STAT3 Mutations Research Articles

High prevalence of low-allele-fraction somatic mutations in STAT3 in peripheral blood CD8+ cells in multiple sclerosis patients and controls.

Somatic mutations have a central role in cancer, but there are also a few rare autoimmune diseases in which somatic mutations play a major role. We have recently shown that nonsynonymous somatic mutations with low allele fractions are preferentially detectable in CD8+ cells and that the STAT3 gene is a promising target for screening. Here, we analyzed somatic mutations in the STAT3 SH2 domain in peripheral blood CD8+ cells in a set of 94 multiple sclerosis (MS) patients and 99 matched controls. PCR amplicons targeting the exons 20 and 21 of STAT3 were prepared and sequenced using the Illumina MiSeq instrument with 2x300bp reads. We designed a novel variant calling method, optimized for large number of samples, high sequencing depth (>25,000x) and small target genomic area. Overall, we discovered 64 STAT3 somatic mutations in the 193 donors, of which 63 were non-synonymous and 77% have been previously reported in cancer or lymphoproliferative disease. The overall median variant allele fraction was 0.065% (range 0.007-1.2%), without significant difference between MS and controls (p = 0.82). There were 26 (28%) MS patients vs. 24 (24%) controls with mutations (p = 0.62). Two or more mutations were found in 9 MS patients vs. 2 controls (p = 0.03, pcorr = 0.12). Carriership of mutations associated with older age and lower neutrophil counts. These results demonstrate that STAT3 SH2 domain is a hotspot for somatic mutations in CD8+ cells with a prevalence of 26% among the participants. There were no significant differences in the mutation prevalences between MS patients and controls. Further research is needed to elucidate the role of antigenic stimuli in the expansion of the mutant clones. Furthermore, the high discovered prevalence of STAT3 somatic mutations makes it feasible to analyze these mutations directly in tissue-infiltrating CD8+ cells in autoimmune diseases.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation

The association between cancer and autoimmune disease is unexplained, exemplified by Tcell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ Tcell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ Tcell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subsetalso expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ Tcells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ Tcell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Immunogenetics associated with severe coccidioidomycosis.

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, less than 1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled patients with DCM and performed whole-exome sequencing. In an exploratory set of 67 patients with DCM, 2 had haploinsufficient STAT3 mutations, and defects in β-glucan sensing and response were seen in 34 of 67 cases. Damaging CLEC7A and PLCG2 variants were associated with impaired production of β-glucan-stimulated TNF-α from PBMCs compared with healthy controls. Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were overrepresented in DCM, including CLEC7A Y238* and PLCG2 R268W. A validation cohort of 111 patients with DCM confirmed the PLCG2 R268W, CLEC7A I223S, and CLEC7A Y238* variants. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived hydrogen peroxide [H2O2] in transfected cells. Heterozygous DUOX1 or DUOXA1 variants that impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired β-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.

Identification of Genetic Subtypes in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma subtype and is characterized by heterogeneous clinical trajectories. While certain genetic abnormalities are highly prevalent, heterogeneity exists also at the molecular level. Yet, unlike diffuse large B-cell lymphoma, where genetic subtypes have been described in multiple studies, the taxonomy of FL has not been clearly established to date. While clusters defined by STAT6 mutations or aberrant somatic hypermutation have been described in FL by Crouch et al. (Blood Advances, 2022), its landscape of molecular alterations has been insufficiently dissected into biologically distinct subgroups. The recognition of such subtypes may pave the way for treatment individualization. Here, we sought to define novel genetic subtypes in FL by means of targeted DNA sequencing and clustering. Methods: Samples of FL grade 1 to 3A (i.e. classic FL) were accrued from biorepositories of participating centres in Canada, Northern Europe and Australia, as well as from two prospective clinical trials (E4402 and E2408). We performed hybridization-based targeted DNA-sequencing of 57 genes that are recurrently mutated in FL. Moreover, we included targeted DNA sequencing data from a previously published dataset (PMID 27959929). All cases were analyzed using a uniform computational pipeline. Samples were removed from the analysis if the mean on-target coverage was <50X. Single nucleotide variants were identified using Mutect2 and we applied a variant allele fraction cut-off of 0.1. Bernoulli mixture-model clustering was applied using variants as features for the identification of genetic subtypes. Cluster stability was assessed by bootstrap consensus. Clusters were tested for their association with outcome measures. As a further means to annotate genetic clusters, we generated genome-wide methylation data using the Illumina EPIC platform. Results: Following quality control, the total number of pre-treatment samples available for clustering was 715 (543 from formalin-fixed and paraffin-embedded tissue samples and 172 from fresh-frozen). The average number of mutations and mutated genes per sample were 5.9 (range 1-20) and 4.8 (range 0-14), respectively. An analysis of interactions between somatic mutations revealed both expected (e.g. mutual exclusivity of CREBBP and EP300 mutations) and unexpected patterns (e.g. co-occurring mutations of GNA13 and MEF2B), overall justifying the pursuit of unsupervised clustering. After consensus clustering, the optimal clustering solution resolved 5 genetically distinct groups, henceforth referred to as C1 to C5 (Figure). The C1 cluster was characterized by variants in genes involved in the mTOR signaling pathway (ATP6AP1, RRAGC, ATP6V1B2); the C2 cluster by low mutation burden; the C3 cluster by mutations involving TNFAIP3, TP53 and MYD88; the C4 cluster by GNA13 and MEF2B mutations; and the C5 cluster by STAT6, CREBBP, EZH2 and TNFRSF14 mutations. The number of mutations located in RGYW or WRCY motifs, suggestive of somatic hypermutation, was highest in the C4 and the C3 clusters (P < 0.001). Applying a previously reported methylation-based clock (epiCMIT) to the subset of samples with methylation data (n=345), we found that samples in the C2 cluster had the lowest proliferative history. Concordantly, the C2 cluster was characterized by a lower degree of global hypomethylation and lower average methylation of CpG islands, whereas samples in clusters C1, C3, C4 and C5 were characterized by not just longer proliferative histories, but also by higher mean methylation in CpG islands. In terms of clinical associations, the C2 cluster was significantly associated with limited-stage disease presentation. In an exploratory outcome analysis of 165 patients treated with immunochemotherapy, the C3 and C5 clusters were associated with inferior time to progression. Conclusion: Here, we identified novel genetic subtypes that are distinguishable not just by unique sets of mutations, but also by their clinical associations and methylation profiles. The novel genetic profiles shed light on core pathways distinctly tied to each subtype. Targeted DNA sequencing of a limited gene panel represents a potentially accessible method of subtyping FL in the clinical setting, with implications for the understanding of responses to targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PRDM1 Deletion and STAT3 Mutations Cooperatively Promote CD8+ T-Cell and NK-Cell Growth in Vitro

PRDM1 loss and STAT3 activating mutations commonly co-occur in several non-Hodgkin lymphomas, including ALK negative Anaplastic Large Cell Lymphoma (ALCL) and NK-cell lymphoma (NKCL). PRDM1 is a tumor suppressor gene in NKCL, and also plays important roles in the homeostasis and differentiation of CD8+ T-cells. STAT3 encodes a critical transcription factor involved in specific cytokine-mediated JAK/STAT pathway activation in lymphocytes, controlling proliferation, apoptosis, and other cellular functions. However, it remains unclear whether these two abnormalities cooperate during lymphomagenesis. Here, we introduced PRDM1 deletion and STAT3 mutants into primary human T- or NK-cells isolated from PBMCs of healthy donors and performed functional studies on the genetically modified cells. When either of the two common STAT3 mutants, STAT3Y640F or STAT3D661Y, was transduced into T- or NK-cells, the transduced cells showed short term growth advantage but could not persist, indicating that STAT3 activating mutations may provide stimulatory signals to T- and NK-cells initially, but inhibitory pathways could be activated that prevent long-term sustainable growth. When we knocked out PRDM1 first and then transduced the cells with the STAT3 mutants, the double modified T- or NK-cells showed sustained competitive growth advantage during in vitro culture. The effects were specific to STAT3 mutants as transduction of wild-type (WT) STAT3 or the STAT5BN642H mutant did not improve cell growth. These findings suggest that PRDM1 loss and STAT3 mutations synergistically promote growth in primary T- and NK-cells. We further showed that PRDM1-null, STAT3Y640F-transduced NK-cells displayed better survival and growth compared to PRDM1-deleted or WT cells with feeder cell stimulation. More importantly, the double mutant NK-cells were more independent of feeder cell stimulation, and they were able to expand when supplemented with a single cytokine (IL-15 or IL-2). This feature may be relevant to lymphoma development in vivo where stimulatory cytokines may be limiting. In addition, these double mutant NK-cells had reduced expression of exhaustion markers Tim-3 and Tigit. Similarly, genetically modified CD8+ T cells with PRDM1 knockout and STAT3-mutant were found to express much lower levels of PD-1, Tim-3, and Tigit, an important feature that may promote their growth in vivo where ligands of these co-inhibitory molecules may be expressed in the microenvironment. These double mutant CD8+ T cells exhibited greater stem memory (Tscm) phenotype, marked by CD45RA+, CD62L+, and CCR7+, suggesting the switch to a memory rather than an effector program. Taken together, our in vitro experiments using primary human cells have provided evidences for the functional cooperation between PRDM1 loss and STAT3 activating mutation in promoting CD8+ T- and NK-cell growth. Some features shown by the double mutant cells may facilitate their survival and growth more significantly in vivo. We are currently investigating the adoptive transfer of modified mouse or human cells into littermates or NSG-Tg(huIL15) mice respectively to observe the in vivo development and evolution of the transferred cells. The detailed mechanistic interactions between these two genetic abnormalities in lymphoma development will be further investigated.

Towards a Unified Genetic Classification System for Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell neoplasm and the most common lymphoid malignancy worldwide. DLBCL, NOS is a genetically and biologically complex entity that remains uniformly treated at diagnosis despite the growing appreciation for subgroups with molecular distinctions. There are various approaches for assigning DLBCL into one or more molecular subgroups using either gene expression and/or mutation patterns. Several genetic classification systems have been proposed, each sharing commonalities. Distinctions between the definitions of individual classes and the ability to reproducibly assign individual patient samples to subgroups using each system remains a significant barrier to their implementation, further emphasized by up to 50% of patients that remain unclassified. The disparities among the genetic features considered by each classification scheme further limits the ability to consolidate, let alone adopt, these for research and clinical studies. We aimed to develop a classification system that unifies and extends the robustness of existing classification algorithms. Methods: We assembled whole genome sequencing (WGS) data from 900 tumors from DLBCL, Burkitt (BL), follicular (FL) and other mature B-cell neoplasms to comprehensively determine the set of significantly mutated genes (SMG) and common targets of aberrant somatic hypermutation (aSHM). These were intersected with the features used by the current DLBCL classification systems and a curated set of lymphoma genes to tabulate the mutation matrix. Common structural rearrangements involving oncogenes were determined from WGS data and the copy number state of focal high-level recurrent alterations was united with simple somatic mutations. For genes containing mutation hot-spots each was considered separately. Results: Clustering all cases using non-negative matrix factorization (NMF) analysis resulted in a solution with 7 distinct genetic subgroups (Figure 1). Many of these directly resemble clusters in the existing systems with either expanded coverage or refinement of a subgroup into two. First, the BNZ cluster was highly enriched for any aberration in BCL6, 3'UTR mutations in NFKBIZ, and truncating mutations in NOTCH2. Unlike the similar BN2/C1/NOTCH2 groups defined previously, the NFKBIZ mutations were weighted more highly than those of NOTCH2 in defining the BNZ subgroup. The cluster we named MCaP resembled the MCD/C5/MYD88 group. Tumors classified in this subgroup had evidence of aSHM with frequent non-coding mutations in PIM1,CD44, GRHPR, PIM2, and BCL11A among others. Another cluster, MP3, was enriched for coding mutations or aSHM in the MYC TSS (MYC_aSHM), which were generally correspondent with IG::MYC rearrangements. MP3 tumors were characterized by TP53 mutations, including hotspots, and significantly depleted for aberrations in BCL2, BCL6 and lower mutation rates at aSHM targets SGK1, PAX5, CIITA, RHOH and others. Notably, tumors in this subgroup were enriched for IRF4 translocations, suggesting potential overlap with large B-cell lymphoma with IRF4 rearrangement. Our approach further refined both the the ST2/C4 and EZB/C3/BCL2 classes into two smaller subgroups, aSEL/aSCI and EGB/ETB, respectively. The EGB tumors were enriched for GNAI2 mutations, and MYC translocations involving the BCL6 enhancer, and were more often associated with MEF2B hotspot mutations. Notably, ETB was significantly enriched for mutations in the lysine acetyltransferase (KAT) domain in CREBBP, while EGB was significantly enriched for non-KAT domain mutations. ETB tumors were further characterized by mutations in STAT6 and FOXO1 hotspot regions, MYC rearrangements with other non-IG partners, and TNFRSF14 mutations, largely resembling the mutational profile of follicular lymphomas. Conclusions: We present here a refined classification of DLBCL genetic subgroups that combines previously described classification algorithms and extends it to allow classification of more tumors into additional subgroups that preserve the major divisions of existing systems. This represents an important advancement that will facilitate further understanding of genomic complexity in this disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Evolution of Cutaneous T Cell Lymphoma Revealed at Single Cell Resolution

Background: Patients with Cutaneous T Cell Lymphoma (CTCL) subtypes Mycosis Fungoides and Sézary Syndrome (MF/SS) who progress to advanced stage disease have a 20% 5-year overall survival rate. Current treatments for CTCL range from localized therapies for skin lesions to systemic treatments, including histone deacetylase inhibitors, chemotherapy, and immunotherapy. While many treatments are initially effective, patients often develop resistance to ongoing treatment. Though recent studies have comprehensively profiled the genomic landscape of CTCL, no clear driver mutation has been identified and the molecular factors that drive MF/SS progression remain undefined. To address these outstanding questions, we collected longitudinal tumor and normal samples from 10 MF/SS patients and subjected them to high resolution genome and transcriptome analyses (exome, whole genome and single-cell RNA sequencing, eWGS and scRNAseq) to define the clonal evolution and temporal progression of CTCL. Methods: We collected 52 serial tumor samples (peripheral blood, skin and lymph node biopsies, 49-1050 days apart) and matched, purified non-malignant cells from 10 MF/SS patients (3 - 8 specimens per patient) representative of patients seen at the Washington University School of Medicine Cutaneous Lymphoma clinic (mean age 56.1 years, 50% female, 60% Caucasian and 40% African American). All were diagnosed with advanced MF and/or SS and half of first specimens were collected within 60 days of diagnosis. Subsequent specimens were collected at progression or lack of response to therapy, on average 20 months between specimens (1.4 - 60, SD = 11.8); patients had received 1-11 different CTCL therapies. For eWGS, specimens were purified by flow cytometry as follows, tumor: CD3+CD4+CD7- and normal: CD4+CD7+ (non-malignant T cells) or CD3-CD14+ (monocytes) or CD3-CD56+ (NK cells). Specimens were subjected to scRNAseq, exome sequencing plus low coverage whole genome sequencing (eWGS), and patient specific TCR clonotype assignment (scRepertoire). Results: Clustering and cell type inference of scRNAseq (UMAP/Seurat/singleR) from 92,496 total cells showed canonical CTCL marker genes and high clonal proportions (1-100%) of patient specific TCR clonotypes. Somatic genome copy number variants (CNV) from eWGS (cnvkit) and scRNAseq (copykat) and deleterious/damaging single nucleotide variants (SNV) (eWGS, GATK/Mutect2; scRNAseq, cb_sniffer) were identified in CTCL versus matched, non-malignant cells. To segregate early genomic events from those that were progression-associated, we developed an integrative analysis pipeline to prioritize somatic CNV and SNV that were detected in samples collected at timepoints with lack of response to therapy and/or progression in ≥30% of patients. This novel approach revealed alterations in T-cell activation pathways as early events that were maintained in subsequent CTCL samples. Loss of some chromatin-modifying enzymes were early events, while gains of others (DNMT3A, EZH2, KMT2C) were acquired with therapy resistance/progression (Fig. 1A). Copy number amplifications of STAT3/STAT5 are frequent and we demonstrate a novel-to-CTCL single nucleotide mutation in STAT3 that has gain-of-function activity. Progression-associated genomic changes were enriched in cell cycle (CDK genes), DNA damage/repair (TP53BP1, RAD51C), RAS and Rho signaling pathways, and transcriptional regulators (FOXO genes). Expression of mutant alleles was detectable in scRNAseq and variant levels fluctuated with progression or lack of therapy response (Fig. 1B). Conclusion: To date, there have been limited studies defining temporal changes in the genomes and transcriptomes of CTCL due in part to low tumor burden in the skin and blood. Using innovative sequencing and informatic approaches, we identified recurrent alterations in CDK11A, DNMT3A, EZH2, FOXO1, RAD51C, STAT3/STAT5, and TP53BP1, among others, with clonal evolution in CTCL. Many of these have therapeutic implications. Taken together, these findings represent new insights into MF/SS progression, reveal new markers for response assessment, and identify new targets for therapeutic development. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Comprehensive, Multiomics Analysis of Natural Killer-Cell Malignancies

Natural killer/T cell lymphoma (NKTCL) and aggressive NK cell leukemia (ANKL) are rare mature NK cell malignancies which show a geographic predilection for Asia and Central/South America and are strongly associated with EBV infection. Despite recent advances in chemotherapeutical agents, the prognosis (especially for ANKL) remain poor. On the contrary, NK cell proliferations of a more indolent nature also exist. NK-large granular lymphocytic leukemia (NK-LGLL) is a disorder of NK cells with indolent clinical manifestation. Additionally, chronic active Epstein-Barr virus infection (CAEBV) affecting NK cells can have variable presentation, between indolent and aggressive. Due to their rarity, a comprehensive understanding of the events differentiating the wide spectrum of NK malignancies remains elusive. Here, we use a multi-omics approach to investigate the genetic differences underlying the different clinical manifestations of NK malignancies. To discover the pathogenesis behind indolent and aggressive NK malignancies, we studied whole-genome/exome sequencing and whole transcriptome sequencing (RNA-seq) data from over 200 NK-cases collected from 11 previous publications. We investigated driver genes from 145 WES and WGS samples, and transcriptomes from 91 samples. Single-cell RNA-seq analysis was performed to identify and characterize distinct NK-cell subsets. We used pooled CRISPR screens with a single-cell RNA sequencing readout (CROP-seq) to investigate the effect of knocking out the most frequently mutated genes on the transcriptome of NKTCL cells under the effect of commonly used chemotherapeutic agents. We also aim to construct a comprehensive host-EBV interaction map to elucidate the role of EBV in the pathogenesis of NK malignancies We validated previously found driver genes STAT3 and DDX3X as the most frequently mutated genes in NK malignancies, being mutated in 15% and 14% of samples, respectively. STAT3 mutations were found only in NKTCL and ANKL samples. Additionally, our combinatorial analysis allowed for identification of previously unappreciated putative driver genes, MSN and HLA-A, which were mutated in 8% and 4% of samples, respectively. We also discovered associations between important genes, such as significant co-occurrence of mutations in STAT3 and novel driver gene MSN (p <0.05). Samples originating from different diseases were intermingled in non-negative matrix factorization (NMF) analyses, highlighting the effect of mutational profile, rather than disease type, on sample clustering. Using RNA-seq, differentially expressed genes (Q-value < 0.01) were identified between ANKL, NKTCL and healthy samples. Gene set enrichment analysis revealed differential enrichment and depletion of biological processes in ANKL and NKTCL. Upregulation of chemokine signalling and macrophage-associated processes in NKTCL reflected the inflammatory microenvironment of the disease. Of 17 putative driver genes, CRISPR knockout of STAT3 and TP53 led to most transcriptional changes. Knockout of STAT3 caused upregulation of HLA I and II molecules and downregulation of IL10 signaling, thus promoting tumor cell immune evasion. Our preliminary results indicate that NK malignancies are, contrary to previous knowledge, a spectrum of manifestations rather than distinct diseases. Our results concerning the heterogeneous genetic landscape of NK malignancies can lead to better and faster diagnosis as well as improved treatment stratification in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Somatic Mutations in T Cells in Patients with Hematological Diseases

Somatic mutations accumulate in cells during aging, and the mutation rate and spectrum vary between tissues. Mutation accumulation can be associated with inflammation: for example, myeloid driver gene mutations in hematopoietic stem cells (clonal hematopoiesis, CH), has been linked with atherosclerosis. Somatic mutations can also accumulate in T cells, where they are connected with immune dysregulation in large granular lymphocytic leukemia, characterized by autoimmunity and STAT3 mutations in T cells. However, the mutation spectrum in T cells in other hematological disorders is not yet fully discovered. We hypothesized that T cell lineage specific somatic mutations are associated with T cell clonality in various diseases involving the hematopoietic system. We systematically characterized the spectrum of somatic mutations in CD4+ and CD8+ T cells in altogether 207 samples from 95 patients with chronic graft-versus-host disease (cGVHD, number of patients: 40), myelodysplastic syndrome (MDS, n = 15), immune thrombocytopenia (n = 2), aplastic anemia (AA, n = 21, previously published), and immunodeficiency (n = 17, previously published). Age at sampling was similar between disease cohorts. DNA was extracted from bead separated CD4+ and CD8+ cells from peripheral blood or bone marrow samples. We applied a custom-made targeted NGS panel, consisting of 2533 immune-related genes. Variant calling was performed using Mutect2. In variant filtering, we used 42 healthy T cell and skin samples as panel of normals. To explore the association between somatic mutations and clonal T cell expansions, we performed TCRb deep sequencing from 148 matched patient samples. With mean sequencing depth of 382x, we identified 888 non-synonymous somatic variants in total. 784 (86%) of the variants were only found in either CD4+ or CD8+ fraction. 52 variants were shared between individual patient's CD4+ and CD8+ T cells, suggesting that they were acquired prior to T cell lineage differentiation. On the average, we observed 3.8 fraction-specific variants per sample. Mean variant allele frequency (VAF) was 4.5% and 12.7% for fraction-specific and shared variants, respectively. Variant types included missense (91%), nonsense (5.1%), splice-site (1.7%), frameshift (1.3%), and non-stop (0.2%) mutations. The most common mutational signatures were age-associated Signature 1 and defective DNA mismatch repair associated Signature 15. Immunodeficiency patients had lower number of fraction-specific variants compared to AA, MDS and cGVHD patients (p = 0.027). In all cohorts, CD8+ T cells had acquired more variants than CD4+ T cells (p = 0.00012; mean [median] for CD8: 4.4 [4] and CD4: 3.2 [2]). The highest fraction-specific VAF in each sample was associated with the largest T cell clone size in CD8+ but not in CD4+ samples (R = 0.59, p = 3.1e-09 and p = ns, respectively). In CD8+ T cells, age was associated with number of fraction-specific variants (R = 0.38, p = 1.1e-4) but not with the highest CD8-specific VAF (p = ns). Among the variants shared between individual patients' CD4+ and CD8+ T cells, we found mutations in DNMT3A (n = 3) and TET2 (n = 2) and ASXL1 (n = 1), the most recurrently mutated CH genes. In addition to shared mutations, in 12% of patients had fraction-specific myeloid driver gene mutations (e.g. DNMT3A, TET2, KRAS, NRAS) in CD4+ or CD8+ T cells. To identify pathways enriched for putative driver mutations, we performed analyses of fraction-specific T cell variants with OncodriveFM. The most significantly mutated gene ontology pathways included T cell co-stimulation, leukocyte migration, Ras protein signal transduction, and positive regulation of MAP kinase activity. In conclusion, T cell fraction-specific somatic mutations are common in patients with various hematological diseases, and they are enriched in pathways important for T cell function. CD8+ T cells accumulate more mutations than CD4+ T cells. In CD8+ T cells, the number of somatic mutations was associated with age, reflecting stochastic accumulation of mutations. Based on mutational signatures, somatic mutations in T cells are mainly result of endogenous mutational processes. In addition, mutational clone size CD8+ T cells was significantly associated with the largest TCR clone, suggesting that mutations have accumulated in expanded CD8+ T cell clones. Further studies are warranted to determine the functional effects of somatic mutations in T cells.

Oncogenic JAK2/STAT Signaling Molecules Activation Is Associated with PD-L1 Upregulation in Acute Myeloid Leukemia

Background: Increased evidence suggests the PD-1/PD-L1 axis is involved in the oncogenesis of myeloid neoplasms through promoting cancer immune escape, which suggests potential benefits by blockade of this axis in these neoplasms by immune checkpoint inhibitors. However, the ongoing clinical trials on PD-1/PD-L1 blocker in MDS/AML have reported controversial results, unlike the promising therapeutic efficacy shown in solid tumors. Identifying reliable biomarkers and scoring of PD-L1 expression and targeting the subgroup of patients who might benefit the most from PD-1/PD-L1 blockade are promising directions. Janus kinase 2 (JAK2) pathway activation has been shown to enhance PD-L1 expression in malignancies by in vitro studies. Here, we investigated PD-L1 expression associated with JAK2/STAT oncogenic pathway in AML patients using the combined positive score (CPS) algorithm. Methods: Immunohistochemical staining (IHC) for PD-L1 and pSTAT3 was performed on the marrow biopsies of 31 AML patients (39 cases), including 11 patients (17 cases) with JAK2/STAT mutation and 20 patients (22 cases) with wild-type JAK2/STAT. For PD-L1 IHC, the cases were evaluated for variable staining intensity (weak, moderate, strong) and membrane staining pattern (partial, complete) in tumor and mononuclear immune cells. The PD-L1 CPS was calculated by summing the number of all PD-L1-stained cells and dividing by the total number of tumor cells, multiplied by 100. The pSTAT3 was evaluated by percentage of positive tumor cells among the total tumor cells. The status of JAK2 / STAT mutations and other molecular abnormalities was evaluated by next generation sequencing (NGS) study. Results: Among patients of JAK2/STAT mutations, nine with JAK2 V617F exon 14 mutations, one with JAK2 I540-E543delinsRG exon 12 mutation and one with STAT5B N642H mutation. Evaluation of PD-L1 expression by IHC revealed four major cell staining patterns in our study cohort: 1. Negative (Figure 1A); 2. Partial weak membrane staining (Figure 1B, CPS<10); 3. Partial-complete, weak-moderate membrane staining (Figure 1C, CPS≥10); 4. Complete, moderate-strong membrane staining (Figure 1D, CPS≥10). Patterns 3-4 were classified as positive PD-L1 staining with a CPS cut-off of 10 (CPS≥10). Comparison of the PD-L1 CPS score in the JAK2/STAT wild-type versus the JAK2/STAT mutant groups revealed significantly higher PD-L1 CPS score in the JAK2/STAT mutant group (Figure 1K, P<0.0001, CPS≥10). One patient showed wild-type JAK2 at initial AML diagnosis. At two subsequent relapses, gain of JAK2 exon 12 (I540-E543delinsRG) mutation with increased VAF from 17% to 41% was detected. Interestingly, PD-L1 expression was correspondingly increased with CPS from 25 to 75 in these subsequent biopsies (Figure 1E, F). In another case with wild-type JAK2, STAT5B N642H mutation was detected and strong PD-L1 expression with CPS of 80 was obtained (Figure 1G). To address if JAK2/STAT3 axis is involved in PD-L1 upregulation, we assessed STAT3 phosphorylation by IHC in JAK2 wild-type and mutated groups. The results revealed significantly increased p-STAT3 expression in the JAK2 mutant compared with the wild-type cases, supporting the idea that oncogenic JAK2 mutation leads to STAT3 phosphorylation (Figure 1H-J). Furthermore, p-STAT3 expression showed a significant positive correlation with the PD-L1 CPS score (Figure 1H, r=0.8663, P<0.0001). Conclusion: In the AML patients with oncogenic JAK2 or its downstream signaling molecule STAT5B mutations, there is significantly enhanced PD-L1 expression. In addition, we proposed that the PD-L1 CPS positivity cut-off score of 10 being used as a quantitative measure of PD-L1 expression in this heterogeneous myeloid neoplasm. In the patient with relapsing AML, PD-L1 expression showed a positive correlation with increased JAK2 mutant VAF. Independent of the oncogenic JAK2 mutation, PD-L1 expression upregulation was also noted with STAT5B N642H mutation. Lastly, we demonstrated enhanced p-STAT3 expression in JAK2 mutant cases and a positive correlation between p-STAT3 and PD-L1 expression. Taken together, these data support the model that JAK2/STAT pathway activation is associated with PD-L1 upregulation in AML patients (Figure 1I), together with the proposed PD-L1 CPS cut-off of 10, can serve as scanning biomarkers to select relevant subgroups of AML patients for PD-1/PD-L1 immunotherapeutic study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic Profiling and Clinicopathological Features in Peruvian Patients with EBV-Positive Diffuse Large B-Cell Lymphomas, NOS

Introduction: Epstein Barr virus-positive (EBV+) diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS) is a newly recognized entity by the World Health Organization. EBV+ DLBCL, NOS is commonly encountered in Latin American countries and carries a dismal prognosis. It has been published studies that have evaluated the genetic profiling in EBV +DLBCL from Asian and Western countries. However, there are lacking of studies from Latin America. This study aimed to characterize and compare the genetic profiling and clinicopathological features in Peruvian EBV+ with EBV- DLBCL patients. Methods: This retrospective cohort study included patients ≥18 years from one institution in Peru from 2010 to 2020. Hematopathologists reviewed pathological samples to confirm the diagnosis of EBV+ DLBCL cases. An EBV+ case was confirmed when the EBER test had a positivity more or equal to 40%. We collected clinicopathological data by reviewing the medical records of the patients. Twenty paraffin-embedded biopsy samples were analyzed and tumor DNA was purified using silica columns (QIAGEN, Valencia, CA, USA). The quantity and quality of the purified DNA were evaluated by spectrophotometry. We compared the genetic profiling with ten EBV+ versus ten EBV- DLBCL samples. Results: Twenty-two candidate genes were selected from previous reports of exome analysis with the diagnosis of DLBC. The median age at diagnosis was similar in both groups (58 years old, range 19-89). There was a male predominance in EBV+ (57%) compared to EBV- DLBCL cases (43%). Sixty-eight percent of patients with EBV+ had advanced disease (Ann arbor stage III-IV), compared to 48% of EBV- DLBCL patients. The non-germinal center was more common in EBV+ (60%) than EBV- cases (40%). All patients received either R-CHOP-like regimens as a first-line treatment. The overall response rate was 91% for EBV- compared to 74% for EBV+ DLBCL. We found mutations in MYC (10%), NOTCH2 (5%), STAT6 (5%) , ANKRD11(5%), TP53 (5%), KMT2A (5%), YY1 (5%), CARD11 (5%), ARID1A (5%) and RHOA (5%) in EBV+ cases, meanwhile, ARID1A (10%) , CCR6 (10%), KMT2A (5%), CARD11(5%), NOTCH2 (5%), TP53 (5%), and ANKRD11(5%) in EBV - DLBCL cases. Conclusions: This is the first Latin American study that have performed the genomic pattern in Peruvian EBV+ in comparison to EBV- DLBCL patients. Overall, our data suggest that Peruvian EBV+ DLBCL have different genetic profiling compared to East and Western countries. Also, we found that EBV+ and EBV- patients share similar drive mutations. Our findings should be validated in a larger prospective cohort of Latin American patients.

Detection of High-Frequency Mutation Genes in HIV-Positive and HIV-Negative Lymphomas

Objective Compare HIV-positive lymphoma with HIV-negative lymphoma mutation genes differences, screen specific mutations genes in HIV-positive lymphoma, analyze high frequency mutation genes in HIV-positive lymphoma patients, and explore the effect of HIV infection on lymphoma gene mutation and disease development. It can provide a theoretical basis for the clincal diagnosis and targeted therapy HIV-positive lymphoma. Methods A total of 7 HIV-positive lymphoma patients and 13 HIV-negative lymphoma patients who were treated in Chongqing University Cancer Hospital from September 2020 to September 2021 were enrolled. Tumor tissue samples were detected by second-generation sequencing technology, and gene mutations in HIV-positive lymphoma patients were analyzed by gene heat map, comparing the gene mutation differences between HIV-positive and HIV-negative lymphoma patients. Results Compared with HIV-negative lymphoma patients, more and more complex gene mutation sites were detected in HIV-positive lymphoma patients. Single nucleotide mutation of MYC gene was more common in HIV-positive lymphoma patients, while deletion of KMT2D gene was more common in HIV-negative lymphoma patients. And TP53 gene is highly mutated in both HIV-positive and HIV-negative lymphoma patients. However, mutations of TET, CCND3, TNFRSF14, ARID1A, TNFAIP3, NFKBIE, CXCR4, STAT3, NOTCH1 and other gene mutation with low mutation abundency occurred only in HIV-positive lymphoma patients. NOTCH2, PRDM1, EZH2, CARD11, EP300, MEF2B, CREBBP and other gene mutations occurred only in HIV-negative lymphoma patients. Conclusion HIV-positive lymphoma patients are more prone to gene mutations, and the gene mutations are more complex and diverse. MYC gene mutations and TP53 gene mutations play an important role in the occurrence and development of HIV-positive lymphoma, while the occurrence and development of HIV-negative lymphoma are more related to KMT2D gene mutations and TP53 gene mutations.

Clinical and Mechanistic Relationship of T-Cell Large Granular LymphocyticLeukemia (TLGL) andmonoclonal Gammopathy of Undetermined Significance(MGUS)

Introduction: The incidental finding of asymptomatic clonal expansion of plasma cells (MGUS), and T cells (TCUS) is not uncommon and its prevalence increases with age. While the propensity of such conditions to progress to multiple myeloma (MM) and TLGL, respectively is not fully elucidated, we observed in our clinical practice an intertwine between TLGL and MGUS, suggesting a possible mechanistic relationship. Per definition, MGUS should be asymptomatic and without cytopenia, the presence of which would upstage this condition. Thus, most often cytopenias in patients (pts) with MGUS and TLGL coexistence have been primarily attributed to the autoimmune sequelae of the T cell clonopathy. However, TLGL can also be asymptomatic in 20-50% of cases. In such a scenario the identification of the culprit etiology of cytopenia is crucial for the selection of proper therapies. Moreover, understanding the relationship between MGUS and TLGL may provide important lessons regarding the nature of the latter. One could stipulate that TLGL cells are the closest natural equivalent to the chimeric antigen receptor T cells (CART) and naturally arise from the immune trigger provided by clonal plasma cells. The initially polyclonal CTL responses may subsequently evolve to a clonal process, in particular upon acquisition of STAT3 mutations, ultimately controlling the MGUS clone. Alternatively, both humoral and CTL responses are triggered by the same offending agent and its persistence leads to the selection of dominant MGUS and TLGL clones as markers of an overshooting immune response. Methods/Results: We accrued a cohort of 280 pts with TLGL and explored both deep clinical and genomic phenotypes on 171 informative pts for whom complete data was available. Overall, pts had a median age of 67 years (IQR 58-73) with almost 1.09 M:F ratio. Among them, 23% had a concomitant plasma cell dyscrasia, with the majority being MGUS (19%), whereas only 2% and 1% of pts presented with Waldenstrom's macroglobulinemia/lymphoplasmacytic lymphoma (WM-LPL) and MM, respectively. Of note, no MGUS was found in TLGL pts <50 years, suggesting other pathogenetic drivers in younger cases. Overall, TLGL/MGUS had IgG (63%), IgM (25%) and IgA (9%) monoclonal protein phenotypes. In addition, polyclonal hypergammaglobulinemia was present in another 12% of pts. Whereas the overall expected MM evolution is 1-2% per year in typical MGUS, at a median follow up time of 12.5 years (8-17) only 1 MGUS/TLGL case progressed to MM as opposed to the estimated 3-4 pts. Furthermore, while the median overall survival (OS) of pts with MGUS has been reported as 8 years in historical cohorts, in our TLGL/MGUS cases reached 19 years. Looking at clinical characteristics, 13.5% of pts were asymptomatic, 61% had anemia, 53% neutropenia, and 34.5% thrombocytopenia. Anemia (76% vs 58%, p=.04), and bilineage cytopenia (38% vs 22%, p=.04) were more common in pts with co-associated MGUS. Gender, age distribution and disease-specific features such as TLGL counts (median 1785 vs 1680/µL), presence of STAT3 mutations (26% vs 33%) and co-occurring rheumatoid arthritis (15% vs 14%) did not differ between the two groups. Overall response rate (ORR) to T cell-directed therapy achieved 87% in pts with TLGL vs 64% in pts with concomitant MGUS (p=.0017). Indeed, because of the higher refractoriness rate in the latter group (median line of therapies 4 vs 3 in non-MGUS cases), 6 of these immunosuppression-refractory pts received bortezomib-dexamethasone resulting in a 67% ORR with a median time-to-next-treatment of 6.5 months (3-11). These observations argue against TLGL being the culprit of cytopenia in all TLGL/MGUS, suggesting that the monoclonal protein may have a specificity against myeloid or erythroid precursors. Conclusion: Our study shed light on the intertwining relation of the TLGL/MGUS clinical dyad. Rather than a coincidental finding in older pts, such clinical presentation may represent the first paradigmatic example of natural CART cells, analogous to those currently used in MM clinical practice. Finally, the etiology of cytopenias in TLGL need to be cautiously investigated, not automatically assuming a relation to TLGL. To further dissect such complicated dynamics, we are investigating a large cohort of MGUS pts for the presence of TCUS/TLGL and assessing the specificity of polyclonal and monoclonal proteins in MGUS pts with cytopenia using 2 million 12-mers peptide arrays.

Intense Immunosuppressive Therapy (anti-thymocyte globulin, alemtuzumab) As Salvage Therapy in Refractory T-Cell Large Granular Lymphocytic Leukemia and Pure Red Cell Aplasia

Background T-cell directed immunosuppression (IS: methotrexate, cyclosporine, and cyclophosphamide) is the main treatment modality applied for the therapy of immune single-lineage cytopenias including idiopathic neutropenia and pure red cell aplasia (PRCA). These two conditions are likely T-cell mediated and are often associated with T-cell large granular lymphocytic leukemia (T-LGL). The latter is thought to result from an originally polyclonal process evolving to a clonal proliferation of cytotoxic T-lymphocytes. Generally, response to 1st line therapies is about 30%, whereas rates as high as 80-90% can be achieved with 2nd, and 3rd lines. However, a significant fraction of patients remains refractory to multiple therapies and constitutes a management challenge. These patients remain transfusion dependent and/or have severely depressed neutrophil counts refractory to granulocyte-colony stimulating factor (G-CSF). In this scenario, intense IS with anti-thymocyte globulin (ATG) and alemtuzumab is often considered a salvage option, but no systematic experience exists and only a few controlled clinical trials have been conducted. Particularly, we previously reported an overall response rate (ORR) of 63% (n=23) with low dose alemtuzumab (10 mg sc q week x10) and 2/2 with ATG (40 mg/kg/d x 4 days) in patients with PRCA and/or LGL. Previous experience showed an ORR of 74% in a phase II study of 19 T-LGL patients treated with alemtuzumab, whereas a literature meta-analysis showed an ORR of 33% in 24 patients with PRCA treated with ATG. To better rationalize salvage therapy for refractory immune cytopenias, we explored the effectiveness of these agents at our tertiary referral center. Design and Methods We retrospectively examined treatment outcomes in 296 patients with LGL (113 had neutropenia and 73 anemia), and 78 patients with PRCA (+/- LGL). The study cohort had severe refractory cytopenia despite multiple treatment regimens and received salvage therapy with ATG and/or alemtuzumab. Complete response (CR) was defined as hemoglobin >10 g/dL, and absolute neutrophil count >1.0x109/L. Partial response (PR) was defined not meeting CR criteria. Response to therapy was evaluated while on therapy (alemtuzumab) and 4 months post-therapy (ATG). Overall response rate (ORR) was calculated as the percentage of patients with both CR and PR over the total number of patients who received the drug. The response rate was not available for 4 and 3 cases in the ATG and alemtuzumab cohorts respectively. Results A total of 62 patients who received salvage therapy with ATG (N=23) and alemtuzumab (N=39) for refractory cytopenia associated with LGL (N=38) and without LGL (N=24) were identified. Overall, the median age at disease onset was 58 years (IQR 45-69) with a 1.2 male to female ratio. On average, patients received 4 treatment modalities prior to ATG/alemtuzumab to which they did not respond or became refractory. CR rate (CRR) for patients treated with ATG was 42% (8/19) and ORR 89% (17/19). The CRR and ORR was 22% (8/36) and 81% (29/36), respectively in patients treated with alemtuzumab. No significant difference was observed in terms of ORR in patients treated with alemtuzumab compared to ATG (P = 0.45). In T-LGL cases, the presence of a STAT3 mutation did not impact an ORR to either alemtuzumab (31 vs 45% in STAT3 wild type) or ATG-treated cases (54 vs 38%). There was no significant difference in the LGL and PRCA (+/- LGL) cohorts treated with alemtuzumab (P = 0.89) and ATG (P = 0.13). Overall, no treatment-related death was registered, and toxicities were manageable. Alemtuzumab was held if absolute lymphocyte count was <200/µL. Of note, only one patient had CMV re-activation in our cohort. Conclusion Our data shows that alemtuzumab and ATG represent salvage IS regimens highly effective for the treatment of T-LGL and PRCA refractory to multiple lines of therapy with ORR higher if compared to historical data. This therapy resulted in a durable response in both T-LGL and PRCA (+/- LGL) with a good safety profile. The use of low dose alemtuzumab greatly decreases the complication rate and tolerability.

Aberrant tolerogenic functions and proinflammatory skew of dendritic cells in STAT1 gain-of-function patients may contribute to autoimmunity and fungal susceptibility

STAT1 gain-of-function (GOF) mutations underlie an inborn error of immunity hallmarked by chronic mucocutaneous candidiasis (CMC). Beyond the fungal susceptibility, attributed to Th17 failure, over half of the reported patients suffer from autoimmune manifestations, mechanism of which has not been explained yet. We hypothesized that the STAT1 mutations would affect dendritic cells' (DCs) properties and alter their inflammatory and tolerogenic functions. To test the hypothesis, we generated monocyte-derived DCs (moDCs) and tolerogenic DCs (tDCs). Functional and signaling studies, co-culture experiments and RNA sequencing demonstrated that STAT1 GOF DCs were profoundly altered in their phenotype and functions, characterized by loss of tolerogenic functions, proinflammatory skew and decreased capacity to induce Th17. Cytokine signaling, autophagy and metabolic processes were identified as the most prominently altered cellular processes. The results suggest that DCs are directly involved in STAT1 GOF-associated immune pathology, possibly contributing to both autoimmune manifestations and the failure of antifungal defense.

Evaluation of Breast Implant-Associated Anaplastic Large Cell Lymphoma With Whole Exome and Genome Sequencing.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare malignancy originating from the periprosthetic capsule of a textured, most often macrotextured, breast implant. Identified in women whose indications for breast implants can be either aesthetic or reconstructive, the genomic underpinnings of this disease are only beginning to be elucidated. The aim of this study was to evaluate the exomes, and in some cases the entire genome, of patients with BIA-ALCL. Specific attention was paid to copy number alterations, chromosomal translocations, and other genomic abnormalities overrepresented in patients with BIA-ALCL. Whole-exome sequencing was performed on 6 patients, and whole-genome sequencing on 3 patients, with the Illumina NovaSeq 6000 sequencer. Data were analyzed with the Illumina DRAGEN Bio-IT Platform and the ChromoSeq pipeline. The Pathseq Genome Analysis Toolkit pipeline was used to detect the presence of microbial genomes in the sequenced samples. Two cases with STAT3 mutations and 2 cases with NRAS mutations were noted. A critically deleted 7-Mb region was identified at the 11q22.3 region of chromosome 11, and multiple nonrecurrent chromosomal rearrangements were identified by whole-genome sequencing. Recurrent gene-level rearrangements, however, were not identified. None of the samples showed evidence of potential microbial pathogens. Although no recurrent mutations were identified, this study identified mutations in genes not previously reported with BIA-ALCL or other forms of ALCL. Furthermore, not previously reported with BIA-ALCL, 11q22.3 deletions were consistent across whole-genome sequencing cases and present in some exomes.

Uncommon Variant of CD4+ T-cell Large Granular Lymphocytic Leukemia

Abstract Introduction/Objective T-cell large granular lymphocytic leukemia (T-LGL) is a T-cell lymphoma with persistently increased large granular lymphocytes in peripheral blood. The majority of T-LGL is associated with CD8+ T-cells. However, the CD4+ variant is uncommonly seen. Methods/Case Report The patient is a 49-year-old female with a history of diabetes mellites and anemia who presented with shortness of breath, lower extremities edema, and fatigue. Physical examination showed a significantly distended abdomen. CBC results showed hemoglobin 6.9 g/dL, WBC 18.2 × 109/L with 88% lymphocytes, and PLT 190,000. The peripheral blood smear presented lymphocytosis with LGL cells, microcytic anemia, and increased target cells. Bone marrow biopsy showed lymphoid infiltrate as an intravascular pattern confirmed by immunochemical stains. A complete workup including flow cytometric, cytogenetics, and molecular studies was performed on the bone marrow. The lymphoma phenotype by flow shows positivity for CD2, CD3, CD4, CD5 (bright), CD7 (partial/dim), CD56, CD57 (majority), TCRαβ. T-cell gene rearrangement was detected and was further confirmed by PCR with the detection of clonal TCR beta gene rearrangements on molecular genetics analysis. However, STAT3 was not detected. The patient was diagnosed with CD4+ T-LGL leukemia. Results (if a Case Study enter NA) NA. Conclusion CD4+CD8- immunophenotype is an uncommon variant of T-LGL, which has a high incidence of activating STAT5B mutation, while CD8+CD4- T-LGLL tends to have STAT3 mutation. Clinically, those two subtypes behave similarly. Flow cytometry is the golden method to confirm the diagnosis. More research is needed to clarify the essence of CD4+ T-LGLL.

NOVEL HETEROZYGOUS SINGLE NUCLEOTIDE POLYMORPHISM IN STAT-3 GENE IN PATIENT WITH SUSPECTED HYPER IGE SYNDROME

<h3>Introduction</h3> Hyper IgE syndrome (HIES) is a rare genetic immune deficiency disorder affecting multiple organs. Recurrent skin infections, abscesses, atopic dermatitis, and hypereosinophilia are common. Mutations in STAT-3 gene is associated with HIES. We present a novel heterozygous point mutation in STAT-3 gene in a patient with hyper IgE and recurrent abscesses. <h3>Case Description</h3> A 22-year-old male presented with severe atopic dermatitis and recurrent abscesses. Initial labs revealed very high serum IgE 41,559 U/ml and eosinophil count of 1290/cmm. CBC showed mild anemia. Infectious work up was all negative. Patient had normal CMP, troponin, spirometry, tryptase, Vit B12 and ECHO. Skin biopsy was suggestive of eosinophilic fasciitis. Bone marrow biopsy and flow cytometry were normal. CT scans showed inguinal and axillary lymphadenopathy and borderline hepatomegaly. He was hospitalized multiple times for I/D of his recurrent abscesses. He was treated with systemic steroids and started on Bactrim prophylaxis. In addition, Dupilumab was started for his eczema which improved his skin. He longer has abscesses. After Dupilumab his IgE level significantly decreased from >50,000 U/ml to 14,790 U/ml. HIES panel revealed a novel heterozygous point mutation in STAT-3 gene, Exon 19 c1673G>A (p.Gly558Asp). Due to patient's supporting clinical phenotype HIES was suspected. Further genetic and functional studies to confirm this variant are planned. <h3>Discussion</h3> HIES is a complex disease, and it is challenging to confirm the diagnosis. Several mutations in STAT-3 have been described but more studies are still warranted to identify different mutations. Dupilumab may improve patient's atopic dermatitis in suspected cases.

RECURRENT APHTHOUS STOMATITIS AND ORAL CANDIDIASIS DUE TO STAT1 MUTATION

<h3>Introduction</h3> Chronic mucocutaneous candidiasis (CMC) often presents with chronic <i>Candida</i> infections of the skin, nails or mucous membranes in the absence of invasive fungal infections. Various genetic defects are associated with CMC. Here, we describe a case of recurrent aphthous stomatitis and chronic oral candidiasis due to gain-of-function (GOF) mutation in STAT1. <h3>Case Description</h3> A 21-year-old male presented to the Allergy and Immunology clinic with long-standing history of recurrent aphthous ulcers and oral candidiasis. He had previously undergone evaluation as a child with normal immunoglobulin levels, normal antibody response to vaccines and normal delayed-type hypersensitivity skin test to <i>Candida</i> antigen, but low absolute NK cells. Targeted genetic testing was negative for APECED and IPEX. He continued to experience frequent aphthous ulcers and oral thrush that can be debilitating at times, with one episode resulting in hospital admission due to severe mucosal pain and PO intolerance. Repeat immunology evaluation was similar to prior. He had low absolute NK cells, and NK cell function study using chromium release assay was notably decreased. A comprehensive primary immunodeficiency next generation sequencing panel identified a pathogenic heterozygous c.800C>T (p.A267V) mutation in STAT1. <h3>Discussion</h3> While fungal infection of the oral mucosa is a main clinical feature of CMC, aphthous stomatitis can be found frequently in patients with STAT1 GOF mutations as well. These patients also frequently have decreased NK cells and NK cell function. It is important to be aware of these additional phenotypes in the evaluation of a patient with CMC.

40. Clonal evolution of Cutaneous T Cell Lymphoma (CTCL) revealed at single cell resolution

Patients with CTCL subtypes Mycosis Fungoides and Sézary Syndrome (MF/SS) frequently progress to advanced stage disease, which has a 20% 5-year survival. The molecular factors that drive MF/SS progression remain undefined. To address this, we subjected 41 longitudinal tumor samples (peripheral blood, skin and lymph node biopsies, 49-1050 days apart) and matched, purified non-malignant cells from 10 MF/SS patients to single-cell (sc)RNA-seq and exome+whole genome sequencing (eWGS). and patient-specific TCR clonotype assignment (scRepertoire). Clustering and cell type inference of scRNAseq (UMAP/Seurat/singleR) showed canonical CTCL marker genes and high clonal proportions (1-100%) of patient-specific TCR clonotypes. Somatic genome copy number variants (CNV) from scRNAseq (copykat) and eWGS (cnvkit), and deleterious/damaging single nucleotide variants (SNV) (eWGS, GATK/Mutect2) were identified in CTCL versus matched, non-malignant cells. To segregate early genomic events from progression-associated, we developed an integrative analysis pipeline to prioritize somatic CNV/SNV in genes with altered expression in >=30% of patients. This novel approach revealed alterations in NF-KB and T-cell receptor pathways (NFKB2, STAT3/5, TOX) as early events that were maintained in subsequent CTCL samples. We demonstrate gain-of-function activity in a novel-to-CTCL mutation in STAT3. Losses of some chromatin-modifying enzymes (DNMT3A, KDM6B) are early events, while gains of others (EZH2) are acquired with progression. Strikingly, progression-associated mutations were enriched in cell metabolism, growth, and motility genes, some of which are new to MF/SS: HIF1A, ITPR3, GTPase/RAS (IQGAP1/2, RASA1), and PI kinase (AKT1, MTOR, PIK3R1). These findings represent new insights into MF/SS progression and identify new precision medicine targets for currently available therapies.