STAT3 Mutations Research Articles

Pathogenetic Subtypes of Pure Red Cell Aplasia and Therapeutic Management Strategies : A Single Centre Experience in the United Kingdom of 75 Patients over 10 Years

Introduction Pure red cell aplasia (PRCA) is a syndrome characterised by a severe anemia, reticulocytopenia and severe depletion of erythroid precursors in an otherwise unremarkable bone marrow. PRCA arises congenitally in Diamond-Blackfan Anemia and usually presents in childhood. Acquired PRCA can occur at any age, can be transient due to infections such as from parvovirus B19, or more chronic, secondary to autoimmune conditions, malignancies especially thymoma, erythropoiesis stimulating agents, , pregnancy and ABO incompatible hematopoetic stem cell transplantation. PRCA is also known to arise from drug and toxin exposure. Haematological malignancy/clonal disorders for e.g. CLL, MGUS, T-LGLs have well known associations with PRCA. PRCA can also be a feature of myelodysplastic syndrome and can herald evolution to aplastic anemia. Despite the heterogeneity in aetiology, the mainstay of treatment remains immunosuppression with steroids and/or ciclosporin. Here, we report our single centre experience of a large cohort of 75 patients with PRCA, referred to our centre over a 10 year period. Methods Patients with pure red cell aplasia referred to the Department of Hematological Medicine at King's College Hospital were identified by interrogation of the Electronic Patient Record (EPR) and pathology systems. The search terms used were a clinical or pathological diagnosis of ‘pure red cell aplasia’ or ‘PRCA’. Our hospital receives diagnostic samples from the South East of England, therefore data was collected only for patients with clinical information available. Patients (n=33) who developed PRCA post-allogeneic stem cell transplant were excluded. Results A total of 75 patients with both a pathological diagnosis of PRCA and available clinical information were identified, 31 (41.3%) were female. Median age was 51 years (range 9 - 84). The majority of patients had underlying conditions known to be associated with PRCA. Of the haematological conditions, PRCA was most seen in association with MDS (Table 1). Of the 75 patients in our cohort, 82% (61/75) required treatment for PRCA and 57% (43/75) required two or more lines of therapy. In total 25.3% (19/75) patients received corticosteroids as first line therapy. Of those, 75% showed a response, with 50% achieved a complete response. The response rate was less favourable (57%) in those patients in whom ciclosporin was used as first line therapy. Most common second line therapy was Ciclosporin (22.3%) followed by Cyclophosphamide (4%), Sirolimus (2.7%) and Anti-Thymocyte Globulin (1.33%). Of the cohort with an underlying MDS, 52% (11/21) responded to treatment, compared to 65% of those with idiopathic PRCA, likely reflective of the recalcitrant nature of PRCA in MDS. Other therapies received were IVIG, erythropoietin and disease-directed therapy,e.g lenalidomide for MDS with 5q- and thymectomy. Median follow of patients were 42 months.The overall survival of PRCA since last follow up was 50% for the cohort as a whole and 63% for those without an associated malignancy or clonal disorder. D iscussion In this large single centre experience of PRCA, we describe a lower incidence of idiopathic PRCA, lower median age despite a large proportion of MDS and increased incidence in male as compared to published literature. Response to initial therapy was seen in the majority of idiopathic PRCA patients, however response was lower in patients with an underlying malignancy. Of note we identified STAT3 mutations in two patients, highlighting the role of molecular testing and keeping a broad differential diagnosis when presented with this rare condition.

Clinical Features and Outcomes in Large Granular Lymphocyte Leukemia-Associated Pure Red Cell Aplasia with STAT3 Mutation

Background Large granular lymphocyte leukemia (LGLL) is a rare disease frequently complicated with pure red cell aplasia (PRCA) [1, 2]. STAT3 mutations have been describled in 30-40% of LGLL patients [3]. The aim of this work was to evaluate whether STAT3 mutations might be associated with specific clinical features and outcomes in LGLL-associated PRCA. Methods and results From January 2016 to July 2023, 81 patients with LGLL-associated PRCA were enrolled in the China Eastern Cooperation Group for Anemia (CECGA) database (ChiCTR2100043485). As an initial step in the STAT3 mutational analysis, we screened all patients with Sanger sequencing or Next-generation sequencing (NGS). The initial dose of CsA was 3-5mg/kg/day, and the serum concentration was adjusted to be 150-200ng/mL based on adverse reactions. After 12 months of maintenance, the dosage was slowly reduced. Cyclophosphamide combined with prednisone (CP) regimen was used as a salvage therapy for patients who failed to CsA. The initial dose of cyclophosphamide and prednisone were 100mg/day and 0.5-1mg/kg/day, respectively. Among the 81 cases, 26% of them were positive for STAT3 mutations. The clinical characteristics of patients with or without STAT3 mutation were summarized in Table 1. Patients with STAT3 mutations had a higher reticulocyte percentage (0.88% vs 0.28%, P=0.039) and red cell distribution width-coefficient of variation (18.8% vs 15.8%, P=0.008) than patients without STAT3 mutations. Y640F mutation were found in 9 of 21 cases. Subgroup analysis showed that patients with Y640F mutation were associated with younger age (44 vs 65 years old, P=0.007) and higer lymphocyte percentage in peripheral blood (63.7% vs 34.4%, P=0.033). Deep sequencing of rearranged T-cell receptor Vβ complementarity-determining region 3 by NGS was conducted in 61 patients. The predominant V-gene of LGLL clonotypes belonged to the TRBV06 family gene was detected in 12 (25%) patients. The expression of TRBV06 family gene was a litter lower in patients with STAT3 mutation than non-mutant groups (8% vs 31%, P=0.189). The complete response rate (CRR) [31% (5/16) vs 33% (19/58), P=0.909] and overall response rate (ORR) [56% (9/16) vs 50% (29/58), P=0.658] of cyclosporine (CsA) treatment were similar in patients with STAT3 mutations or not. In STAT3 mutant group, the CRR [54% (7/13) vs 31% (5/16), P=0.274] and ORR [85% (11/13) vs 56% (9/16), P=0.130] of CP regimen tended to be better than CsA . 6 patients (67%) relapsed among the 9 patients who responded to CsA in STAT3 mutant group. In patients without STAT3 mutation, 19 of 29 (66%) CsA-responders relapsed. There was no siginificant difference in the relapse-free survival between the two group (Figure 1). Discussion/Conclusions In summary, STAT3 mutation was frequently recognized in LGLL-associated PRCA, and the hotspot site was Y640F. Patients with STAT3 mutations responded to CsA as well as those without the mutation. CP regimen could be used as a salvage therapy for patients who failed to CsA. Reference 1. Balasubramanian SK, Sadaps M, Thota S, et al. Rational management approach to pure red cell aplasia. Haematologica. 2018, 103(2): 221-230. 2. Wu X, Cheng L, Liu X, et al. Clinical characteristics and outcomes of 100 adult patients with pure red cell aplasia. Ann Hematol. 2022, 101(7):1493-1498. 3. Barilà G, Teramo A, Calabretto G, et al. Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients. Leukemia. 2020, 34: 1116-1124.

Ruxolitinib Promotes Clinical Responses in Large Granular Lymphocytic Leukemia Via Suppression of JAK/STAT-Dependent Inflammatory Cascades

Background: Large granular lymphocytic leukemias (LGL) are T or NK-cell lymphoproliferative disorders that are characterized by myelosuppression and frequent mutations in STAT3. Commonly used agents include low doses of methotrexate or cyclophosphamide. Among 5 patients (pts) with LGL treated on our phase II study of ruxolitinib in T-cell lymphoma (Moskowitz, et al. Blood 2021), 2 achieved partial response (PR) and 2 achieved stable disease, suggesting oncogenic dependence on JAK/STAT pathway activity in LGL. Methods: To further explore the efficacy and mechanism of JAK1/2 blockade in LGL, we expanded our phase II study to include 23 pts with relapsed/refractory LGL. This sample size was chosen to allow for narrowing of the 95% confidence interval to 54-87% (Wilson score method) if similar efficacy was observed. Pts received ruxolitinib 20mg PO BID continuously. Responses were assessed after cycles 2, 5, and every 3 cycles thereafter using the response assessment from the ECOG-5998 study for LGL. Furthermore, stable disease (SD) was defined as improvement in cytopenia(s) by 1 grade (without meeting criteria for PR) for a minimum of 6 months. Overall response rate (ORR) included complete response (CR) plus PR; clinical benefit rate (CBR) included ORR plus SD. In parallel, we performed single-cell RNA sequencing (scRNA-seq) on paired C1D1 and C1D8 peripheral blood mononuclear cells (PBMCs) from 7 patients (5 PR, 1 SD, and 1 non-responder) and plasma proteomic profiling on 5 patients (3 PR, 1 SD, and 1 POD). Results: Among 23 pts, median age was 58 (31-86), 13 (57%) were male, 19 (83%) were white, 1 (4%) was black. Median number of prior therapies was 2 (range 0-5). 12/22 (54%) had STAT3 mutations (of whom 1 also had a JAK1 mutation); 1 had a JAK2 mutation, 1 pt was missing sequencing. Baseline cytopenias included anemia (n=14, 61%), thrombocytopenia (n=12, 52%), and/or neutropenia (n=16, 70%). Among 20 pts evaluable for response (3 too early), there were 5 (25%) CRs, 6 (30%) PRs, and 4 (20%) SD. ORR and CBR were 55% and 75% respectively. Median event free survival (EFS) was not reached and was 68% (95% CI: 50-93) at 14 months ( Fig. 1A). STAT3 mutation status predicted for improved EFS (14-month EFS 100% vs 40%, p=0.007). After a median duration of treatment of 13.1 months, there were no unexpected toxicities. Severe adverse events included febrile neutropenia (n=3, 13%) and herpes zoster (n=1, 4%). To understand the mechanism by which ruxolitinib improved hematologic parameters in LGL, we performed unbiased proteomic analysis of peripheral blood plasma samples using the OLink 96 Inflammation and Immune Response Platforms. Ruxolitinib led to a decrease in several soluble mediators known to be driven by interferon-driven JAK/STAT activation, including CXCL9, CXCL10, and PD-L1, as well as TNF ( Fig. 1B). Several of these factors have been shown to suppress hematopoietic stem cell function, suggesting a JAK/STAT-dependent paracrine mechanism by which LGL leads to myelosuppression. ScRNA-seq of PBMCs identified LGL cells based on expression of cytotoxic genes, including NKG7, CST7, CCL5, GZMB and PRF1. Analysis of cell type proportions within the peripheral blood showed that patients achieving a PR to Ruxolitinib showed a decrease in the proportion of circulating myeloid cells. Gene set enrichment analysis (GSEA) of LGL cells revealed substantial downregulation of both JAK/STAT signaling and TNF signaling in LGL cells in PR but not SD patients, whereas persistently elevated TNF signaling was seen not only in LGL cells but also circulating myeloid and naïve T-cells in SD patients, suggesting that LGL cells might promote bone marrow suppression via paracrine effects on the bystander host immune system. Network analysis of LGL cells and non-malignant peripheral blood immune cells is ongoing. Conclusions: Ruxolitinib induced durable responses in pts with LGL and therefore may represent an option for pts requiring treatment. STAT3 mutation status was predictive of EFS and responses were associated with JAK/STAT pathway down-regulation, demonstrating on-target effects. Integrated immune profiling suggests that ruxolitinib may interrupt JAK/STAT-dependent paracrine inflammatory signals that promote bone marrow suppression. An ongoing phase II study of ruxolitinib in LGL will aid in clarifying optimal dosing and confirming both efficacy and mechanism of action (NCT05592015).

Multi-Dimensional Molecular and Tumor-Microenvironment Analysis of Classic Hodgkin Lymphoma

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P < .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients <45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

Insights into Large Granular Lymphocytic Leukemia-Associated Hemolytic Anemia: Clinical Associations, Therapeutic Responses, and Optimal Management Strategies from a Large Institutional Study

Introduction Large granular lymphocytic leukemia (LGLL) is characterized by a clonal proliferation of cytotoxic CD8+ T or CD3− natural killer (NK) cells. The disease typically presents with neutropenia in about 39-62% of patients, and anemia in up to 50% 1. While most cases of anemia in LGLL resemble pure red cell aplasia and present as reticulocytopenic and malproductive, we have also observed rare instances of hemolytic anemia (HA) with a high reticulocyte count. This unique and atypical presentation has prompted us to investigate the clinical features of LGLL with HA, aiming to gain insights into the pathogenesis of both conditions. In light of this, we conducted a comprehensive and systematic study of hemolysis within the context of LGLL. Methods We retrospectively examined clinical and molecular features of a large series of LGLL cases (n=262) diagnosed and managed at the Cleveland Clinic Foundation from 1998 to 2023 to identify those experiencing hemolysis. LGLL diagnosis was based on the presence of i) elevated LGL count (>0.5 × 10^9/L), ii) clonal TCR rearrangement, iii) VB expansion, iv) flow cytometric detection of aberrant CTL or NK cell proliferation and v) STAT3/5 mutation and vi) LGLL infiltration of the marrow. A diagnosis was established with >4/6 criteria fulfilled. HA diagnosis was based on evidence at any time during clinical course of laboratory signs of hemolysis. Results Overall, the median age at diagnosis was 63 years (IQR: 55-72) with a M:F ratio of 0.5. Majority of patients had been diagnosed with T-LGLL (n=225), whereas 22 had NK-LGLL. In 32/262 cases (12%), clinical and laboratory features were asserted to establish the diagnosis of HA. Of these, 22 (68%) had T-LGLL and 8 (25%) had NK-LGLL (Table 1). When comparing HA patients to the rest of the cohort, we observed a higher proportion of NK-LGLL cases with HA (25% vs 8%, p < 0.001). HA presented as the initial sign of the disease in majority of patients (88%), and 40% of them required transfusion support based on the degree of anemia and hemodynamics. The direct anti-globin test was positive in 30% of cases. Warm autoimmune HA (AIHA) was observed in 23% of patients, cold agglutinin disease in 7%, and one patient had both. Among 7 patients with IgG AIHA, 5 were also positive for anti-C3b, C3d antibodies. Two-thirds of the patients had multilineage cytopenia. Coexisting B-cell dyscrasia was identified in 13 cases, more frequently in HA than in those without (40% vs 26%, p=0.048), and autoimmune disorders in 8 cases (25%). HA in LGLL was also more likely to be associated with presence of splenomegaly (59% vs 21%, p<0.00001) and polyclonal hypergammaglobulinemia (22% vs 8%, p=0.02). Notably, STAT3 mutations were present in a higher proportion of HA patients (38% vs 22%, p=0.02), whereas median LGLL clone size (%) was similar between groups (68% vs 53%, p=0.5). Overall, 77% of HA patients received treatment, and 16% had still evidence of hemolysis at a median follow-up of 82 months. The majority (47%) were treated with steroids with a positive, albeit transient response (7 out of 15 treated). Other first-line treatments and their corresponding median duration of response (months) included cyclosporine (n=7, 72 months), cyclophosphamide (n=5, 72 months), IVIG (n=1, 16 months), and methotrexate (n=11, 21 months). Subsequent lines of therapy included campath (n=9), cyclosporine (CSA) (n=4), cyclophosphamide (n=9), intravenous immunoglobulin (IVIG) (n=2), MTX (n=4), rituximab (n=1), sirolimus (n=1), tacrolimus (n=2), and tofacitinib (n=2). Splenectomy was performed as definitive treatment for refractory cytopenia in 9 patients, with a durable response in 5 patients at last follow-up ( Fig.1). Conclusions Based on this large series, our study illustrates that LGLL represents a clonal lymphoproliferative disorder that is closely linked to HA. Notably, NK-LGLL cases exhibited a stronger association with HA. Our observations indicate that treatment with steroids and other therapeutic agents resulted in positive responses in a fraction of patients, while splenectomy demonstrated the most enduring response. These significant findings contribute substantially to advancing our comprehension of LGLL-associated HA and its optimal management strategies. AIHA in patients with LGLL points towards a global immune dysfunction in this disease beyond clonal cytotoxic T/NK cell expansion.

Genetic Susceptibility to Fungal Infections.

Reports of fungal infections have increased over the past decades, making them a major threat to human health. In this study, we review the effects of genetic defects on susceptibility to fungal diseases. To identify all relevant literature, we searched Google Scholar, PubMed, and Scopus and profiled studies published between 2008 and 2021. The results of several studies conducted on this subject have shown the significant effects of genetic variations such as hyper-IgE syndrome, Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome, dectin-1 deficiency, CARD9 mutations, STAT1 mutations, and IL17 mutationson the host immune system's response, which has an important impact on susceptibility to fungal infections. The underlying immune system-related genetic profile affects the susceptibility of individuals to different fungal infections; therefore, this subject should be further studied for better treatment of fungal diseases.

The mechanism underlying the inverse proportion between serum IgE concentration and Stat3 transcriptional activity depends on Stat3 mutation type

Abnormalities in the IL-6/STAT3 signaling pathway can sometimes result in extremely high levels of IgE concentration in serum, however, the regulatory role of STAT3 in IgE production is elusive. We used several genetically modified mice with distinctive germline Stat3 transcriptional activity to assess the influence of Stat3 on the biochemical characteristics of IgE and found that the IgE concentration in serum is inversely proportional to Stat3 transcriptional activity. Intriguingly, the serum IgE concentration is directly proportional to IgE-producing B cells in Stat3-GOF mice but inversely proportional in mice carrying Stat3 mutations with reduced transcriptional activity. For reduced Stat3 transcriptional activity induced high levels of IgE in the mice, IL-4/Stat6 signaling is indispensable for IgE production, but it was observed that an increased IgE concentration was accompanied by reduced IL-4/Stat6 signaling and lessened IgE-producing B cells, which implies that an increase in IgE concentration may result from an extended half-life of IgE but not an increasing number of IgE-producing cells.

Stat3 Has a Different Role in Axon Growth During Development Than It Does in Axon Regeneration After Injury.

Signal transducer and activator of transcription 3 (STAT3) is essential for neural development and regeneration as a key transcription factor and mitochondrial activator. However, the mechanism of Stat3 in axon development and regeneration has not been fully understood. In this study, using zebrafish posterior lateral line (PLL) axons, we demonstrate that Stat3 plays distinct roles in PLL axon embryonic growth and regeneration. Our experiments indicate that stat3 is required for PLL axon extension. In stat3 mutant zebrafish, the PLL axon ends were stalled at the level of the cloaca, and expression of stat3 rescues the PLL axon growth in a cell-autonomous manner. Jak/Stat signaling inhibition did not affect PLL axon growth indicating Jak/Stat was dispensable for PLL axon growth. In addition, we found that Stat3 was co-localized with mitochondria in PLL axons and important for the mitochondrial membrane potential and ATPase activity. The PLL axon growth defect of stat3 mutants was mimicked and rescued by rotenone and DCHC treatment, respectively, which suggests that Stat3 regulates PLL axon growth through mitochondrial Stat3. By contrast, mutation of stat3 or Jak/Stat signaling inhibition retarded PLL axon regeneration. Meanwhile, we also found Schwann cell migration was also inhibited in stat3 mutants. Taken together, Stat3 is required for embryonic PLL axon growth by regulating the ATP synthesis efficiency of mitochondria, whereas Stat3 stimulates PLL axon regeneration by regulating Schwann cell migration via Jak/Stat signaling. Our findings show a new mechanism of Stat3 in axon growth and regeneration.

Interventional pulmonary procedures and their outcomes in patients with STAT3 hyper IgE syndrome

BackgroundSTAT3 hyperimmunoglobulin E syndrome (STAT3-HIES) also referred to as autosomal dominant HIES (AD-HIES) is an inborn error of immunity characterized by the classic triad of eczema, frequent opportunistic infections, and elevated serum IgE levels. As a consequence of lung sequels due to repeated infections and impaired tissue healing, patients may require interventional pulmonary procedures.MethodFour patients with dominant-negative STAT3 mutations who had received interventional pulmonary procedures were enrolled. The demographic, clinical, and molecular characteristics were gathered through a medical record search. All reported STAT3-HIES patients in the literature requiring pulmonary procedures as part of their treatment were reviewed.ResultRecurrent episodes of pneumonia and lung abscess were the most prevalent symptoms. The most common non-immunological features were scoliosis, failure to thrive, and dental problems such as primary teeth retention and disseminated decays. Bronchiectasis, lung abscess, pneumatocele, and cavitary lesion were the most prevalent finding on high-resolution computed tomography at the earliest recording. All patients underwent pulmonary surgery and two of them experienced complications.ConclusionPatients with STAT3-HIES have marked pulmonary infection susceptibility which may necessitate thoracic surgeries. Since surgical procedures involve a high risk of complication, surgical options are recommended to be utilized only in cases of drug resistance or emergencies.

TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

POSTER: TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

POSTER: TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

Novel insights into the pathogenesis of follicular lymphoma by molecular profiling of localized and systemic disease forms

Knowledge on the pathogenesis of FL is mainly based on data derived from advanced/systemic stages of FL (sFL) and only small cohorts of localized FL (lFL) have been characterized intensively so far. Comprehensive analysis with profiling of somatic copy number alterations (SCNA) and whole exome sequencing (WES) was performed in 147 lFL and 122 sFL. Putative targets were analyzed for gene and protein expression. Overall, lFL and sFL, as well as BCL2 translocation-positive (BCL2+) and –negative (BCL2−) FL showed overlapping features in SCNA and mutational profiles. Significant differences between lFL and sFL, however, were detected for SCNA frequencies, e.g., in 18q-gains (14% lFL vs. 36% sFL; p = 0.0003). Although rare in lFL, gains in 18q21 were associated with inferior progression-free survival (PFS). The mutational landscape of lFL and sFL included typical genetic lesions. However, ARID1A mutations were significantly more often detected in sFL (29%) compared to lFL (6%, p = 0.0001). In BCL2 + FL mutations in KMT2D, BCL2, ABL2, IGLL5 and ARID1A were enriched, while STAT6 mutations more frequently occurred in BCL2- FL. Although the landscape of lFL and sFL showed overlapping features, molecular profiling revealed novel insights and identified gains in 18q21 as prognostic marker in lFL.

High Epstein-Barr virus capsid antigen IgG level associates with the carriership of CD8+ T cell somatic mutations in the STAT3 SH2 domain

High carrier prevalence of STAT3 SH2 domain somatic mutations was recently discovered in CD8+ T cells. We found these low-allele-fraction clones in 26% of donors, without difference between multiple sclerosis (MS) patients and controls. Here we tested whether anti-viral antibodies associate with the carriership of these mutant clones. We compared antibody responses against common viruses in mutation carriers vs. non-carriers. Plasma samples of 152 donors (92 MS patients, 60 controls) were analyzed for antibodies against cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus-6A and parvovirus B19. The mutation carrier status associated with EBV VCA IgG level (p = 0.005) and remained significant after logistic regression (p = 0.036). This association was contributed similarly by MS patients and controls. These results suggest that EBV contributes to the generation or growth of these clones. The pathogenic role of the STAT3 mutant clones in MS is presently unclear, but their detailed characterization warrants further study.

Context-dependent T-cell Receptor Gene Repertoire Profiles in Proliferations of T Large Granular Lymphocytes.

T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.

Single-cell multi-omics sequencing reveals the immunological disturbance underlying STAT3-V637M Hyper-IgE syndrome

Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by, among others, the excessive production of IgE and repetitive bacterial/fungal infections. Mutations in STAT3, a transcription factor that orchestrates immune responses, may cause HIES, but the underlying mechanisms are not fully understood. Here, we used multi-omic approaches to comprehensively decipher the immune disturbance in a male HIES patient harboring STAT3-V637M. In his peripheral blood mononuclear cell (PBMC) we found significant clonal expansion of CD8 T cells (with increased CD8 subunits expression, potentially enhancing responsiveness to MHC I molecules), but not in his CD4 T cells and B cells. Although his B cells exhibited a higher potential in producing immunoglobulin, elevated SPIC binding might bias the products toward IgE isotype. Immune checkpoint inhibitors, including CTLA4, LAG3, were overexpressed in his PBMC-CD4 T cells, accompanied by reduced CD28 and IL6ST (gp130) expression. In his CD4 T cells, integrative analyses predicted upstream transcription factors (including ETV6, KLF13, and RORA) for LAG3, IL6ST, and CD28, respectively. The down-regulation of phagocytosis and nitric oxide synthesis-related genes in his PBMC-monocytes seem to be the culprit of his disseminated bacterial/fungal infection. Counterintuitively, in his PBMC we predicted increased STAT3 binding in both naïve and mature CD4 compartments, although this was not observed in most of his PBMC. In his bronchoalveolar lavage fluid (BALF), we found two macrophage subtypes with anti-bacterial properties, which were identified by CXCL8/S100A8/S100A9, or SOD2, respectively. Together, we described how the immune cell landscape was disturbed in STAT3-V637M HIES, providing a resource for further studies.

Establishment and characterization of a novel breast implant-associated anaplastic large cell lymphoma cell line and PDX model (BIA-XR1) with a unique KRAS mutation

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is an uncommon T-cell lymphoma type with distinct clinical, molecular and genetic features. Establishment of BIA-ALCL cell lines and patient-derived xenograft (PDX) models are essential experimental tools to investigate the molecular pathogenesis of the disease. We characterized a novel BIA-ALCL cell line and PDX model, named BIA-XR1, derived from a patient with textured breast implant who developed lymphoma. Next-generation sequencing revealed a STAT3 mutation, commonly detected in BIA-ALCL, and a unique KRAS mutation reported for the first time in this lymphoma type. Both JAK/STAT3 and RAS/MEK/ERK oncogenic pathways were activated in BIA-XR1, which are targetable with clinically available agents.

Genomic landscape of T-large granular lymphocyte leukemia and chronic lymphoproliferative disorder of NK cells: a single institution experience

LGLL is a rare and chronic lymphoproliferative disorder including T-LGLL and CLPD-NK. Here, we investigated the genomic profiles of LGLL with a focus on STAT3 and STAT5B mutations in a cohort of 49 patients (41 T-LGLL, 8 CLPD-NK). Our study indicated that STAT3 was identified in 38.8% (19/49) of all patients, while STAT5B occurred in only 8.2% (4/49) of patients. We found that STAT3 mutations were associated with lower ANC in T-LGLL patients. The average number of pathogenic/likely pathogenic mutations in STAT3/STAT5B-mutated patients was significantly higher than that in WT patients (1.78 ± 1.17 vs 0.65 ± 1.36, p = 0.0032). Additionally, TET2-only mutated T-LGLL (n = 5) had a significant reduction in platelet values compared with the WT (n = 16) or STAT3-only mutated T-LGLL (n = 12) (p < 0.05). In conclusion, we compared the somatic mutational landscape between STAT3/STAT5B WT and mutated patients and correlate with their distinct clinical characteristics.

SDH mutations, as potential predictor of chemotherapy prognosis in small cell lung cancer patients

PurposeSmall cell lung cancer (SCLC) is an aggressive and rapidly progressive malignant tumor characterized by a poor prognosis. Chemotherapy remains the primary treatment in clinical practice; however, reliable biomarkers for predicting chemotherapy outcomes are scarce.MethodsIn this study, 78 SCLC patients were stratified into “good” or “poor” prognosis cohorts based on their overall survival (OS) following surgery and chemotherapeutic treatment. Next-generation sequencing was employed to analyze the mutation status of 315 tumorigenesis-associated genes in tumor tissues obtained from the patients. The random forest (RF) method, validated by the support vector machine (SVM), was utilized to identify single nucleotide mutations (SNVs) with predictive power. To verify the prognosis effect of SNVs, samples from the cbioportal database were utilized.ResultsThe SVM and RF methods confirmed that 20 genes positively contributed to prognosis prediction, displaying an area under the validation curve with a value of 0.89. In the corresponding OS analysis, all patients with SDH, STAT3 and PDCD1LG2 mutations were in the poor prognosis cohort (15/15, 100%). Analysis of public databases further confirms that SDH mutations are significantly associated with worse OS.ConclusionOur results provide a potential stratification of chemotherapy prognosis in SCLC patients, and have certain guiding significance for subsequent precise targeted therapy.

Distinct mutational pattern of T-cell large granular lymphocyte leukemia combined with pure red cell aplasia: low mutational burden of STAT3

T-cell large granular lymphocyte leukemia (T-LGL) is often accompanied by pure red cell aplasia (PRCA). A high depth of next generation sequencing (NGS) was used for detection of the mutational profiles in T-LGL alone (n = 25) and T-LGL combined with PRCA (n = 16). Beside STAT3 mutation (41.5%), the frequently mutated genes included KMT2D (17.1%), TERT (12.2%), SUZ12 (9.8%), BCOR (7.3%), DNMT3A (7.3%), and RUNX1 (7.3%). Mutations of the TERT promoter showed a good response to treatment. 3 of 41 (7.3%) T-LGL patients with diverse gene mutations were revealed as T-LGL combined with myelodysplastic syndrome (MDS) after review of bone marrow slide. T-LGL combined with PRCA showed unique features (low VAF level of STAT3 mutation, low lymphocyte count, old age). Low ANC was detected in a STAT3 mutant with a low level of VAF, suggesting that even the low mutational burden of STAT3 is sufficient for reduction of ANC. In retrospective analysis of 591 patients without T-LGL, one MDS patient with STAT3 mutation was revealed to have subclinical T-LGL. T-LGL combined with PRCA may be classified as unique subtype of T-LGL. High depth NGS can enable sensitive detection of concomitant MDS in T-LGL. Mutation of the TERT promoter may indicate good response to treatment of T-LGL, thus, its addition to an NGS panel may be recommended.

HSV-1 and HHV-6 Meningoencephalitis in a Child with STAT1 Mutation

Introduction: STAT1 is a critical signal transducer for both the interferon gamma receptor and IFN-alpha/beta receptors and is required for responses to herpesviruses. STAT1 defects vary in severity and clinical presentation. STAT1 loss of function (LOF) mutations with Mendelian Susceptibility to Mycobacterial Disease (MSMD) and STAT1 gain of function (GOF) mutations have been described with a wide clinical spectrum, including nontuberculous mycobacteria, disseminated fungal, bacterial, and viral infections in addition to immune dysregulation. We present a 10-year-old female with HSV-1 and HHV-6 meningoencephalitis that was found to have a variant in STAT1. MethodsRetrospective chart review was conducted. Laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogens and antigens, NK cell functional assays, quantitative immunoglobulins, toll like receptor function defects, genetic evaluation by next generation sequencing, and assays for further evaluation for MSMD, T helper IL17, STAT GOF. ResultsA previously healthy 10-year-old Caucasian female presented with fever, headache, and new-onset seizure with status epilepticus. She was diagnosed with HSV-1 and HHV-6 meningoencephalitis, as CSF PCR panel was positive for HSV-1 and HHV-6. Brain MRI showed findings compatible with HSV-1 encephalitis. She was treated with a 14-day course of acyclovir with no noted neurological sequelae. Infection history was unremarkable except for one episode of otitis media as a child and a couple upper respiratory viral illnesses. Denied prior history of HSV infection. No family history of immunodeficiency disorders. Both parents reported remote history of herpetic gingivostomatitis. Laboratory immune evaluation was remarkable for decreased lymphocyte proliferative response to Candida antigen. Next generation sequencing revealed a variant of uncertain significance in STAT1 (c.295A>G; p.Ile99Val) and did not detect TLR3 or UNC93B1 mutations. ConclusionThe clinical phenotype caused by STAT1 mutations is highly diverse, and there is overlap between GOF and LOF phenotypes, making it difficult to clinically diagnose. Although HSV gingivostomatitis has been reported in a patient with STAT1 GOF, invasive disease, such as meningoencephalitis, has not been reported in those with variants in STAT1.