Large Granular Lymphocyte Leukemia Research Articles

DR-01, a Non-Fucosylated Anti-CD94 Antibody, Depletes Leukemic Cells in Ex Vivo and In Vivo Models of Large Granular Lymphocyte Leukemia

Large granular lymphocyte leukemia (LGLL) is a rare leukemia more frequently occurring in the elderly population with three major subtypes: T-LGLL, chronic lymphoproliferative disorder of NK cells (CLPD-NK) and aggressive NK cell leukemia (ANKL). Over 50% of patients develop complications including severe neutropenia, thrombocytopenia and anemia, thus predisposing patients to opportunistic infections. In addition, treatments are limited to standard immunosuppressive therapy such as methotrexate, cyclosporine or cyclophosphamide, with complete response rates of only 50% or less for each agent. Thus, novel and effective therapies for LGLL patients are critically needed. Dren Bio is developing DR-01, a non-ligand blocking, non-fucosylated monoclonal antibody targeting CD94. CD94 is a type II transmembrane receptor identified on mature, cytotoxic lymphocytes such as healthy NK and terminally differentiated CD8 T cells. In the context of disease, CD94 is known to be upregulated on LGLL cells. DR-01 functions through depletion of target cells via antibody-mediated cellular cytotoxicity (ADCC) by means of fratricide, a method in which the same cell type induces ADCC on each other. Through characterization of DR-01 using various ELISA and cell-based assays, DR-01 was determined to bind with nanomolar affinity to CD16a (EC50: 0.04µg/mL) and to CD94 on healthy human donor NK cells, cynomolgus monkey NK cells and LGLL cells (EC50: 0.5-1nM). DR-01 induces potent, dose-dependent depletion of CD94+ cells in ex vivo ADCC assays (IC50: 1ng/mL) of peripheral blood mononuclear cells (PBMCs) from healthy and diseased individuals. Furthermore, DR-01 has been shown to be well tolerated in a 4-week dose-range finding and Good Laboratory Practice (GLP) study in cynomolgus monkeys from 5-100mg/kg on a weekly dosing regimen. A 12-color flow cytometry panel was applied to a cohort of patient LGLL PBMCs to profile CD94 expression and other cytotoxic markers associated with the disease, including CD16, CD57 and functional binding partners of CD94 such as NKG2A and NKG2C. CD94 was identified on 97% (37/38) of patient samples screened, ranging from 400 to 725,000 receptors per cell. All patient leukemic cells expressed variable levels of CD16 and CD57. NKG2A and NKG2C were present in over 5% of total leukemic cells in 37% (14/38) and 21% (8/38) of patient samples, respectively. In addition, DR-01 was able to deplete LGL leukemic cells with high potency and in a dose-dependent manner in a functional ADCC assay (IC50:1-10ng/mL). The mechanism of fratricide was demonstrated through degranulation of the leukemic population via expression of CD107a. Cytokine profiling results are pending. To demonstrate in vivo efficacy of DR-01 in a disease model of LGLL, humanized NSG-Tg(hIL-15) mice engrafted with patient LGLL splenocytes were utilized. Optimal splenocyte engraftment (5-20% human CD45+ cells in blood) was achieved within 3 weeks post-inoculation with 10 million PBMCs per pre-sublethally irradiated mouse. The engrafted leukemic population partially preserved the CD94 expression profile from the patient leukemic cells and was successfully depleted (>90%) in blood, bone marrow, spleen and liver 1-week post-treatment with a single dose of DR-01 at 5mg/kg. Overall, DR-01 is a non-fucosylated anti-CD94 antibody with high affinity and potent leukemic cell depleting ability ex vivo and in vivo. The favorable toxicity profile of DR-01 in cynomolgus monkeys precede clinical studies in LGLL patients. Additional profiling and evaluation of DR-01 in LGLL patient samples ex vivo as part of a pre-phase I study is ongoing. A phase I/II trial has been initiated to assess the safety and efficacy of DR-01 in previously treated LGLL patients.

Efficacy of Short-Duration Alemtuzumab in T-Cell Large Granular Lymphocytic Leukemia: Potential for a Response-Adapted Strategy

Introduction T-cell large granular lymphocytic leukemia (T-LGL) is a chronic incurable malignancy where once refractory to immunosuppressive therapy, few treatment options and no optimal treatment consensus exists. Alemtuzumab, 10 milligrams (mg) intravenously (IV) administered daily for a total of 10 doses, has been shown to be effective for relapsed or refractory (R/R) T-LGL (Dumitriu, et al. Lancet Haematol 2016); however, it can be associated with severe infusion reactions and infections. Given the palliative nature of T-LGL-directed treatments and the increasing potential for toxicity with cumulative alemtuzumab exposure, we conducted a retrospective analysis to describe the efficacy and safety of abbreviated alemtuzumab regimens in R/R T-LGL patients. Methods We identified patients ≥18 years with R/R T-LGL who were treated with short-duration (defined as ≤ 5 doses) alemtuzumab between 2012 and 2022 from the University of Washington lymphoma database. Patients who received prior alemtuzumab were excluded. The primary endpoint was overall hematologic response rate (ORR). Secondary efficacy endpoints included hematologic response rate at 3 and 6 months, rate of transfusion independence, progression-free and overall survival. Safety endpoints included rate of infusion reactions and infections, specifically Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reactivation. Results Nine patients meeting criteria were identified (Table 1). Five patients received alemtuzumab 10 mg IV daily for 5 doses and 4 patients received alemtuzumab 10 mg IV weekly for 4 doses. Median age was 65 (range 34-81) years and the median number of prior regimens was 3 (range 1-5). All (100%) patients were refractory to immunosuppressive therapy. Seven (78%), 1 (11%) and 1 (11%) patients had unilineage, bilineage and trilineage cytopenias, respectively. Five patients (56%) had autoimmune comorbidities and five (56%) required periodic transfusions. No patients had myelodysplasia on bone marrow biopsy. Overall, 6 (67%) patients had a hematologic response with 2 patients achieving a partial response (PR) and 4 patients achieving a complete response (CR). ORR at 3 and 6 months was 56% and 44%, respectively. Two (40%) patients achieved transfusion independence. The 3 patients without a hematologic response were retreated with alemtuzumab 10 mg IV daily for 10 doses with 1 subsequently achieving a CR. At a median follow-up time of 7.2 (range 2.3-36.6) months, 3 out of 6 patients who achieved a response had disease relapse; 8 (89%) patients were alive and 1 had died from COVID-19 pneumonia. Among the 5 patients who received alemtuzumab at 10 mg daily for 5 doses, ORR was 40% with 1 patient achieving PR and 1 patient achieving CR. ORR at 3 and 6 months was 40% and 40%, respectively. One (20%) patient had an infusion reaction. Two patients (40%) had subclinical CMV viremia which resolved with valganciclovir. No cases of EBV viremia were noted. Four patients completed alemtuzumab at 10 mg weekly for 4 doses; ORR was 100% with 1 patient achieving PR and 3 achieving CR. Hematologic response rate at 3 and 6 months was 75% and 50%, respectively. Three (75%) patients relapsed at 3, 4 and 14.2 months, respectively; 2 were retreated 4-6 times with alemtuzumab 10 mg IV weekly for 2 to 6 doses each time, with both achieving hematologic responses with each course of treatment. One (25%) patient experienced an infusion reaction. No cases of CMV or EBV viremia were noted. Conclusions Short-duration alemtuzumab is effective and well tolerated in R/R T-LGL with responses observed in 67% overall and no cases of CMV viremia detected in the weekly dosing group. Alemtuzumab given at 10 mg IV weekly for 4 doses or until a response is noted followed by retreatment upon relapse was particularly effective; this abbreviated, response-adapted alemtuzumab strategy should be prospectively explored in this patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Splenectomy for Immune Cytopenias: Treatment Outcomes and Predictors of Response

The pathogenesis of immune cytopenias involves diverse processes including production, inhibition, and destruction of specific blood cell types mediated by antibodies and/or T cells. Classic examples of immune-mediated cytopenias are immune thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA), and autoimmune neutropenia (AIN) as well as T cell-mediated idiopathic neutropenia and pure red cell aplasia. Both antibody and autoimmune T cell responses may often coexist, and assignment of the proper pathogenic importance, albeit difficult, is crucial for rational therapeutic approaches. Clonal processes such as T-cell large granular lymphocytic leukemia (T-LGL) or various B cell dyscrasias may also occur. While the results of splenectomy have been studied in individual conditions such as Evans syndrome (ES), AIN, and AIHA, the availability of new therapies and improvement of surgery outcomes have provided an impetus to update the older and smaller series. We retrospectively reviewed a cohort of 339 adult patients who underwent splenectomy at the Cleveland Clinic Foundation between 2002-2020 (Fig.1A), for whom the main indication for the procedure was: ITP (n= 235), AIHA (n=68), ES (n=26) and AIN (n=10). Among these subtypes, there were 15 patients with an established diagnosis of T-LGL. Our aim was to determine the hematologic response of patients who receive splenectomy for immune cytopenia following treatment failure with conventional therapies and identify factors that were associated with responses. Overall, the median age of the patients was 55 years (IQR:68-36), M:F ratio=0.98, with a median time from diagnosis to splenectomy of 11 months (IQR:31-3). Prior to the procedure, all patients had failed multiple lines of medical therapies (mode 1, range1-4). The median preoperative platelet count was 19 (IQR:44-5) x109/L, median preoperative hemoglobin (Hb) of 9 (IQR:10-8) g/dl and median preoperative ANC of 0.28 (IQR:1.19-0.23) x109/L. A total of 306 patients underwent laparoscopic splenectomy, while 33 patients had an open approach. In 16 cases (5%), we recorded an intraoperative conversion from laparoscopic to open splenectomy. The mean spleen weight was 477 gr (SD±687). The average operative time was 2.3 hours, with four days median hospital length of stay (range:1-50). The overall rate of postoperative complications with 30-day readmissions was 0.1%, with no mortality. Of note is that up to 20% of the cases had a post-operative diagnosis that was discordant with the original indication for splenectomy. These discordant diagnoses included benign conditions such as Felty's syndrome (n=2), as well as hematologic malignancies such as mature B-cell neoplasm (28), T-cell lymphoma (n=8), NK-LGL (n=1), and T-LGL (n=15). The overall response rate (ORR) to splenectomy was 74%, of which 86% complete response (CR) and 14% partial response (PR) with relapse occurring in 12% of cases. In the subset of thrombocytopenic patients, CR reached 69% of patients (P<.0001) as early as one week following the procedure. At 1-year, the mean platelet count was 246 (SD±170) x 109/L and relapse occurred in 13% of patients. Conversely, in anemic patients, a CR was observed only in 55% (P<.0001) within two weeks following splenectomy. At 1-year, the mean Hb level was 11.7 (SD±1.3) g/dl, with relapse occurring in 6% of patients. Comparatively, 80% of patients who underwent splenectomy for neutropenia responded at 26 weeks post-splenectomy with relapse in 10% of cases. In known and newly diagnosed patients with LGL (n=16), the response with regard to the cytopenia resulting in splenectomy indication was 81%. The response in Felty's syndrome was 100%. When analyzing predictors of response, failed pharmacologic therapies with either intravenous immunoglobulin (IVIg) or rituximab was predictive of non-response to splenectomy (Fig.1B). No significant differences in spleen weight between responders and non-responders were observed. Our results show a significant and durable response to splenectomy in immune-mediated cytopenia of various etiologies refractory to conventional therapies indicating that, in selected patients, splenectomy remains a rational salvage treatment with a low complication rate. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Outcomes of Patients Referred for Asymptomatic Neutropenia at a Single Academic Center: A Focus on Racial Disparities

Introduction Asymptomatic neutropenia is a common referral to Hematology. Differentiating between a normal variant and an underlying hematologic disorder is further complicated by discrepancies in blood cell count reference ranges that differ among institutions. In the following study, we aim to describe the demographic characteristics and laboratory and clinical outcomes of patients referred for asymptomatic neutropenia to an academic classical hematology practice. Methods In this retrospective cohort study, we included all adult patients referred for evaluation of asymptomatic neutropenia to the Classical Hematology Clinic at Oregon Health & Science University from 2018 through 2021. Asymptomatic neutropenia was defined as an absolute neutrophil count (ANC) below the internal reference range of 1.8 x 109/L, without a history of abnormal infection. Patients who had other cytopenias were included in the study as long as the primary referral reason was asymptomatic neutropenia. Patient demographics, pertinent medical history, baseline laboratory values, diagnostic testing and results, and subsequent clinical diagnoses were obtained through individual chart review. A clinically significant outcome was defined as a newly diagnosed hematologic disorder, including both pre-malignant and malignant conditions. Standard descriptive statistics were used to summarize baseline and outcome characteristics. Results We identified 102 patients who were referred over the 3-year period and who met the study inclusion criteria. The median age was 56 years (range: 19 - 96), 10% of patients identified as Asian, 18% as Black, and 68% as White (Table 1), which was disproportionate to local population demographics in Portland Oregon (9% Asian, 6% Black, and 75% White). The average ANC at referral was 1.0 x 109/L. 55% of patients had an ANC between 1.8 x 109/L - 1.0 x 109/L, 34% had an ANC between 1.0 x 109/L - 0.5 x 109/L and 11% had an ANC less than 0.5 x 109/L. The average number of tests performed at referral, including repeat complete blood cell count with differential, was 6. Bone marrow biopsies were obtained in 15 patients who had an average ANC 0.6 x 109/L (standard deviation 0.46). The most common clinical outcome was idiopathic persistent neutropenia in 50% of patients, with resolution of neutropenia in occurring in 28% of patients. Among patients with an ANC > 1.0 x 109/L, only 1 patient was found to have a clinically relevant underlying disorder (HIV infection). In total, twenty-three percent of patients were found to have a clinically significant outcome with mean ANC of 0.65 x 109/L: Myelodysplastic syndrome (n=6), Immune thrombocytopenic purpura (n=4), Large granular lymphocytic leukemia (LGL) (n=3), Chronic lymphocytic leukemia (n=2), monoclonal gammopathy of undetermined significance (MGUS) (n=2), Hairy cell leukemia (n=1), B cell lymphoma (n=1), Multiple myeloma (n=1), and Acute Myeloid Leukemia (n=1). Only two of these patients with clinically significant findings (MGUS and LGL leukemia) were Black. Among Black patients who underwent testing for Duffy antigens, nearly 100% of patients were positive for the Duffy null phenotype [Fy(a-b-)], though one patient was [Fy(a-b+)]. In Non-Black patients tested, only 25% were positive for this phenotype. Conclusion Among patients referred to our institution for asymptomatic neutropenia, none of the patients with ANC > 1.0 x 109/L had an underlying hematologic disorder detectable by standard peripheral blood neutropenia workup. Black individuals were disproportionately represented, more likely to harbor the Duffy null phenotype, and less likely to have a clinically significant outcome. Our findings elucidate the need for standardized reference ranges that include demographic variants and are more representative of the community they serve, as current thresholds may inadvertently flag benign as abnormal, leading to significant emotional and financial implications, as well as inequitable treatment decision-making. Abbreviations: ANC, absolute neutrophil count; CBC, complete blood cell count; MDS, Myelodysplastic syndrome; ITP, Immune thrombocytopenic purpura; LGL, Large granular lymphocytic leukemia; CLL, Chronic lymphocytic leukemia; MGUS, monoclonal gammopathy of undetermined significance; AML, Acute myeloid leukemia Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Incidence, Clinical Associations, and Co-Mutation Patterns of UBA1 Mutations in MDS

Background Mutations in UBA1 are associated with VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, an adult-onset inflammatory disorder (Beck DB et al. NEJM 2020). Approximately 40% of VEXAS patients are also diagnosed with myelodysplastic syndromes (MDS). However, the prevalence and characteristics of UBA1 mutations in MDS are unknown. We hypothesize that UBA1 mutations underlie a proportion of MDS cases with systemic inflammation. Methods Molecular data were reviewed from a representative diagnostic and treatment-naive MDS cohort (n= 3,328) ascertained through the International Working Group for MDS (Bernard E et al NEJM Evid 2022). We prioritized 375 cases for UBA1 genotyping based on: 1) male gender; and 2) no identified driver mutations or few mutations, enriched in DTA (DNMT3A, TET2, ASXL1) genes; or 3) unclassifiable (MDS-U or MDS/MPN-U) per 2016 WHO Classification. We identified UBA1 hotspot mutations (M41T/V/L) with digital droplet PCR. Resulting UBA1 status was integrated with cytogenetic and sequencing data from 121 genes involved in myeloid pathogenesis to identify patterns of co-mutation. Clinical associations of UBA1 mutations were evaluated and where available, clinical history was reviewed for inflammatory conditions. Results We identified 30 UBA1 M41T/V/L mutations (median number mutant droplets: 1301; range: 4-12166) in 8% of the cohort (n=28 of 375 patients). This included 15 patients with M41T (c.122T>C), 7 with M41V (c.121A>G), 4 with M41L (c.121A>C), and 2 patients with more than one mutation (1 with M41V/T, and 1 with M41L/V). In 79% of UBA1-mutant cases (22/28), the karyotype was normal. Loss of the Y chromosome was observed in 6 cases. Three cases had MDS-associated cytogenetic abnormalities: del(11q), del(13q) and focal del(20q). In 43% of UBA1-mutant cases (12/28), UBA1 mutations were the sole genetic abnormalities. For a further 43% we observed co-mutation with DTA genes (DNMT3A, TET2, ASXL1) with median variant allele fraction (VAF) of 0.21 (0.02-0.56). SF3B1 co-mutations with VAF >30% were observed in 3 cases. Most UBA1-mutant cases were classified per 2016 WHO as MDS-SLD/MLD (n=18), but two cases were MDS with excess blasts 1, and one case was CMML-1 with 9% blasts. The median age at diagnosis was 72 years (44-89). No significant difference was found when comparing clinical parameters (blast counts, complete blood counts, age) between UBA1 mutant and wildtype MDS MDS. Clinical history was readily available for 4 UBA1-mutant patients. Inflammatory symptoms were reported in 3 patients, localized in the lungs (n=2), skin (n=2) and cartilage (n=1). Two patients also had a diagnosis of inflammatory/rheumatological disease (vasculitis and relapsing polychondritis), two patients had thrombosis, one had a history of monoclonal gammopathy of undetermined significance. The 4th patient, without inflammatory symptoms, had a prior diagnosis of T-cell large granular lymphocytic leukemia. Follow-up for transformation to acute myeloid leukemia (AML) and overall survival was available for 310 and 339 patients, respectively. No UBA1-mutant patients transformed to AML (n=24; median follow-up 2.6 years). There was no difference in overall survival between UBA1-mutant (n=27) and wildtype (n=312) patients (log-rank test p = 0.7), however, when subsetting for patients with no mutations detected by targeted DNA sequencing (UBA1-mutant n=7; wildtype n=45), UBA1-mutant patients had worse overall survival (median 5.2 vs 7.3 years; log-rank test p = 0.03). ConclusionUBA1 mutation in isolation or in combination with other mutations implicated in myeloid pathogenesis account for a significant proportion (8%) of patients diagnosed and treated as MDS but without established disease classification. Ongoing work includes 1) clinical review of inflammatory symptoms for the remaining UBA1-mutant cases and 2) profiling a larger cross-section of MDS by a UBA1 targeted panel to characterize mutations beyond the M41 locus.

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.

A Phase 1 Study of KT-333, a Targeted Protein Degrader of STAT3, in Patients with Relapsed or Refractory Lymphomas, Large Granular Lymphocytic Leukemia, and Solid Tumors

A Phase 1 Study of KT-333, a Targeted Protein Degrader of STAT3, in Patients with Relapsed or Refractory Lymphomas, Large Granular Lymphocytic Leukemia, and Solid Tumors

Epigenome-wide analysis of T-cell large granular lymphocytic leukemia identifies BCL11B as a potential biomarker

BackgroundThe molecular pathogenesis of T-cell large granular lymphocytic leukemia (T-LGLL), a mature T-cell leukemia arising commonly from T-cell receptor αβ-positive CD8+ memory cytotoxic T cells, is only partly understood. The role of deregulated methylation in T-LGLL is not well known. We analyzed the epigenetic profile of T-LGLL cells of 11 patients compared to their normal counterparts by array-based DNA methylation profiling. For identification of molecular events driving the pathogenesis of T-LGLL, we compared the differentially methylated loci between the T-LGLL cases and normal T cells with chromatin segmentation data of benign T cells from the BLUEPRINT project. Moreover, we analyzed gene expression data of T-LGLL and benign T cells and validated the results by pyrosequencing in an extended cohort of 17 patients, including five patients with sequential samples.ResultsWe identified dysregulation of DNA methylation associated with altered gene expression in T-LGLL. Since T-LGLL is a rare disease, the samples size is low. But as confirmed for each sample, hypermethylation of T-LGLL cells at various CpG sites located at enhancer regions is a hallmark of this disease. The interaction of BLC11B and C14orf64 as suggested by in silico data analysis could provide a novel pathogenetic mechanism that needs further experimental investigation.ConclusionsDNA methylation is altered in T-LGLL cells compared to benign T cells. In particular, BCL11B is highly significant differentially methylated in T-LGLL cells. Although our results have to be validated in a larger patient cohort, BCL11B could be considered as a potential biomarker for this leukemia. In addition, altered gene expression and hypermethylation of enhancer regions could serve as potential mechanisms for treatment of this disease. Gene interactions of dysregulated genes, like BLC11B and C14orf64, may play an important role in pathogenic mechanisms and should be further analyzed.

Safety of mRNA COVID-19 Vaccines in Patients with Inborn Errors of Immunity: an Italian Multicentric Study

PurposeLittle is known about vaccine safety in inborn errors of immunity (IEI) patients during the current vaccination campaign for COVID-19. To better investigate the reactogenicity and adverse event profile after two, three, and four doses of mRNA vaccines, we conducted an observational, multicentric study on 342 PID patients from four Italian Referral Centres.MethodsWe conducted a survey on self-reported adverse reactions in IEI patients who received mRNA vaccine by administering a questionnaire after each dose.ResultsOver the whole study period, none of the patients needed hospitalization or had hypersensitivity reactions, including anaphylaxis and delayed injection site reaction. After two vaccination doses, 35.4% of patients showed only local reactogenicity-related symptoms (RrS), 44.4% reported both systemic and local RrS, and 5% reported only systemic RrS. In more than 60% of cases, local or systemic RrS were mild. After the first and second booster doses, patients showed fewer adverse events (AEs) than after the first vaccination course. Patients aged 50 years and older reported adverse events and RrS less frequently. Among AEs requiring treatment, one common variable immune deficiency patient affected by T cell large granular lymphocytic leukemia developed neutropenia and one patient had Bell’s paralysis perhaps during herpes zoster reactivation.ConclusionAlthough our follow-up period is relatively short, the safety data we reported are reassuring. This data would help to contrast the vaccine hesitancy often manifested by patients with IEI and to better inform their healthcare providers.

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.

Clinical Study of the Relationship between Sjögren Syndrome and T-Cell Large Granular Lymphocytic Leukemia: Single-Center Experience.

The relationship between Sjögren syndrome (SS) and T-cell large granular lymphocytic (T-LGL) leukemia remains unclear. In this paper, we report for the first time a large case series of 21 patients with primary and secondary SS associated with T-LGL leukemia. Our results suggest the importance of considering T-LGL leukemia in the diagnostic evaluation of SS patients, particularly when neutropenia occurs. We also postulate that elevated antinuclear antibody titers in patients with T-LGL leukemia indicate the need for the clinical assessment of SS. To assess whether SS affects the frequency of the signal transducer and activator of transcription 3 (STAT3) gene mutations in T-LGL leukemia, we examined STAT3 mutations by next-generation sequencing in two cohorts of patients: with SS-associated T-LGL leukemia and T-LGL leukemia in the setting of rheumatic diseases but without SS. While our results suggest that SS, per se, is not associated with an increased frequency of STAT3 mutations in T-LGL leukemia, further studies are needed to better assess the role of the STAT pathway in the development of concomitant SS and T-LGL leukemia.

LGL Clonal Expansion and Unexplained Cytopenia: Two Clues Don't Make an Evidence.

Clonal expansions of large granular lymphocytes (LGL) have been reported in a wide spectrum of conditions, with LGL leukemia (LGLL) being the most extreme. However, the boundaries between LGLL and LGL clones are often subtle, and both conditions can be detected in several clinical scenarios, particularly in patients with cytopenias. The intricate overlap of LGL clonal expansion with other disease entities characterized by unexplained cytopenias makes their classification challenging. Indeed, precisely assigning whether cytopenias might be related to inadequate hematopoiesis (i.e., LGL as a marginal finding) rather than immune-mediated mechanisms (i.e., LGLL) is far from being an easy task. As LGL clones acquire different pathogenetic roles and relevance according to their diverse clinical settings, their detection in the landscape of bone marrow failures and myeloid neoplasms has recently raised growing clinical interest. In this regard, the current availability of different diagnostic techniques, including next generation sequencing, shed light on the relationship between LGL clones and cytopenias, paving the way towards a better disease classification for precision medicine treatments. Herein, we discuss the clinical relevance of LGL clones in the diagnostic algorithm to be followed in patients presenting with cytopenias, offering a foundation for rational management approaches.

ALENTUZUMAB AS THIRD LINE TREATMENT IN PURE RED SERIE APLASIA - A SUCCESSFUL CASE REPORT

Pure red cell aplasia (PRCA) is a rare hematologic disorder characterized by reticulocytopenic, normocytic anemia with absence of red cell precursors in bone marrow, normal granulopoiesis, and megakaryopoiesis. The disease may be associated with viral infections, large granular lymphocytic leukemia, chronic lymphocytic leukemia, or thymoma, but the most common form is idiopathic. Refractory PRCA may respond to more profound immunosuppression with anti-CD52. To describe a refractory PRCA patient treated with anti-CD52 humanized monoclonal antibody, alemtuzumab, with good response. A 52-year-old male patient with hemophilia A, previously treated with cryoprecipitate and currently using prophylactic Factor VIII, presents to healthcare with severe asthenia, no bleeding episodes during the period. Laboratory evaluation: Hb 3.4 g/dL; Ht 10.6% VCM 95; WBC 6.490 × 10 9 /L; Platelets 196 × 10 9 /L, intense reticulocytopenia (25 × 10 9 /L). He had normal renal function, liver function, LDH, vitamin B12, folic acid, iron profile; serology with positive anti-HCV. Bone marrow evaluation showed 50-60% cellularity with intense hypoplasia of the erythroid series, granulocytic series with no changes in morphology or maturation pattern, and normal megakaryocytes with no signs of dysplasia. After investigation with immunoglobulin dosing, chest CT, flow cytometry for LGL, and serology for parvovirus, all normal, the diagnosis of constitutional APSV was confirmed. Treatment with cyclosporine (CSA) at a dose of 6mg/kg was initiated, with no reticulocyte response. In addition, he evolved with progressively increasing bilirubin levels (BT 7.4 BD 5.6) concomitant with a significant increase in Hepatitis C viral load (380,667 IU/mL Log: 5.58). Immunosuppression was discontinued, treatment with sofosbuvir and simeprevir was initiated, with good viral response. However, he required red blood cell transfusions every 2-3 weeks; new therapy was restarted with CSA and prednisone. The patient did not tolerate this treatment and developed severe thrombocytopenia and no improvement in hemoglobin levels. Considering the high risk of bleeding, CSA was discontinued. Platelet count normalized but he continued to require mensal RBC transfusion. At this point, second-line therapy with IV Alentuzumab, 3 mg dose followed by 6 weekly 15 mg doses (total 96 mg), coupled with infectious prophylaxis, has been proposed. Response occurred as early as the third infusion, as documented by a rapid increase in reticulocyte count. Within seven weeks the patient had a complete response (Hb 12.4 g/dL, Vg 35.3% and reticulocytes 170 × 10 9 /L). He had no toxicity-related intercurrences and remained with a complete response after six months of treatment. Unfortunately, a further progressive drop in hemoglobin after nine months from the end of the first treatment was observed. We opted to restart Alentuzumab at a dose of 15 mg/month, and follow with this maintenance dose. After the third application, the patient again showed a complete response (Hb 14.3 g/dL and VG 40.5%). Currently, the patient continues to use the medication every 45 days with a maintained hemoglobin level. Small case series cite Alentuzumab for refractory APSV as a treatment possibility with 63% overall response. We suggest use in refractory cases, followed by maintenance dosing to avoid relapse in long-term follow-up.

Inherited Bone Marrow Failure Syndromes – Challenges and Updates

Abstract: Congenital or inherited bone marrow failure syndromes are hereditary syndromes of diverse nature which are characterized by inadequate production of blood cells causing cytopenias. Failure of bone marrow can be limited to one or more lineages of blood cells, along with symptoms specified to lineage, through it can affect all cell lineages leading to clinical picture like aplastic anemia. These syndromes are genetic diseases of heterogenous nature caused by germline mutations affecting basic pathways of cell including telomerase biology, biogenesis of ribosomes, structural proteins, and repair of DNA. Common inherited bone marrow failure syndromes consist of Schwachman – Diamond syndrome, Diamond – Blackfan anemia, Fanconi anemia, and Dyskeratosis Congenita. These syndromes have different prognosis and tendency to develop solid or hematological malignancies. Therefore, the adequate diagnosis of these disorders and their differentiation from other bone marrow failure syndromes and/or other etiologies of the boe marrow failure is very significant for surveillance and management of patients. Acquired causes may also lead to bone marrow failure including radiations, chemicals, drugs, immune diseases, viral infections, myelodysplastic syndromes, large granular lymphocytic leukemia, or paroxysmal nocturnal hemoglobinuria (PNH). Inherited bone marrow failure syndromes are heritable and affects family members as well, therefore need genetic counselling. In this review, differential diagnosis, various causes, and their pathogenesis are discussed for better understanding of inherited bone marrow failure syndromes.

All that glitters is not LGL Leukemia.

LGL disorders are rare hematological neoplasias with remarkable phenotypic, genotypic and clinical heterogeneity. Despite these constraints, many achievements have been recently accomplished in understanding the aberrant pathways involved in the LGL leukemogenesis. In particular, compelling evidence implicates STAT signaling as a crucial player of the abnormal cell survival. As interest increases in mapping hematological malignancies by molecular genetics, the relevance of STAT gene mutations in LGL disorders has emerged thanks to their association with discrete clinical features. STAT3 and STAT5b mutations are recognized as the most common gain-of-function genetic lesions up to now identified in T-LGL leukemia (T-LGLL) and are actually regarded as the hallmark of this disorder, also contributing to further refine its subclassification. However, from a clinical perspective, the relationships between T-LGLL and other borderline and overlapping conditions, including reactive cell expansions, clonal hematopoiesis of indeterminate potential (CHIP) and unrelated clonopathies are not fully established, sometimes making the diagnosis of T cell malignancy challenging. In this review specifically focused on the topic of clonality of T-LGL disorders we will discuss the rationale of the appropriate steps to aid in distinguishing LGLL from its mimics, also attempting to provide new clues to stimulate further investigations designed to move this field forward.

Tγδ LGLL identifies a subset with more symptomatic disease: analysis of an international cohort of 137 patients

AbstractTγδ large granular lymphocyte leukemia (LGLL) is a rare variant of T-cell LGLL (T-LGLL) that has been less investigated as compared with the more frequent Tαβ LGLL, particularly in terms of frequency of STAT3 and STAT5b mutations. In this study, we characterized the clinical and biological features of 137 patients affected by Tγδ LGLL; data were retrospectively collected from 1997 to 2020 at 8 referral centers. Neutropenia and anemia were the most relevant clinical features, being present in 54.2% and 49.6% of cases, respectively, including severe neutropenia and anemia in ∼20% of cases each. Among the various treatments, cyclosporine A was shown to provide the best response rates. DNA samples of 97 and 94 cases were available for STAT3 and STAT5b mutation analysis, with 38.1% and 4.2% of cases being mutated, respectively. Clinical and biological features of our series of Tγδ cases were also compared with a recently published Tαβ cohort including 129 cases. Though no differences in STAT3 and STAT5b mutational frequency were found, Tγδ cases more frequently presented with neutropenia (P = .0161), anemia (P < .0001), severe anemia (P = .0065), and thrombocytopenia (P = .0187). Moreover, Vδ2− cases displayed higher frequency of symptomatic disease. Overall, Tγδ cases displayed reduced survival with respect to Tαβ cases (P = .0017). Although there was no difference in STAT3 mutation frequency, our results showed that Tγδ LGLL represents a subset of T-LGLL characterized by more frequent symptoms and reduced survival as compared with Tαβ LGLL.

STAT3 mutations in "gray-zone" cases of T-cell large granular lymphocytic leukemia associated with autoimmune rheumatic diseases.

A persistently increased T-cell large granular lymphocyte (T-LGL) count in the blood of more than 2 × 109/L for at least 6 months is necessary for a reliable diagnosis of T-LGL leukemia. In cases with LGL counts of approximately 0.5–2 × 109/L, a diagnosis of T-LGL leukemia can be made if clonal rearrangement of T-cell receptor (TCR) genes is present and if the patient shows typical manifestations of T-LGL leukemia, such as cytopenia, splenomegaly, or concomitant autoimmune disease. However, in cases with LGL counts of less than 0.5 × 109/L, the diagnosis of T-LGL leukemia is questionable (termed as “gray-zone” cases). Although mutations in signal transducer and activator of transcription 3 (STAT3) gene are the molecular hallmark of T-LGL leukemia, their diagnostic value in the “gray-zone” cases of T-LGL leukemia has not been evaluated – our study has been aimed to examine the prevalence of STAT3 mutations in these cases. Herein, we describe 25 patients with autoimmune rheumatic diseases, neutropenia, clonal rearrangement of TCR genes, and circulating LGL count of less than 0.5 × 109/L. Splenomegaly was observed in 19 (76%) patients. Mutations in the STAT3 were detected in 56% of patients using next-generation sequencing. Importantly, in 3 patients, no involvement of the blood and bone marrow by malignant LGLs was noted, but examination of splenic tissue revealed infiltration by clonal cytotoxic T-lymphocytes within the red pulp, with greater prominence in the cords. We suggest using the term “splenic variant of T-LGL leukemia” for such cases.

Clinicopathologic and immunophenotypic features in dogs with presumptive large granular lymphocyte leukaemia.

Large granular lymphocytic leukaemia (LGLL) has been described in a range of species but has been most commonly reported in humans and dogs. In both species, this neoplasia exhibits diversity in both phenotype and biological behaviour with phenotype only partially predicting behaviour. There is currently little knowledge of concurrent haematological and serum biochemistry features or concurrent occurrence of distinct neoplasia in canine LGLL cases. This study presents a canine case series and defines haematological parameters, novel serum biochemistry findings and phenotype of the large granular lymphocytes in an Australian case series. Neutrophilia was the most common haematological abnormality, identified in 43% of dogs, and 84% of dogs with biochemistry data available had elevated serum gamma-glutamyl transferase. Five of the 40 dogs in this study exhibited concurrent neoplasia during the period of the study, demonstrating this is a relatively common clinical outcome in canine LGLL cases. In agreement with previous canine and human studies, the most common LGLL phenotype in dogs is CD3+, CD4- and CD8+. Further work is needed to define the variables predictive of the biological behaviour of LGLL in dogs.

T-cell clonality testing for the diagnosis of T-cell large granular lymphocytic leukemia: Are we identifying pathology or incidental clones?

T-cell clonality testing by T-cell receptor (TCR) gene rearrangement is key to the diagnosis of T-cell lymphoproliferative disorders such as T-cell large granular lymphocytic (T-LGL) leukemia. Benign clonal T-cell expansions, however, are commonly found in patients without identifiable disease, a condition referred to as T-cell clones of uncertain significance (T-CUS). In practice, T-cell clonality testing is performed for a range of reasons and results are often challenging to interpret given the overlap between benign and malignant clonal T-cell proliferations and uncertainties in the management of T-CUS. We conducted a 5-year retrospective cohort study of 211 consecutive patients who underwent PCR-based T-cell clonality testing for suspected T-LGL leukemia at our institution to characterize the use of T-cell clonality testing and its impact on patient management. Overall, 46.4% (n=98) of individuals tested had a clonal T-cell population identified. Patients with a monoclonal T-cell population were more likely to be older, have rheumatoid arthritis and have higher lymphocyte counts compared to patients with polyclonal populations. The majority of patients eventually diagnosed and treated for T-LGL leukemia had rheumatoid arthritis and lower neutrophil counts compared to untreated patients with monoclonal T-cell populations. A diagnosis of T-LGL leukemia was made in only a minority of patients (n=48, 22.7%), and only a small proportion were treated (n=17, 8.1%). Our study suggests that T-cell clonality testing most commonly identifies incidental T-cell clones with only a minority of patients receiving a diagnosis of T-LGL leukemia and fewer requiring active treatment. These finding indicate an opportunity to improve utilization of T-cell clonality testing in clinical practice to better target patients where the results of testing would impact clinical management.