Frequency and perturbations of various peripheral blood cell populations before and after eculizumab treatment in paroxysmal nocturnal hemoglobinuria.

While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations. We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment. We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis. In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.

Extended experience with a non-cytotoxic DNMT1-targeting regimen of decitabine to treat myeloid malignancies.

The nucleoside analogue decitabine can deplete the epigenetic regulator DNA methyltransferase 1 (DNMT1), an effect that occurs, and is saturated at, low concentrations/doses. A reason to pursue this molecular-targeted effect instead of the DNA damage/cytotoxicity produced with high concentrations/doses, is that non-cytotoxic DNMT1-depletion can cytoreduce even p53-null myeloid malignancies while sparing normal haematopoiesis. We thus identified minimum doses of decitabine (0·1-0·2 mg/kg) that deplete DNMT1 without off-target anti-metabolite effects/cytotoxicity, and then administered these well-tolerated doses frequently 1-2X/week to increase S-phase dependent DNMT1-depletion, and used a Myeloid Malignancy Registry to evaluate long-term outcomes in 69 patients treated this way. Consistent with the scientific rationale, treatment was well-tolerated and durable responses were produced (~40%) in genetically heterogeneous disease and the very elderly.

Large granular lymphocytic leukaemia after solid organ and haematopoietic stem cell transplantation.

T-cell large granular lymphocytic leukaemia (T-LGLL) is a chronic clonal lymphoproliferative disorder of cytotoxic T lymphocytes which commonly occurs in older patients and is often associated with autoimmune diseases. Among 246 patients with T-LGLL seen at our institution over the last 10 years, we encountered 15 cases following solid organ or haematopoietic stem cell transplantation. Here, we studied the clinical characterization of these cases and compared them to de novo T-LGLL. This experience represented a clear picture of the intricate nature of the disease manifestation and the complexities of several immune mechanisms triggering the clonal expansion.

Distinctive and common features of moderate aplastic anaemia.

The therapy algorithm for severe aplastic anaemia (sAA) is established but moderate AA (mAA), which likely reflects a more diverse pathogenic mechanism, often represents a treatment/management conundrum. A cohort of AA patients (n = 325) was queried for those with non-severe disease using stringent criteria including bone marrow hypocellularity and chronic persistence of moderately depressed blood counts. As a result, we have identified and analyzed pathological and clinical features in 85 mAA patients. Progression to sAA and direct clonal evolution (paroxysmal nocturnal haemoglobinuria/acute myeloid leukaemia; PNH/AML) occurred in 16%, 11% and 1% of mAA cases respectively. Of the mAA patients who received immunosuppressive therapy, 67% responded irrespective of time of initiation of therapy while conservatively managed patients showed no spontaneous remissions. Genomic analysis of mAA identified evidence of clonal haematopoiesis with both persisting and remitting patterns at low allelic frequencies; with more pronounced mutational burden in sAA. Most of the mAA patients have autoimmune pathogenesis similar to those with sAA, but mAA contains a mix of patients with diverse aetiologies. Although progression rates differed between mAA and sAA (P = 0·003), cumulative incidences of mortalities were only marginally different (P = 0·095). Our results provide guidance for diagnosis/management of mAA, a condition for which no current standard of care is established.

Laparoscopic splenectomy for immune thrombocytopenia (ITP): long-term outcomes of a modern cohort.

BACKGROUND: The advent of newer second-line medical therapies (SLMT) for immune thrombocytopenia (ITP) has contributed to decreased rates of splenectomy, following a trend to avoid or delay surgery. We aimed to characterize the long-term outcomes of laparoscopic splenectomy (LS) for ITP at our institution, examining differences in LS efficiency when performed before or after SLMTs. METHODS: Adults with primary ITP who underwent LS between 2002 and 2016 were identified. Retrospective review of electronic medical records was supplemented with telephone interviews. Treatment response was defined according to current guidelines as complete responders (CR), responders (R), and non-responders (NR). Kaplan-Meier estimates assessed relapse-free rates, and predictors of long-term response were investigated using logistic regression. RESULTS: 109 patients met inclusion criteria, from which 42% were treated with an SLMT before referral to LS. LS was completed in all cases, with no conversions or intraoperative complications. The perioperative morbidity was 7.3%, including 3 deep vein and 2 portal vein thrombosis, one reoperation for bleeding, and no mortalities. Splenectomy was initially effective in 99 patients (CR + R = 90.8%), and 10 patients were NR. At a median 62-month follow-up, 25 patients relapsed, resulting in a 68% CR + R rate. Proportion of CR + R was similar in patients who previously received SLMT and those who did not (61 vs. 76.7%, p = 0.08). CR + R patients were younger (45 vs. 53, p = 0.03), had higher preoperative platelet counts (36 vs. 19, p = 0.01), and experienced a higher increment in platelet counts during hospital stay (117 vs. 38, p < 0.001) as well as 30-days postoperatively (329 vs. 124, p < 0.001). Only a robust response in platelet count at 30-days postoperatively was independently associated with long-term response (OR 1.005, p = 0.006). CONCLUSION: LS was curative in 68% of patients, with no statistically significant difference when performed before or after SLMTs. Outcomes remain challenging to predict preoperatively, with only a robust increase in platelet counts on short term being associated with long-term response.

STAT3 mutations indicate the presence of subclinical T-cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients.

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

Pulmonary vascular disease in mice xenografted with human BM progenitors from patients with pulmonary arterial hypertension.

Hematopoietic myeloid progenitors released into the circulation are able to promote vascular remodeling through endothelium activation and injury. Endothelial injury is central to the development of pulmonary arterial hypertension (PAH), a proliferative vasculopathy of the pulmonary circulation, but the origin of vascular injury is unknown. In the present study, mice transplanted with BM-derived CD133(+) progenitor cells from patients with PAH, but not from healthy controls, exhibited morbidity and/or death due to features of PAH: in situ thrombi and endothelial injury, angioproliferative remodeling, and right ventricular hypertrophy and failure. Myeloid progenitors from patients with heritable and/or idiopathic PAH all produced disease in xenografted mice. Analyses of hematopoietic transcription factors and colony formation revealed underlying abnormalities of progenitors that skewed differentiation toward the myeloid-erythroid lineage. The results of the present study suggest a causal role for hematopoietic stem cell abnormalities in vascular injury, right ventricular hypertrophy, and morbidity associated with PAH.

Hypoxia-inducible factors in human pulmonary arterial hypertension: a link to the intrinsic myeloid abnormalities.

Pulmonary arterial hypertension (PAH) is a proliferative vasculopathy characterized by high circulating CD34(+)CD133(+) proangiogenic progenitors, and endothelial cells that have pathologic expression of hypoxia-inducible factor 1 α (HIF-1α). Here, CD34(+)CD133(+) progenitor cell numbers are shown to be higher in PAH bone marrow, blood, and pulmonary arteries than in healthy controls. The HIF-inducible myeloid-activating factors erythropoietin, stem cell factor (SCF), and hepatocyte growth factor (HGF) are also present at higher than normal levels in PAH blood, and related to disease severity. Primary endothelial cells harvested from human PAH lungs produce greater HGF and progenitor recruitment factor stromal-derived factor 1 α (SDF-1α) than control lung endothelial cells, and thus may contribute to bone marrow activation. Even though PAH patients had normal numbers of circulating blood elements, hematopoietic alterations in myeloid and erythroid lineages and reticulin fibrosis identified a subclinical myeloproliferative process. Unexpectedly, evaluation of bone marrow progenitors and reticulin in nonaffected family members of patients with familial PAH revealed similar myeloid abnormalities. Altogether, the results show that PAH is linked to myeloid abnormalities, some of which may be related to increased production of HIF-inducible factors by diseased pulmonary vasculature, but findings in nonaffected family suggest myeloid abnormalities may be intrinsic to the disease process.

Clonal drift demonstrates unexpected dynamics of the T-cell repertoire in T-large granular lymphocyte leukemia.

T-cell large granular lymphocyte leukemia (T-LGLL) is characterized by chronic lymphoproliferation of cytotoxic T lymphocytes (CTLs) and is associated with lineage-restricted cytopenias. Introduction of T-cell receptor (TCR) variable ß-chain (Vß) monoclonal antibodies has facilitated identification and enumeration of clonal CTLs by flow cytometry. A highly skewed TCR Vß repertoire identified by flow cytometry is strongly associated with monoclonal CDR3 regions by quantitative sequencing and positive TCRγ rearrangement assays. Therefore, Vß expansions can serve as surrogate markers of CTL clonality to assess clonal kinetics in T-LGLL. We analyzed the TCR repertoire in 143 patients, 71 of which were available for serial measurements over 6 to 96 months. Although the majority (38/71, 54%) maintained a consistent monoclonal expansion, many (26/71, 37%) unexpectedly displayed a change in the dominant clone, whereby the original CTL clone contracted and another emerged as demonstrated by Vß typing. Our results demonstrate that the T-cell repertoire is more dynamic in T-LGLL than recognized previously, illustrating the heterogeneity of disorders under this categorization.

Efficacy of rabbit anti-thymocyte globulin in severe aplastic anemia.

BACKGROUND: A combination of horse anti-thymocyte globulin and cyclosporine produces responses in 60-70% of patients with severe aplastic anemia. We performed a phase II study of rabbit anti-thymocyte globulin and cyclosporine as first-line therapy for severe aplastic anemia. DESIGN AND METHODS: Twenty patients with severe aplastic anemia treated with rabbit anti-thymocyte globulin were compared to 67 historical control cases with matched clinical characteristics treated with horse anti-thymocyte globulin. RESULTS: Response rates at 3, 6 and 12 months were similar for patients treated with rabbit anti-thymocyte globulin or horse anti-thymocyte globulin: 40% versus 55% (P=0.43), 45% versus 58% (P=0.44) and 50% versus 58% (P=0.61), respectively. No differences in early mortality rates or overall survival were observed. We then performed multivariable analyses of response at 6 months and overall survival and identified the presence of a paroxysmal nocturnal hemoglobinuria clone (P=0.01) and a pretreatment absolute reticulocyte count greater than 30×10(9)/L (P=0.007) as independent predictors of response and younger age (P=0.003), higher pretreatment absolute neutrophil (P=0.02) and absolute lymphocyte counts (P=0.03) as independent predictors of overall survival. None of the immunogenetic polymorphisms studied was predictive of response to immunosupressive therapy. CONCLUSIONS: Despite reports suggesting differences in biological activity of different anti-thymocyte globulin preparations, rabbit and horse anti-thymocyte globulin appear to have a similar efficacy for up-front treatment of severe aplastic anemia. Clinicaltrial.gov: NCT01231841).

Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial.

BACKGROUND: Chronic immune thrombocytopenic purpura (ITP) is characterised by accelerated platelet destruction and decreased platelet production. Short-term administration of the thrombopoiesis-stimulating protein, romiplostim, has been shown to increase platelet counts in most patients with chronic ITP. We assessed the long-term administration of romiplostim in splenectomised and non-splenectomised patients with ITP. METHODS: In two parallel trials, 63 splenectomised and 62 non-splenectomised patients with ITP and a mean of three platelet counts 30x10(9)/L or less were randomly assigned 2:1 to subcutaneous injections of romiplostim (n=42 in splenectomised study and n=41 in non-splenectomised study) or placebo (n=21 in both studies) every week for 24 weeks. Doses of study drug were adjusted to maintain platelet counts of 50x10(9)/L to 200x10(9)/L. The primary objectives were to assess the efficacy of romiplostim as measured by a durable platelet response (platelet count > or =50x10(9)/L during 6 or more of the last 8 weeks of treatment) and treatment safety. Analysis was per protocol. These studies are registered with ClinicalTrials.gov, numbers NCT00102323 and NCT00102336. FINDINGS: A durable platelet response was achieved by 16 of 42 splenectomised patients given romplostim versus none of 21 given placebo (difference in proportion of patients responding 38% [95% CI 23.4-52.8], p=0.0013), and by 25 of 41 non-splenectomised patients given romplostim versus one of 21 given placebo (56% [38.7-73.7], p<0.0001). The overall platelet response rate (either durable or transient platelet response) was noted in 88% (36/41) of non-splenectomised and 79% (33/42) of splenectomised patients given romiplostim compared with 14% (three of 21) of non-splenectomised and no splenectomised patients given placebo (p<0.0001). Patients given romiplostim achieved platelet counts of 50x10(9)/L or more on a mean of 13.8 (SE 0.9) weeks (mean 12.3 [1.2] weeks in splenectomised group vs 15.2 [1.2] weeks in non-splenectomised group) compared with 0.8 (0.4) weeks for those given placebo (0.2 [0.1] weeks vs 1.3 [0.8] weeks). 87% (20/23) of patients given romiplostim (12/12 splenectomised and eight of 11 non-splenectomised patients) reduced or discontinued concurrent therapy compared with 38% (six of 16) of those given placebo (one of six splenectomised and five of ten non-splenectomised patients). Adverse events were much the same in patients given romiplostim and placebo. No antibodies against romiplostim or thrombopoietin were detected. INTERPRETATION: Romiplostim was well tolerated, and increased and maintained platelet counts in splenectomised and non-splenectomised patients with ITP. Many patients were able to reduce or discontinue other ITP medications. Stimulation of platelet production by romiplostim may provide a new therapeutic option for patients with ITP.

AMG 531, a thrombopoiesis-stimulating protein, for chronic ITP.

BACKGROUND: Most current treatments for chronic immune thrombocytopenic purpura (ITP) act by decreasing platelet destruction. In a phase 1-2 study, we administered a thrombopoiesis-stimulating protein, AMG 531, to patients with ITP. METHODS: In phase 1, 24 patients who had received at least one treatment for ITP were assigned to escalating-dose cohorts of 4 patients each and given two identical doses of AMG 531 (0.2 to 10 microg per kilogram of body weight). In phase 2, 21 patients were randomly assigned to receive six weekly subcutaneous injections of AMG 531 (1, 3, or 6 microg per kilogram) or placebo. The primary objective was to assess the safety of AMG 531; the secondary objective was to evaluate platelet counts during and after treatment. RESULTS: No major adverse events that could be attributed directly to AMG 531 occurred during the treatment period; 4 of 41 patients had transient post-treatment worsening of thrombocytopenia. In phase 1, a platelet count that was within the targeted range (50,000 to 450,000 per cubic millimeter) and at least twice the baseline count was achieved in 4 of 12 patients given 3, 6, or 10 mug of AMG 531 per kilogram. Overall, a platelet count of at least 50,000 per cubic millimeter was achieved in 7 of 12 patients, including 3 with counts exceeding 450,000 per cubic millimeter. Increases in the platelet count were dose-dependent; mean peak counts were 163,000, 309,000, and 746,000 per cubic millimeter with 3, 6, and 10 microg of AMG 531 per kilogram [corrected], respectively. In phase 2, the targeted platelet range was achieved in 10 of 16 patients treated with 1 or 3 mug of AMG 531 per kilogram per week for 6 weeks. Mean peak counts were 135,000, 241,000, and 81,000 per cubic millimeter in the groups that received the 1-mug dose, the 3-mug dose, and placebo, respectively. CONCLUSIONS: AMG 531 caused no major adverse events and increased platelet counts in patients with ITP. (ClinicalTrials.gov number, NCT00111475 [ClinicalTrials.gov].).

Therapeutic implications of variable expression of CD52 on clonal cytotoxic T cells in CD8+ large granular lymphocyte leukemia.

BACKGROUND: T-cell large granular lymphocytic leukemia is a clonal proliferation of cytotoxic T-lymphocytes which often results in severe cytopenia. Current treatment options favor chronic immunosuppression. Alemtuzumab, a humanized monoclonal antibody against glycophosphatidylinositol-anchored CD52, is approved for patients refractory to therapy in other lymphoid malignancies. DESIGN AND METHODS: We retrospectively examined treatment outcomes in 59 patients with CD8+ T-cell large granular lymphocytic leukemia, 41 of whom required therapy. Eight patients with severe refractory cytopenia despite multiple treatment regimens had been treated with subcutaneous alemtuzumab as salvage therapy. Flow cytometry was used to monitor expression of glycophosphatidylinositol-anchored CD52, CD55, and CD59 as well as to characterize T-cell clonal expansions by T-cell receptor variable beta-chain (Vbeta) repertoire. RESULTS: Analysis of the effects of alemtuzumab revealed remissions with restoration of platelets in one of one patient, red blood cell transfusion independence in three of five patients and improvement of neutropenia in one of three, resulting in an overall response rate of 50% (4/8 patients). Clonal large granular lymphocytes exhibited decreased CD52 expression post-therapy in patients refractory to treatment. Samples of large granular lymphocytes collected prior to therapy also unexpectedly had a significant proportion of CD52-negative cells while a healthy control population had no such CD52 deficiency (p=0.026). CONCLUSIONS: While alemtuzumab may be highly effective in large granular lymphocytic leukemia, prospective serial monitoring for the presence of CD52-deficient clonal cytotoxic T-lymphocytes should be a component of clinical trials investigating the efficacy of this drug. CD52 deficiency may explain lack of response to alemtuzumab, and such therapy may confer a survival advantage to glycophosphatidylinositol-negative clonal cytotoxic T-lymphocytes.

Clonal predominance of CD8(+) T cells in patients with unexplained neutropenia.

OBJECTIVE: T-cell-mediated autoimmunity may be involved in some cases of idiopathic neutropenia. We hypothesized that a precise T-cell receptor repertoire analysis may uncover cytotoxic T-cell (CTL) expansions that are less pronounced than those seen in T large granular lymphocyte leukemia (T-LGL), but are pathophysiologically analogous and thus can serve as markers of a T-cell-mediated process. MATERIALS AND METHODS: Using rational algorithms for T-cell receptor analysis and in vivo tracking of CTL responses previously established in our laboratory, we studied patients with unexplained chronic neutropenia (n = 20), T-LGL (n = 15), and healthy controls (n = 12). We further investigated the involvement of soluble inhibitory factors by coculture assays. To determine the level of immune activation, we studied interferon-gamma expression in CD8(+)cells using Taqman polymerase chain reaction. RESULTS: Fifteen expanded (immunodominant) CTL clones were detected in 12 of 20 patients. In comparison to LGL leukemia, these clones were less immunodominant, but clearly discernible from subclinical lymphoproliferations in controls. As a surrogate of cytotoxic activity, we found markedly increased production of interferon-gamma in most of the neutropenia patients, irrespective of the presence of immunodominant CTL clones. CONCLUSIONS: These results suggest that, while immunodominant CTL clones are detectable in a proportion of patients only, CTL-mediated pathophysiology may be a general mechanism operating in idiopathic neutropenia. Oligogoclonal CTL expansions in chronic neutropenia may indicate an ongoing autoimmune process, while highly polarized monoclonalities in a subset of neutropenic LGL patients may represent the "extreme" end of the clonal continuum.

Outcomes of splenectomy in T-cell large granular lymphocyte leukemia with splenomegaly and cytopenia.

OBJECTIVE: T-cell large granular lymphocyte leukemia (T-LGL) is a chronic clonal lymphoproliferation of cytotoxic T cells often complicated by cytopenia. Because the outcomes of splenectomy in patients with T-LGL have been only reported sporadically, we objectively assessed the outcomes of splenectomy. MATERIALS AND METHODS: When a cohort of 56 T-LGL patients was analyzed, patients with splenomegaly (n = 34) and had higher frequency of bi- and pancytopenia than patients with no splenomegaly (70% vs 27%; p = 0.001). We identified 15 patients who, in their clinical course, underwent splenectomy and studied their hematological and clinical outcomes. RESULTS: Indications for splenectomy included symptomatic splenomegaly and/or severe refractory cytopenia. Median spleen weight was 1300 g, consistent with diagnosis of splenomegaly; T-cell receptor (TCR)-gamma rearrangement and typical T-LGL were detected by immunophenotype in all specimens. There was no surgery-related mortality, with the median follow-up and survival of 719 and 498 days, respectively. Two patients died due to causes possibly related to the splenectomized state and/or primary disease. All patients showed lineage-specific hematologic response and achieved transfusion independence; however, precise molecular analysis of TCR and variable chain Vbeta flow cytometry showed persistence of the LGL clones. CONCLUSION: We conclude that splenectomy constitutes a viable and safe therapeutic option for patients with T-LGL, splenomegaly, and refractory cytopenia.

Subclonal STAT3 mutations solidify clonal dominance.

T large granular lymphocyte leukemia (T-LGLL) is a clonal lymphoproliferative disorder that can arise in the context of pathologic or physiologic cytotoxic T-cell (CTL) responses. STAT3 mutations are often absent in typical T-LGLL, suggesting that in a significant fraction of patients, antigen-driven expansion alone can maintain LGL clone persistence. We set out to determine the relationship between activating STAT3 hits and CTL clonal selection at presentation and in response to therapy. Thus, a group of patients with T-LGLL were serially subjected to deep next-generation sequencing (NGS) of the T-cell receptor (TCR) Vß complementarity-determining region 3 (CDR3) and STAT3 to recapitulate clonal hierarchy and dynamics. The results of this complex analysis demonstrate that STAT3 mutations produce either a sweeping or linear subclone within a monoclonal CTL population either early or during the course of disease. Therapy can extinguish a LGL clone, silence it, or adapt mechanisms to escape elimination. LGL clones can persist on elimination of STAT3 subclones, and alternate STAT3-negative CTL clones can replace therapy-sensitive CTL clones. LGL clones can evolve and are fueled by a nonextinguished antigenic drive. STAT3 mutations can accelerate this process or render CTL clones semiautonomous and not reliant on physiologic stimulation.

High-resolution genomic arrays facilitate detection of novel cryptic chromosomal lesions in myelodysplastic syndromes.

OBJECTIVE: Unbalanced chromosomal aberrations are common in myelodysplastic syndromes and have prognostic implications. An increased frequency of cytogenetic changes may reflect an inherent chromosomal instability due to failure of DNA repair. Therefore, it is likely that chromosomal defects in myelodysplastic syndromes may be more frequent than predicted by metaphase cytogenetics and new cryptic lesions may be revealed by precise analysis methods. METHODS: We used a novel high-resolution karyotyping technique, array-based comparative genomic hybridization, to investigate the frequency of cryptic chromosomal lesions in a cohort of 38 well-characterized myelodysplastic syndromes patients; results were confirmed by microsatellite quantitative PCR or single nucleotide polymorphism analysis. RESULTS: As compared to metaphase karyotyping, chromosomal abnormalities detected by array-based analysis were encountered more frequently and in a higher proportion of patients. For example, chromosomal defects were found in patients with a normal karyotype by traditional cytogenetics. In addition to verifying common abnormalities, previously cryptic defects were found in new regions of the genome. Cryptic changes often overlapped chromosomes and regions frequently identified as abnormal by metaphase cytogenetics. CONCLUSION: The results underscore the instability of the myelodysplastic syndromes genome and highlight the utility of array-based karyotyping to study cryptic chromosomal changes which may provide new diagnostic information.

Clinical features and treatment outcomes in large granular lymphocytic leukemia (LGLL).

Large granular lymphocytic leukemia (LGLL) represents a clonal/oligoclonal lymphoproliferation of cytotoxic T and natural killer cells often associated with STAT3 mutations. When symptomatic, due to mostly anemia and neutropenia, therapy choices are often empirically-based, because only few clinical trials and systematic studies have been performed. Incorporating new molecular and flow cytometry parameters, we identified 204 patients fulfilling uniform criteria for LGLL diagnoses and analyzed clinical course with median follow-up of 36 months, including responses to treatments. While selection of initial treatment was dictated by clinical features, the initial responses, as well as overall responses to methotrexate (MTX), cyclosporine (CsA), and cyclophosphamide (CTX), were similar at 40-50% across drugs. Sequential use of these drugs resulted in responses in most cases: only 10-20% required salvage therapies such as ATG, Campath, tofacitinib, splenectomy or abatacept. MTX yielded the most durable responses. STAT3-mutated patients required therapy more frequently and had better overall survival.