR. The CD8+ T EM/EMRA cells that were expanded in CML diagnosis were suppressed following TKI treatment and decreased following TKI discontinuation. To track anti-CML T cells, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 from the TCRb-sequenced samples. As a validation of our classifier, we noticed that anti-PR1 T cells were more prevalent in CML than in healthy ( P adj&amp;lt;0.0001) or melanoma ( P adj&amp;lt;0.0001), more expanded than similar anti-viral T cells ( P adj&amp;lt;0.0001) and enriched in bone marrow samples compared to peripheral blood ( P adj&amp;lt;0.0001). From different TKIs, dasatinib expanded anti-PR1 T cells the most ( P adj&amp;lt;0.01). In the scRNA+TCRαβ-seq data, anti-PR1 T cells were enriched in the mature, cytotoxic CD8+ T EMRA phenotype ( P adj&amp;lt;0.0001). In a patient maintaining TFR, anti-PR1 T cells had a highly cytotoxic profile ( GZMB, PRF1) throughout the therapy with upregulated NK receptors like FCGR3A/CD16 and KIR2DL2, even more so than T cells targeting other targets e.g., different viruses. In comparison, the anti-PR1 T cells in a patient with early relapse had an exhausted profile with upregulation of memory marker GZMK and exhaustion markers like LAG3 and HAVCR2/TIM3. Conclusions: With our in-depth analysis of cellular and molecular immune responses in CML, we identified the active NK cells and anti-PR1 T cells that could help maintain TFR in patients discontinuing TKI treatment. Different immune-checkpoint blockade therapies such as anti-TIM3 could be considered as future strategies to improve the TFR rates.

Inflammation Shapes the Transcriptional Landscape of the Bone Marrow Microenvironment in Post-Transplantation Leukemia Relapses

Allogeneic Hematopoietic Cell Transplantation (allo-HCT) represents the most successful therapeutic option for many patients suffering from Acute Myeloid Leukemia (AML). Nevertheless, leukemic cells often find means to evade control from the donor-derived immune system and re-emerge. To date, three alternative and frequent mechanisms by which AML cells modify their features and escape immune control have been described: genetic loss of the mismatched HLA haplotype (HLA loss), downregulation of HLA class II molecules and upregulation of inhibitory ligands, all ultimately leading to abolished leukemia recognition by T lymphocytes. However, increasing evidence suggests that leukemic cells weave a much broader net of interactions, involving several other immune cell types within the bone marrow microenvironment. Still, how this complex crosstalk between malignant cells and the bone marrow niche may shape the features of leukemia post-transplant relapse remains unknown. By exploiting the high resolution provided by single-cell RNA-sequencing (scRNA-seq), we provide a detailed insight of the human bone marrow immune microenvironment and the changes that occur following allo-HCT, both when disease reemergence is prevented, and when it occurs. Using cryopreserved BM aspirates, we profiled a total of 145,420 mononuclear cells from 25 adult AML patients at the time of disease relapse; 56,831 cells from 5 post-transplant patients in complete remission (CR), each tested at two different timepoints (+90 and +365 days) and 45,279 cells from 6 healthy controls (HC). First, scRNA-seq allowed us to recapitulate known leukemia-intrinsic features of each relapse modality, such as HLA loss, down-regulation of HLA class II molecules and upregulation of inhibitory T cell ligands. Moreover, combining genotype and Copy Number Variation (CNV) inference algorithms, we were able to separate malignant cells from their healthy counterparts. Interestingly, HLA loss relapses exhibited a more immature profile, with a significantly higher leukemia stem cell signature (LSC17) (p-value &amp;lt; 0.0001, Wilcoxon rank-sum test) (Ng SW et al., Nature 2016), hinting that the hematopoietic cell of origin impacts on the mechanism of post-transplantation relapse. Leukemic cells also featured a high inflammation-associated gene score (iScore) (Lasry A et al., Nat Cancer 2023), as also evident in non-malignant HSPCs from relapsed patients, which showed an increased IFN-α signature compared to HC and CR. However, AML samples that couldn't be categorized by a known mechanism of relapse displayed a significantly lower iScore, suggesting that leukemia escape in these patients might not have had an immune-related driver (Figure 1a). We next sought to characterize specific BM immune compartments. We overall detected a lower frequency of CD4 T cells in relapsed patients as compared to HC and CR patients (p-value = 0.04, Wilcoxon rank-sum test). We annotated 5 major Natural Killer (NK) cell clusters (immature CD56 dim, mature CD56 dim, CD56 bright, proliferating NK, NKT cells) and a smaller subset of immature CD56 bright NK cells characterized by higher expression of IFN-related genes and NK exhaustion markers, which was defined as inflamed NK cluster. Inflamed NK cells were more represented in patients with upregulation of T cell inhibitory ligands compared to other relapsed patients (5.42% of total NK vs. 0.74%, respectively. p-value = 0.03, Wilcoxon rank-sum test), thus suggesting a shared mechanism of dysfunction signature between T and NK cell that may favor leukemia immune evasion (Figure 1b). In conclusion, we generated a comprehensive atlas of the BM immune microenvironment in AML post-transplantation relapses, showing that inflammation shapes immune subset composition and transcriptional profile in the BM, and that leukemia mechanisms of immune evasion are influenced by the surrounding milieu. Overall, while an in depth understanding of whether leukemia relapse is caused by an inflamed microenvironment, or vice versa, might only be achieved through longitudinal sampling from patients before and after transplant, our results here exemplify how the BM niche could be exploited to find novel vulnerabilities in AML relapses, to be specifically targeted by personalized therapeutic approaches.

Single-Cell Transcriptomics Reveals Adaptive NK Cell and Antigen-Specific T Cell Responses Shaped by CMV Viremia Post-Hematopoietic Stem Cell Transplant

Background Impaired immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) poses risks for both disease recurrence and treatment-related complications, such as cytomegalovirus (CMV) reactivation. CMV strongly shapes the immune system, but the development of anti-CMV T cells and CMV-associated adaptive NK cells in vivo is still largely uncharacterized. Understanding these dynamics post-allo-HSCT may have clinical implications, since NK cells mediate a graft-versus-leukemia effect, particularly in myeloid malignancies, and adaptive NK cells have recently been associated with favorable immunotherapy responses in both hematological and solid cancers. Here, with longitudinal sample collection pre and post-allo-HSCT, we aimed to determine how patient and donor-derived NK and T cells synergistically contribute to immune system regeneration, how clonotypes develop and persist, and whether donor/recipient CMV serostatus and CMV reactivation post-allo-HSCT influence NK and T cell phenotypes. Methods We sequenced 32 longitudinal CD45+ sorted peripheral blood samples with single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) from 5 allo-HSCT donor-recipient pairs (pre-transplant sample from donor and recipient and 3-4 follow-up samples per patient over 3 months) and 6 healthy controls (Figure). All recipients were CMV seropositive, while 2 and 3 donors were seropositive and negative, respectively. CMV reactivation occurred in 4 patients during follow-up, on average 32 days post-allo-HSCT. We screened 32 common viral epitopes from post-transplant samples (n=3, 5 samples) with scRNA+TCRαβ+pMHC-seq (Immudex dCODE Dextramers). We then profiled T cells specific to CMV pp65 NLV-multimer with TCRβ-seq (allo-HSCT donor-recipient pairs n=3, 13 samples, healthy n=4), and traced CMV-recognizing TCRs in the scRNA+TCRαβ-seq data. Results We analyzed a total of 197,887 immune cells, including 37,123 NK cells and 59,852 CD8+ T cells. After allo-HSCT, an early increase in classical CD14+ monocytes was observed, followed by expansion of NK and T cell populations. At the time of CMV viremia, NK cell and CD8+ T effector memory/terminally differentiated effector memory (CD8+ T EM/EMRA) populations increased regardless of donor CMV serology. CD56 bright, CD56 dim and adaptive NK cells were present in all patients post-transplant, with patient-specific heterogeneity in phenotype and temporal dynamics. The proportion of adaptive NK cells out of CD45+ cells was higher post-viremia in 2 patients with CMV+ donors (18.9% and 7.4%), compared to 2 patients with CMV- donors (3.1% and 1.7%). Surprisingly, the expansion of adaptive NK cells (3.8%) was also noted in a patient with a CMV- donor without CMV reactivation post-allo-HSCT. After viremia, adaptive NK cells upregulated IFNG production, KLRC2/NKG2C and KLRC3/NKG2E and downregulated KLRC1/NKG2A. CD8+ T cell clonality markedly increased after CMV viremia in all patients with CMV reactivation, as measured by proportion of large and hyperexpanded clones in the scTCRαβ-seq data. Hyperexpanded clones consisted mostly of CD8+ T EM/EMRA phenotypes and were present only in samples collected during or after viremia. With scRNA+TCRαβ+pMHC-seq, pp65 NLV was discovered to be the immunodominant CMV epitope. Characterizing pp65 NLV-specific TCRs in the scRNA+TCRαβ data, CMV-specific CD8+ T cells belonged to highly clonal T EM/EMRA clusters. Further, a subset enriched to a T EMRA phenotype expressing NK cell receptors, including KIR3DL2, KIR2DL3, KLRC2/NKG2C KLRC3/NKG2E, and FCGR3A/CD16, and the exhaustion markers TIGIT, TOX, and CD160 at a higher level than other CD8+ T cells. The donor-derived CMV-specific TCR repertoire evolved in recipients after viremia. For example, in patient 2 we noted an 18.5-fold expansion of a donor-derived CMV-specific clone during viremia that was not originally the most dominant anti-CMV T cell clone in the donor. Conclusions We delineated NK cell and antigen-specific T cell dynamics in the unique setting of allo-HSCT and CMV reactivation, highlighting the manyfold expansion of donor-derived T cell clones and the expansion and evolution in the transcriptional profile of adaptive NK cells during CMV viremia. Our study sheds light on the immunobiology of these two cell types with implications in transplant outcome and translational relevance as cancer immunotherapy.

Expansion, Persistence, and Characteristics of Autologous, Bhv-1100 Armored Memory-like NK Cells Infused Prior to Autologous Stem Cell Transplant in MRD+ Multiple Myeloma Patients: A First-in-Human Trial

Introduction/Background Autologous stem cell transplant (ASCT) improves MRD negativity and prolongs progression-free survival in patients with multiple myeloma (MM) in their first or second remission following induction chemotherapy. MM NK cells are dysfunctional, negatively impacting outcomes. BHV-1100, a novel Antibody Recruiting Molecule ( ARM), binds to CD38 and recruits NK cells for antibody-dependent cell cytotoxicity (ADCC) without inducing fratricide. Allogeneic, cytokine induced memory-like (CIML) NK cells effectively treat myeloid disorders, however, it is not known if autologous CIML NK cells, when coated with BHV-1100, would further improve ASCT outcomes in MM. Methods We designed a first-in-human study of autologous CIML NK cells coated ex-vivo with BHV-1100 for MRD+, MM patients undergoing ASCT following first or second remission. NK cells were isolated from non-mobilized leukapheresis on day -1 (prior to melphalan for HCT) using CD3 depletion followed by CD56 positive selection with Miltenyi's CliniMACS. The NK cells were incubated overnight (12-16 hours) with IL-12 (10ng/ml), IL-15 (100ng/ml), and IL-18 (50ng/ml) to induce CIML differentiation, washed and subsequently coated with BHV-1100 for one hour prior to infusion. The product was infused fresh on D0 after standard melphalan 200 mg/m2 myeloablative conditioning and followed by stem cell infusion. Low dose IL2 (1 mIU/m 2) was administered SQ starting on D+1, QOD for a total of 7 doses. Results This is an ongoing trial (NCT04634435) with a median follow-up of 191 days. We are herein reporting data on the in vivo expansion and functional characterization of ARMored CIML NK for the first 5 patients. CIML NK cells were manufactured with a 100% success rate and infused at a target dose of 5-10x10 6 cells/kg-body weight, 24 hours after 200 mg/m 2 melphalan administration. Patients received between 3.9-6.0x10 6/Kg stem cells. Engraftment based on recovery of neutrophil count occurred on D+12-D+14. There was a 3-fold expansion of NK cells in the peripheral blood from D+7 (from 12% to 42%) to D+28 that persisted until D+60 (25% total PBMC, Fig. 1A). Most expanded NK cells were CD56 dim, CD16 high, KIR high and CD57 high. CD57 and KIR expression increased over time from D+7 to D+60, whereas NKG2A expression decreased, indicating the expansion of mature, activated, and cytotoxic NK cells. Regulatory T cells increased by D+7 (3% vs 15% total PBMC) and returned to baseline after D+14 most likely reflecting the effect of IL-2 treatment. The functional capacity of the infused product was tested in vitro against MOLP8 MM cell line. The BHV-1100 ARMored CIML NK cells showed increased IFN (53% vs 48% at 0H and 53% vs 44% at 24H) and CD107a (Fig.1B) (26% vs 13% at 0H and 34% vs 15% at 24H) production compared to untreated CIML NK cells and the product was stable for 24 hours. Conclusion Autologous, BHV-1100 ARMored CIML NK cells have enhanced anti-MM activity as well as expand and persist in vivo peaking at D+28 after infusion. This represents an innovative approach to boost autologous cancer immunosurveillance in the context of ASCT. Aside from anticipated infusion reactions, no severe/unexpected adverse events were noted; longer follow-up is required to assess safety and efficacy.

Impact of Hemodilution on Measurable Residual Disease Measured Via Flow-Cytometry in Acute Myeloid Leukemia Analyzed Using Subsequent Pulls

Background: Measurable residual disease (MRD) detection plays a crucial role in assessing remission status and prognosis for acute myeloid leukemia (AML) patients. Multiparameter flow cytometry (MFC) and NPM1 gene mutations reverse transcriptase polymerase chain reaction (RT-PCR) are validated and recommended used methods for MRD measurement after two cycles of high-dose induction chemotherapy. However, the accuracy of MRD assessment can be compromised by hemodilution, a phenomenon that occurs during bone marrow (BM) aspiration when peripheral blood (PB) mixes with the BM sample. Due to the lower presence of MRD in PB, this may lead to false-negative results. Although formulas to quantify hemodilution exist, their validation and adoption remain limited, and the exact impact of hemodilution remains unclear. Aim: This prospective study aimed to investigate the impact of hemodilution on MFC-MRD assessment by aspirating three sequential pulls of 2 ml each, instead of the standard 6 ml BM sample, and comparing them with a PB sample collected on the same day. Additionally, we evaluated the performance of different hemodilution formulas and analyzed changes in ratios/percentages of cell populations across the BM pulls and PB samples. Methods: We included 30 AML patients in complete remission, resulting in 40 samples measured after one or two cycles of induction chemotherapy with 46 leukemia-associated immunophenotypes (LAIPs). The first 2 ml of BM were collected as the “first pull”, followed by a second aspirate of another 2 ml to form the “second pull”. This process was repeated to obtain the “third pull”. Precautions were taken during BM aspiration to prevent aspirate loss between pulls, and the aspiration needle remained stationary throughout the procedure. All samples from one time point were analyzed on the same machine by the same operator. The formulas proposed for hemodilution assessment were evaluated using a newly developed tube containing additional markers (CD10, CD16, CD38, and CD138) not present in the standard MFC-MRD assay. Differences in outcomes from the formulas and cell populations across the specimens were analyzed using the non-parametric Friedman's ANOVA test. Moreover, the ability of individual populations or formulas to differentiate between BM and PB samples was assessed using the area under the receiver operating characteristic curve (ROC-AUC) in a binary classification task. Results: Significant decreases in blasts and median MRD% were observed between different pulls and PB samples. The median MRD% in pull 1 (0.06%) was significantly higher than in pull 2 (0.05%), pull 3 (0.04%), and PB (0.01%) ( P&amp;lt;0.001). Pairwise comparisons, adjusted for multiple testing, revealed significant differences between pull 1 and pull 2 ( P=0.025), and between pull 2 to pull 3 ( P=0.025), indicating the effect of hemodilution ( Figure 1A). In the first pull 18/46 LAIPs were above the 0.1% cut-off, compared to 14/46 in the third pull. This resulted in 9 patients being MRD-positive in the first/second pull, compared to 8 patients in the third pull. The evaluated formulas demonstrated progressively higher quantities of diluted samples from pull 1 to PB. Five parameters exhibited an AUC above 0.9, indicating their ability to differentiate between BM and PB samples. These parameters are CD10 (AUC: 0.956), plasma cells (AUC: 0.949), CD16 dim cells (AUC: 0.940), mast cells (AUC: 0.924), and the PB contamination index formula (AUC: 0.905) ( Figure 1B). Conclusion: Hemodilution occurs even after aspirating the initial milliliters of BM, emphasizing the need to consider hemodilution when assessing MRD in AML to avoid false-negative results. We recommend incorporating a hemodilution detection formula into routine practice for AML patients to further evaluate the effect on clinical choices. The results of this study support the examination of the CD16 dim population or mast cell population (CD117hi) for this purpose. Furthermore, we suggest implementing a practical approach that involves numbering each pull of BM during aspiration and taking this into account when giving the final MRD result, thus providing clarity regarding the specific pull being measured. In cases of doubt (e.g., MFC-MRD between 0.07% and 0.09% in a later pull), repeating the BM aspiration is recommended to ensure reliability.

TMEM41A overexpression correlates with poor prognosis and immune alterations in patients with endometrial carcinoma.

Expression levels of transmembrane protein 41A (TMEM41A) are related to the progression of malignant tumors. However, the association between TMEM41A expression and endometrial carcinoma (EC) remains unclear. This study aims to identify the roles of TMEM41A expression in the prognosis of patients with EC and its correlation with EC progression. The TMEM41A expression and its correlation with the survival of patients with EC were assessed. Cox regression analysis was used to identify the prognostic factors, while nomograms were used to examine the association between the prognostic factors and the survival of patients with EC. Finally, the link between TMEM41A level and immune microenvironment and RNA modifications was investigated in EC. TMEM41A was overexpressed in EC. TMEM41A overexpression could diagnose the EC and evaluate the poor prognosis of patients. Overexpression of TMEM41A was associated with clinical stage, age, weight, histological subtype, tumor grade, and survival status of patients with EC. Clinical stage, age, tumor grade, radiotherapy, and TMEM41A overexpression were factors of poor prognosis in patients with EC. The nomograms revealed the correlation between the TMEM41A level and survival time of patients with EC at 1, 3, and 5 years. Furthermore, TMEM41A overexpression was significantly correlated with the level of the stromal score, immune score, estimate score, NK CD56 bright cells, iDC, NK cells, eosinophils, pDC, T cells, TReg, cytotoxic cells, mast cells, Th17 cells, neutrophils, aDC, NK CD56 dim cells, TFH, Th2 cells, CD8 T cells, macrophages, immune cell markers, and RNA modifications. TMEM41A is overexpressed in EC tissues and is associated with the prognosis, immune microenvironment, and RNA modification. Our preliminary studies indicate that overexpression of TMEM41A can potentially serve as a biomarker for EC treatment.

Immune Cell Infiltration Analysis Based on Bioinformatics Reveals Novel Biomarkers of Coronary Artery Disease.

Coronary artery disease (CAD) is a multifactorial immune disease, but research into the specific immune mechanism is still needed. The present study aimed to identify novel immune-related markers of CAD. Three CAD-related datasets (GSE12288, GSE98583, GSE113079) were downloaded from the Gene Expression Integrated Database. Gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and weighted gene co-expression network analysis were performed on the common significantly differentially expressed genes (DEGs) of these three data sets, and the most relevant module genes for CAD obtained. The immune cell infiltration of module genes was evaluated with the CIBERSORT algorithm, and characteristic genes accompanied by their diagnostic effectiveness were screened by the machine-learning algorithm least absolute shrinkage and selection operator (LASSO) regression analysis. The expression levels of characteristic genes were evaluated in the peripheral blood mononuclear cells of CAD patients and healthy controls for verification. A total of 204 upregulated and 339 downregulated DEGs were identified, which were mainly enriched in the following pathways: "Apoptosis", "Th17 cell differentiation", "Th1 and Th2 cell differentiation", "Glycerolipid metabolism", and "Fat digestion and absorption". Five characteristic genes, LMAN1L, DOK4, CHFR, CEL and CCDC28A, were identified by LASSO analysis, and the results of the immune cell infiltration analysis indicated that the proportion of immune infiltrating cells (activated CD8 T cells and CD56 DIM natural killer cells) in the CAD group was lower than that in the control group. The expressions of CHFR, CEL and CCDC28A in the peripheral blood of the healthy controls and CAD patients were significantly different. We identified CHFR, CEL and CCDC28A as potential biomarkers related to immune infiltration in CAD based on public data sets and clinical samples. This finding will contribute to providing a potential target for early noninvasive diagnosis and immunotherapy of CAD.

Tumor infiltrating natural killer cells in soft tissue sarcoma are predominantly CD56 dim: Implications for outcomes

Abstract Natural killer (NK) cells have been shown to be important mediators of anti-tumor responses, including in soft tissue sarcomas (STS). NK cells show significant heterogeneity, depending on maturation, tissue residency, and inflammatory environment. As previous studies have suggested that tumor-infiltrating NK cells (TiNKs) acquire a less cytotoxic CD56 brighttissue resident phenotype, we sought to compare blood versus tumor NK cell phenotype in STS and evaluate any association with survival. Blood and tumor from 27 STS patients undergoing surgery from 2018–2022 were collected prospectively for flow cytometry and retrospectively analyzed. 52% were female, the mean age was 59, and 74% were AJCC stage 3. Increasing absolute number of NK cells in the blood correlated with longer survival (P&amp;lt;0.005, r=0.6). As expected, CD56 dimNK cells predominated in the blood (91.5±5.8% of CD3-CD56+ lymphocytes compared to 80.1±13% in tumor, P&amp;lt;0.005). In contrast, CD56 brightcells were enriched in tumor representing 18.5±13% compared to 8.4±5.8% in blood (P&amp;lt;0.005). Although CD56 brightNK cells were approximately 2.4±3-fold higher in tumor compared to blood, CD56 dimNK cells still represented the majority of TiNKs. Higher proportions of both overall NK cells and CD56 dimNK cells in STS tumors were associated with better metastasis-free survival on Kaplan-Meier analysis (P&amp;lt;0.05). CD56 dimTiNKs had higher NKp46 expression than CD56 brights. In conclusion, both blood and intra-tumoral NK cells appear prognostic in STS. Although the proportion of CD56 brightTiNKs in STS is higher than blood the majority of TiNKs are CD56 dimconsistent with a cytotoxic phenotype. Better characterization of NK subsets in STS is likely to have translational significance.

Machine learning-based prediction of diagnostic markers for Graves’ orbitopathy

PurposeThe pathogenesis of Graves’ orbitopathy/thyroid-associated orbitopathy (TAO) is still unclear, and abnormal DNA methylation in TAO has been reported. Thus, selecting and exploring TAO biomarkers associated with DNA methylation may provide a reference for new therapeutic targets.MethodsThe TAO-associated expression data and methylation data were downloaded from The Gene Expression Omnibus database. Firstly, weighted gene co-expression network analysis was used to obtain the TAO-related genes, which were intersected with differentially methylated genes (DMGs), and differentially expressed genes between TAO samples and normal samples to obtain TAO-associated DMGs (TA-DMGs). Thereafter, the functions of the TA-DMGs were analyzed, and diagnostic markers were screened by least absolute shrinkage and selection operator (Lasso) regression analysis and support vector machine (SVM) analysis. The expression levels and diagnostic values of the diagnostic markers were also analyzed. Furthermore, single gene pathway enrichment analysis was performed for each diagnostic marker separately using gene set enrichment analysis (GSEA) software. Next, we also performed immune infiltration analysis for each sample in the GSE58331 dataset using the single-sample GSEA algorithm, and the correlation between diagnostic markers and differential immune cells was explored. Lastly, the expressions of diagnostic markers were explored by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsA total of 125 TA-DMGs were obtained. The enrichment analysis results indicated that these TA-DMGs were mainly involved in immune-related pathways, such as Th1 and Th2 cell differentiation and the regulation of innate immune response. Moreover, two diagnostic markers, including S100A11 and NKD2, were obtained by Lasso regression analysis and SVM analysis. Single gene pathway enrichment analysis showed that S100A11 was involved in protein polyufmylation, pancreatic-mediated proteolysis, and NKD2 was involved in innate immune response in mucosa, Wnt signaling pathway, etc. Meanwhile, immune cell infiltration analysis screened 12 immune cells, including CD56 dim natural killer cells and Neutrophil cells that significantly differed between TAO and normal samples, with the strongest positive correlation between NKD2 and CD56 dim natural killer cells. Finally, the qRT-PCR illustrated the expressions of NKD2 and S100A11 between normal and TAO.ConclusionNKD2 and S100A11 were screened as biomarkers of TAO and might be regulated by DNA methylation in TAO, providing a new reference for the diagnosis and treatment of TAO patients.

Immature and activated phenotype of blood NK is associated with acute rejection in adult liver transplant.

Natural killer (NK) cells contribute to liver transplant (LTx) rejection. However, the blood-circulating NK cell dynamics of patients who experience acute rejection (AR) are unclear. Herein, we longitudinally profiled the total NK cells and their subsets, along with the expression of activating and inhibitory receptors in sequential peripheral blood mononuclear cells samples, spanning from pre-LTx to the first year post-LTx of 32 patients with AR and 30 patients under a steady immune status. Before transplantation, patients with AR (rejectors) contained a significantly higher proportion of the immature CD56brightCD16- subset and a lower cytolytic CD56dimCD16+ in the total blood-circulating NK cells than patients with steady immunity. Both subsets contained a high NKp30 positive population, and CD56dimCD16+ additionally exhibited a high NKp46 positive ratio. The NKp30 positive ratio in CD56dimCD16+ subset showed the most prominent AR predictive ability pre-LTx, and was an independent risk factor of LTx AR. After transplantation, the blood-circulating NK cells in rejectors maintained a higher CD56brightCD16- and lower CD56dimCD16+ composition than the controls throughout the first year post-LTx. Moreover, both subsets maintained a high NKp30 positive ratio, and CD56dimCD16+ retained a high NKp46 positive ratio. The blood-circulating NK-cell subset composition was consistent during AR, while the expressions of NKp30 and NKp46 were augmented. Collectively, a more immature CD56brightCD16- subset composition and an activated phenotype of high NKp30 expression were the general properties of blood-circulating NK cells in rejected LTx recipients, and the NKp30 positive ratio in CD56dimCD16+ NK subset pre-LTx possessed AR predictive potential.