Somatic STAT3 Mutations in CD8+ T Cells of HTLV-2 Positive Blood Donors

Introduction: T-cell large granular lymphocyte (T-LGL) leukemia is a rare lymphoproliferative disorder with recurrent somatic STAT3 mutations. It has been suggested that viral antigens act as the initial stimuli resulting in clonal expansion of CD8+ cells in the disease. However, less is known whether chronic exposure to viral antigens is associated with acquisition of somatic mutations in CD8+ T cells among individuals without clinically detectable lymphoproliferations. Human T-cell leukemia virus type 2 (HTLV-2) antibody positivity has been detected in patients with T-LGL leukemia. Here, we examined whether CD8+ T cells from HTLV-2 positive healthy blood donors harbor somatic mutations in STAT3 or other immune-associated genes, potentially identifying individuals at risk of subsequent lymphoproliferative diseases. Methods: We analyzed HTLV-2 infected (n=30) and uninfected (n=35) healthy blood donor samples obtained from University of California San Francisco and Vitalant Research Institute, which were enrolled in the United States-based HTLV Outcomes Study (HOST) cohort. All cases had serologic evaluation for HTLV-2 infection at the time of sampling. We examined somatic mutations of STAT3 in CD4+ and CD8+ T-cell populations using ultra-deep targeted amplicon sequencing. In addition, we applied a custom sequencing panel covering the coding regions of 2,533 immune-related genes to characterize a larger spectrum of somatic mutations in CD8+ T cells. Results: Somatic STAT3 mutations were detected in CD8+ but not in CD4+ T cells of four (13.3%, 4/30) HTLV-2 positive healthy blood donors (p.Y640F, p.N647I, p.D661Y, and p.Y657_K658insY with variant allele frequencies of 11.9%, 0.5%, 4.9%, and 1.2%, respectively) using amplicon sequencing. The detected STAT3 mutations have been previously described and reported in T-LGL leukemia. Total white blood cell and lymphocyte counts were similar between STAT3 mutated and non-mutated cases. No STAT3 mutations were discovered in HTLV-2 negative donors with amplicon sequencing. Of the 28 HTLV-2 positive cases, 19 had at least one somatic variant in CD8+ T cells based on the immunogene panel sequencing (n=28). 8 cases had variants in genes previously identified in T-LGLL (STAT3, KMT2D, TYRO3, DIDO1, BCL11B, CACNB2, KRAS, LRBA and FANCA), and 5 cases had variants in genes involved in JAK-STAT signaling (NFKBIA, PIK3R5, MAPK14, EP300, MPL, IFNAR1, IL6ST and IL20RA). Three recurrently mutated genes were detected: VWF, SMAD7 and MXRA5. The four HTLV-2 positive blood donors harboring STAT3 mutations had more somatic mutations (median=6) than HTLV-2 positive donors without STAT3 mutations (median=1, p=0.06). Conclusion: We report the presence of somatic gain-of-function STAT3 mutations in CD8+ T cells of 13% of HTLV-2 positive healthy blood donors. We identified additional somatic mutations in genes associated with JAK-STAT signaling, immune regulation and lymphoproliferation in CD8+ T cells of HTLV-2 positive cases. While STAT3 activation, with or without mutations, is considered as a hallmark of T-LGLL, our results reveal the presence of STAT3 mutations in CD8+ T cells of healthy blood donors harboring HTLV-2 without clinical history of lymphoproliferative disease. Additional research is warranted to elucidate whether HTLV-2 carriers harboring STAT3 and other mutations are at increased risk of subsequent T-LGL leukemia or other lymphoproliferative diseases. Disclosures Mustjoki: Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding; Janpix: Research Funding.

Defective Toll-Like Receptors Driven B Cell Response in Hyper IgE Syndrome Patients With STAT3 Mutations

Autosomal dominant hyper-IgE syndrome (AD-HIES) is a rare inherited primary immunodeficient disease (PIDs), which is caused by STAT3 gene mutations. Previous studies indicated a defective Toll-like receptor (TLR) 9-induced B cell response in AD-HIES patients, including proliferation, and IgG production. However, the other TLRs-mediated B cell responses in AD-HIES patients were not fully elucidated. In this study, we systematically studied the B cell response to TLRs signaling pathways in AD-HIES patients, including proliferation, activation, apoptosis, cytokine, and immunoglobulin production. Our results showed that the TLRs-induced B cell proliferation and activation was significantly impaired in AD-HIES patients. Besides, AD-HIES patients had defects in TLRs-induced B cell class switch, as well as IgG/IgM secretion and IL-10 production in B cells. Taken together, we first systematically reported the deficiency of TLRs driven B cell response in AD-HIES patients, which help to have a better understanding of the pathology of AD-HIES.

Immunosuppressive Therapy in Large Granular Lymphocyte Leukemia-Associated Pure Red Cell Aplasia: Cyclophosphamide Responded Better Than Cyclosporine

Background Large granular lymphocyte leukemia associated pure red cell aplasia (LGLL-PRCA) accounts for a significant portion of secondary PRCA. Cyclosporine (CsA) and cyclophosphamide (CTX) are the main immunosuppressive agents used in treating LGLL-PRCA [1]. Considering the cytotoxicity of CTX, CsA may be proposed as first-line therapy [2]. However, because of the rarity of LGLL-PRCA, long-term responses and relapse rates after CsA and CTX therapy are largely unknown. Methods and results From September 2009 to December 2020, we selected 65 uniformly diagnosed LGLL-PRCA and analyzed clinical features and treatment outcomes of CsA and CTX. In the present study, 43.1% harbored neutropenia (<1.5×10 9/L) and only 1 patient had spondyloarthritis (Table 1). Besides, we found that 9.2% developed recurrent oral ulcer and 18.5% had reduced serum complemet C3 level, both of which were related to abnormal immune status. In our cohort, 44 patients received CsA therapy and 21 patients received CTX therapy. 53.8% (35/65) obtained erythroid lineage response and 26.2% (17/65) achieved complete response. CTX produced higher response rate (81.0% vs 40.9%, P=0.002) and complete response rate (47.6% vs 15.9%, P=0.007) than CsA. We further analyzed related factors influencing efficacy by Binary-Logistic multivariate regression model and found that higher response rate was mainly related to CTX therapy (P=0.02) (Table 2). We detected STAT3 and STAT5b gene in 50 cases. None of the patients had STAT5b mutation and 14 patients had STAT3 mutation. 12 of 14 mutation cases were non-elderly patients (<60 years ). For younger patients, STAT3 mutation was more frequent (85.7% vs 22.2%, P<0.01). In STAT3 mutation group, patients appeared to respond better to CTX than CsA (83.3% vs 37.5%, P=0.138). Up to the last follow-up, 11 patients treated with CsA recurred after CsA reduction or discontinuation, with a median relapse time of 10 (3~80) months after remission. Only 1 patient relpsed after the discontinuation of CTX due to neutropenia and returned to remission status after CTX retreatment. In our research, CsA had higher recurrence rate than CTX without statistical significance (25.0% vs 4.8%, P=0.104). Discussion CsA may be proposed as first-line therapy for LGLL-PRCA [2,3]. CTX also appears to be a good treatment choice, but CTX should not be used for more than 12 months since associated toxicities and the risk for developing myelodysplastic syndromes and acute myeloid leukemia. Therefore, it is necessary to compare the efficacy of CsA or CTX in the treatment of LGLL-PRCA. Deep sequencing analyses of residuals LGLL clones reveals that CTX could eradicate LGLL clones, providing durable response, low relapse rate, whereas CsA is associated with the persistence of leukemic clones, and high frequency of relapse [4]. Our results show that the response rate of CTX is higher than CsA (P=0.02), and the probability of recurrence is relatively low (25.0% vs 4.8%, P=0.104). This is consistent with the results of previous study by Rajala [4]. It was important to note that patients with STAT3 mutations are more likely to respond to MTX [5]. Our study showed that the response rate for CTX was 83.3% in patients with STAT3 mutations, which was seemingly higher than CsA (37.5%), although remained statistically insignificant (P=0.138) that might be due to small number of cases. Conclusions The results of the current study may reflect the real world experience of LGLL-PRCA in whom treated by CTX or CsA, may be limited by its retrospective nature, small cohorts. In preliminary conclusion, LGLL-PRCA could acquired better response to CTX than CsA. Besides, CTX may reduce relapse. References 1. Means RT Jr. Pure red cell aplasia. Blood, 2016, 128(21): 2504~9. 2. Moignet A, Lamy T. Latest Advances in the Diagnosis and Treatment of Large Granular Lymphocytic Leukemia. Am Soc Clin Oncol Educ Book, 2018, 38: 616~25. 3. Go RS, Tefferi A, Li CY, et al. Lymphoproliferative disease of granular T lymphocytes presenting as aplastic anemia. Blood, 2000, 96(10):3644~6. 4. Rajala HLM, Olson T, Clemente MJ, et al. The analysis of clonal diversity and therapy responses using STAT3 mutations as a molecular marker in large granular lymphocytic leukemia. Haematologica, 2015, 100: 91~9. 5. Loughran TP, Zickl L, Olson TL, et al. Immunosuppressive therapy of LGL leukemia: prospective multicenter phase II study by the Eastern Cooperative Oncology Group (E5998). Leukemia, 2015, 29(4): 886~94. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Circular RNA Dysregulation Characterizes Symptomatic T-LGL Leukemia Patients with STAT3 Mutation

T-cell large granular lymphocyte leukemia (T-LGLL) is a rare disease characterized by clonal proliferation of CD3+ lymphocytes whose pathogenesis is not completely understood. LGL resistance to the activation-induced cell death is sustained by anti-apoptotic pathways, mainly due to constitutive activation of the JAK-STAT axis by activating somatic mutations of STAT3 or STAT5B genes or epigenetic disturbances. STAT3 mutations are peculiar to CD8+ T-LGLL and are associated with cytopenias, treatment requirement, and reduced survival. STAT5B lesions are mostly found in CD4+ T-LGLL, a disorder presenting with a mild clinical course. A similar behaviour has been observed also in both STAT wild type (WT) CD8+ and CD4+ forms. The link between STAT3-dependent miR-146b repression and neutropenia exemplifies the role that the pathogenetic effect of somatic mutations can exert through complex axes, also involving non-coding RNAs. Since in the most symptomatic form of LGLL an urgent need to discover new mechanisms in place is demanded, we focused on additional regulatory molecules such as circular RNAs (circRNAs), single-stranded covalently closed RNA molecules generated by backsplicing (Figure 1A). CircRNAs' oncogenic potential is linked to their activity as miRNA sponges, modulators of RNA-binding protein activity, and their ability to encode oncogenic peptides. The study group includes 20 patients recruited at the Hematology Unit of Padua University Hospital (Italy) and diagnosed with T-LGLL according to the WHO criteria, plus 5 healthy controls (CTR). Patient leukemic clones were equally distributed among CD8+ WT and STAT3-mutated and CD4+ WT and STAT5B-mutated. RNA-seq of ribodepleted RNA from immunomagnetically purified CD57+ leukemic clones and CD8+ cells from CTR was obtained to study circRNAs. CircRNAs were identified and quantified from RNA-seq data by CirComPara v0.6.3. Differential expression was assessed by edgeR with robust estimation of dispersion. The circular to linear proportion (CLP), indicating the relative abundance of each circRNA with respect to all the overlapping host gene transcripts, was calculated, and differential CLP across conditions was assessed with CircTest. We detected 68.619 circRNAs and focused on 5.948 with high expression. These were expressed from 2,940 loci, mostly from annotated genes (99.95%). We identified 28 circRNAs derived from genomic regions without known genes. About one-half of circRNA host genes expressed multiple, up to 20, circular isoforms. About 4% of circRNAs had a CLP of at least 0.25, with 95 circRNAs more expressed than the linear counterpart, including circGUSBP2, circSHOC1, circTCEANC, circZNF609, and circKLHL8. Unsupervised analysis of circRNA expression showed a clear separation of T-LGLL from CTR samples, pointing toward circRNA dysregulation in T-LGLL, particularly for the CD8+ STAT3 mutated group, which diverged from the other subtypes (Figure 1B). The direct comparison between the CD8+ STAT3-mutated, the three molecular subtypes as a single group (OTH), and the CTR samples identified differences in circRNA expression specifically linked to the most symptomatic group with STAT3 lesions and those commonly observed in T-LGLL. In STAT3-mutated, 500 circRNAs were dysregulated with an excess (71%) of upregulation compared to CTR (Figure 1C). Most circRNAs commonly altered in both LGLL groups had a more marked dysregulation in patients with STAT3 mutations, as observed for circIKZF2 and a newly discovered intergenic circRNA. Most circRNAs with a significantly varied CLP (96%) showed an increased expression of the circular than linear transcripts in T-LGLL compared to CTR, uncovering genes with an imbalance of circular to linear transcripts proportion in leukemic cells (Figure 1D). The ongoing validation of the differential circRNA expression in an extended cohort of 40 LGLL patients is prioritizing circRNAs for functional investigation. The integration of computational predictions and in vitro functional screening through specific circRNA silencing will attempt to understand the link between circRNAs dysregulated in T-LGLL and disease mechanisms, unveiling possible circRNA-involving axes governed by hyperactivating STAT3 somatic variants that contribute to the malignant LGL phenotypes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Neutropenia and Large Granular Lymphocyte Leukemia: From Pathogenesis to Therapeutic Options

Large granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL or NK cells. Chronic isolated neutropenia represents the clinical hallmark of the disease, being present in up to 80% of cases. New advances were made in the biological characterization of neutropenia in these patients, in particular STAT3 mutations and a discrete immunophenotype are now recognized as relevant features. Nevertheless, the etiology of LGLL-related neutropenia is not completely elucidated and several mechanisms, including humoral abnormalities, bone marrow infiltration/substitution and cell-mediated cytotoxicity might cooperate to its pathogenesis. As a consequence of the multifactorial nature of LGLL-related neutropenia, a targeted therapeutic approach for neutropenic patients has not been developed yet; moreover, specific guidelines based on prospective trials are still lacking, thus making the treatment of this disorder a complex and challenging task. Immunosuppressive therapy represents the current, although poorly effective, therapeutic strategy. The recent identification of a STAT3-mediated miR-146b down-regulation in neutropenic T-LGLL patients emphasized the pathogenetic role of STAT3 activation in neutropenia development. Accordingly, JAK/STAT3 axis inhibition and miR-146b restoration might represent tempting strategies and should be prospectively evaluated for the treatment of neutropenic LGLL patients.

T-Cell Large Granular Lymphocytic Leukemia with Atypical Immunophenotypes: A Single Center Retrospective Analysis of 17 Cases

Purpose T-cell large granular lymphocytic leukemia (T-LGLL) is characterized by expansion of cytotoxic T cells expressing αβ T cell receptor (TCR), CD2, surface CD3, CD8, CD57 as well as cytotoxic molecules. Atypical immunophenotypes of T-LGLL, including γδ TCR and CD4, reported in a small subset, remains to be well-defined. Methods We retrospectively analyzed immunophotypes and clinicopathologic features of 96 T-LGLL cases. Results We found a total of 17 cases with atypical immunophenotypes including 9 TCRγδ + cases and 8 CD4+ TCRαβ + cases. Pure red cell aplasia was less common in atypical immunophenotypes patients compared to that of typical immunophenotypes [0/17 (0%) vs. 26/79 (32.9%), p=0.005]. STAT3 mutations were also less frequent in atypical immunophenotypes cases, although accompanied with marginal significance (p=0.086). Conclusion Patients with atypical immunophenotypes showed a similar survival outcome to that of typical T-LGLL immunophenotypes. Additional efforts were needed to better understand the pathogenesis of these rare atypical immunophenotypes T-LGLL cases.

ALK-Negative Anaplastic Large Cell Lymphoma: Current Concepts and Molecular Pathogenesis of a Heterogeneous Group of Large T-Cell Lymphomas

Anaplastic large cell lymphoma (ALCL) is a subtype of CD30+ large T-cell lymphoma (TCL) that comprises ~2% of all adult non-Hodgkin lymphomas. Based on the presence/absence of the rearrangement and expression of anaplastic lymphoma kinase (ALK), ALCL is divided into ALK+ and ALK-, and both differ clinically and prognostically. This review focuses on the historical points, clinical features, histopathology, differential diagnosis, and relevant cytogenetic and molecular alterations of ALK- ALCL and its subtypes: systemic, primary cutaneous (pc-ALCL), and breast implant-associated (BIA-ALCL). Recent studies have identified recurrent genetic alterations in this TCL. In systemic ALK- ALCL, rearrangements in DUSP22 and TP63 are detected in 30% and 8% of cases, respectively, while the remaining cases are negative for these rearrangements. A similar distribution of these rearrangements is seen in pc-ALCL, whereas none have been detected in BIA-ALCL. Additionally, systemic ALK- ALCL—apart from DUSP22-rearranged cases—harbors JAK1 and/or STAT3 mutations that result in the activation of the JAK/STAT signaling pathway. The JAK1/3 and STAT3 mutations have also been identified in BIA-ALCL but not in pc-ALCL. Although the pathogenesis of these alterations is not fully understood, most of them have prognostic value and open the door to the use of potential targeted therapies for this subtype of TCL.

Clinicobiological Characteristics and Outcomes of Patients with T-Cell Large Granular Lymphocytic Leukemia and Chronic Lymphoproliferative Disorder of Natural Killer Cells from a Single Institution

T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorder of natural killer (NK) cells are two infrequent diseases characterized by clonal expansions of cytotoxic T lymphocytes and NK cells, respectively. Somatic mutations of STAT3 are involved in the pathogenesis of these entities. We describe the clinicobiological features, mutational status of STAT3/STAT5B, treatment and outcome of 131 patients. Neutropenia was the most frequent finding at diagnosis, followed by anemia. Concurrent hematological disorders were diagnosed in 37% of patients and autoimmune conditions and solid tumors in 17% and 15%, respectively. All patients who needed treatment belonged to the CD8+CD57+ group. Remarkably, patients included in the CD4+ group had a higher association with solid tumors (p = 0.037). STAT3 mutations were found in 17% of patients, mainly Y640F and D661Y mutations. Patients carrying STAT3 mutations more frequently presented with anemia, neutropenia, high LDH, high large granular lymphocyte counts and need for treatment (p = 0.0037). Methotrexate was the most frequently used agent (72% of cases). The overall response rate to all treatments was 50%. The 10-year overall survival of this series was 78%, with no differences according to the mutational status of STAT3. We compared the survival of these patients with the general Spanish population and no differences were found, confirming the indolent nature of these hematological malignancies. Our study further extends findings documented by others on the clinical behavior of the disease and the impact of STAT3, and for the first time analyzes survival compared to a matched general Spanish population.

Linking the KIR phenotype with STAT3 and TET2 mutations to identify chronic lymphoproliferative disorders of NK cells

Distinguishing chronic lymphoproliferative disorders of NK cells (CLPD-NK) from reactive NK cell expansions is challenging. We assessed the value of NK receptor phenotyping and targeted high-throughput sequencing in a cohort of 114 consecutive patients with NK cell proliferation, retrospectively assigned to a CLPD-NK group (N=46) and a reactive NK group (N=68). We then developed a NK-clonality score combining flow cytometry and molecular profiling with a positive predictive value of 93%. STAT3 and TET2 mutations were respectively identified in 27% and 34% of the CLPD-NK patients - constituting a new diagnostic hallmark for this disease. TET2-mutated CLPD-NK exhibited preferentially a CD16low phenotype, displayed more frequently a lower platelet count, and were associated with other hematologic malignancies such as myelodysplasia. To explore the mutational clonal hierarchy of CLPD-NK, we performed a whole exome sequencing of sorted, myeloid, T, and NK cells and identified that TET2 mutations were shared by myeloid and NK cells in 3 out of 4 cases. Thus, we hypothesized that TET2 alterations occur early in CLPD-NK disease which could explain a potential link between NK-LGL leukemia and other myeloid malignancies. Finally, we analyzed the transcriptome by RNA-seq of 7 CLPD-NK and evidenced two groups of patients. The first group displayed STAT3 mutations or SOCS3 methylation and overexpressed STAT3 target genes. The second group, including two TET2-mutated cases, significantly under-expressed genes known to be down-regulated in angioimmunoblastic T-cell lymphoma. Our results provide new insights into the pathogenesis of NK cell proliferative disorders and potentially new therapeutic opportunities.

The Multifaced Role of STAT3 in Cancer and Its Implication for Anticancer Therapy

Signal transducer and activator of transcription (STAT) 3 is one of the most complex regulators of transcription. Constitutive activation of STAT3 has been reported in many types of tumors and depends on mechanisms such as hyperactivation of receptors for pro-oncogenic cytokines and growth factors, loss of negative regulation, and excessive cytokine stimulation. In contrast, somatic STAT3 mutations are less frequent in cancer. Several oncogenic targets of STAT3 have been recently identified such as c-myc, c-Jun, PLK-1, Pim1/2, Bcl-2, VEGF, bFGF, and Cten, and inhibitors of STAT3 have been developed for cancer prevention and treatment. However, despite the oncogenic role of STAT3 having been widely demonstrated, an increasing amount of data indicate that STAT3 functions are multifaced and not easy to classify. In fact, the specific cellular role of STAT3 seems to be determined by the integration of multiple signals, by the oncogenic environment, and by the alternative splicing into two distinct isoforms, STAT3α and STAT3β. On the basis of these different conditions, STAT3 can act both as a potent tumor promoter or tumor suppressor factor. This implies that the therapies based on STAT3 modulators should be performed considering the pleiotropic functions of this transcription factor and tailored to the specific tumor type.