A randomized, placebo-controlled clinical trial evaluating olipudase alfa enzyme replacement therapy for chronic acid sphingomyelinase deficiency (ASMD) in adults: One-year results.

PURPOSE: This trial aimed to assess the efficacy and safety of olipudase alfa enzyme replacement therapy for non-central nervous system manifestations of acid sphingomyelinase deficiency (ASMD) in adults. METHODS: A phase 2/3, 52 week, international, double-blind, placebo-controlled trial (ASCEND; NCT02004691/EudraCT 2015-000371-26) enrolled 36 adults with ASMD randomized 1:1 to receive olipudase alfa or placebo intravenously every 2 weeks with intrapatient dose escalation to 3 mg/kg. Primary efficacy endpoints were percent change from baseline to week 52 in percent predicted diffusing capacity of the lung for carbon monoxide and spleen volume (combined with splenomegaly-related score in the United States). Other outcomes included liver volume/function/sphingomyelin content, pulmonary imaging/function, platelet levels, lipid profiles, and pharmacodynamics. RESULTS: Least square mean percent change from baseline to week 52 favored olipudase alfa over placebo for percent predicted diffusing capacity of the lung for carbon monoxide (22% vs 3.0% increases, P = .0004), spleen volume (39% decrease vs 0.5% increase, P < .0001), and liver volume (28% vs 1.5% decreases, P < .0001). Splenomegaly-related score decreased in both groups (P = .64). Other clinical outcomes improved in the olipudase alfa group compared with the placebo group. There were no treatment-related serious adverse events or adverse event-related discontinuations. Most adverse events were mild. CONCLUSION: Olipudase alfa was well tolerated and associated with significant and comprehensive improvements in disease pathology and clinically relevant endpoints compared with placebo in adults with ASMD.

Interobserver variance in myelodysplastic syndromes with less than 5 % bone marrow blasts: unilineage vs. multilineage dysplasia and reproducibility of the threshold of 2 % blasts.

Previous studies have shown the reproducibility of the 2008 World Health Organization (WHO) classification in myelodysplastic syndromes (MDS), especially when multilineage dysplasia or excess of blasts are present. However, there are few data regarding the reproducibility of MDS with unilineage dysplasia. The revised International Prognostic Scoring System R-IPSS described two new morphological categories, distinguishing bone marrow (BM) blast cell count between 0-2 % and >2- < 5 %. This distinction is critical for establishing prognosis, but the reproducibility of this threshold is still not demonstrated. The objectives of our study were to explore the reliability of the 2008 WHO classification, regarding unilineage vs. multilineage dysplasia, by reviewing 110 cases previously diagnosed with MDS, and to study whether the threshold of ≤2 % BM blasts is reproducible among different observers. We used the same methodology as in our previous paper [Font et al. (2013) Ann Hematol 92:19-24], by encouraging investigators to include patients with <5 % BM blasts. Samples were collected from 11 hospitals and were evaluated by 11 morphologists. Each observer evaluated 20 samples, and each sample was analyzed independently by two morphologists. Discordance was observed in 36/108 suitable cases (33 %, kappa test 0.503). Diagnosis of MDS with unilineage dysplasia (refractory cytopenia with unilineage dysplasia (RCUD), refractory anemia with ring sideroblasts (RARS) or unclassifiable MDS) was assessed in 33 patients, by either of the two observers. We combined this series with the cases with RCUD or RARS included in our 2013 paper, thus obtaining 50 cases with unilineage dysplasia by at least one of the observers. The whole series showed very low agreement regarding RCUD (5/23, 21 %) and RARS (5/28, 18 %). Regarding BM blast count, the threshold of ≤2 % was not reproducible (discordance rate 32/108 cases, kappa test 0.277). Our study shows that among MDS WHO 2008 categories, interobserver discordance seems to be high in cases with unilineage dysplasia. We also illustrate that the threshold of ≤2 % BM blasts as settled by the R-IPSS may be not easy to reproduce by morphologists in real practice.

Continued improvement in disease manifestations of acid sphingomyelinase deficiency for adults with up to 2 years of olipudase alfa treatment: open-label extension of the ASCEND trial.

BACKGROUND: Olipudase alfa is a recombinant human acid sphingomyelinase enzyme replacement therapy for non-central-nervous-system manifestations of acid sphingomyelinase deficiency (ASMD). The ASCEND randomized placebo-controlled trial in adults with ASMD demonstrated reductions in sphingomyelin storage, organomegaly, interstitial lung disease and impaired diffusion capacity of the lung (DLCO), during the first year of olipudase alfa treatment. In an ongoing open-label extension of the ASCEND trial, individuals in the placebo group crossed over to olipudase alfa, and those in the olipudase alfa group continued treatment. RESULTS: Thirty-five of 36 participants continued in the extension trial, and 33 completed year 2. Change-from-baseline results are presented as least-square mean percent change ± SEM. Improvements in the cross-over group after 1 year of treatment paralleled those of the olipudase alfa group from the primary analysis, while clinical improvement continued for those receiving olipudase alfa for 2 years. In the cross-over group, percent-predicted DLCO increased by 28.0 ± 6.2%, spleen volume decreased by 36.0 ± 3.0% and liver volume decreased by 30.7 ± 2.5%. For those with 2 years of olipudase alfa treatment, the percent predicted DLCO increased by 28.5 ± 6.2%, spleen volume decreased by 47.0 ± 2.7%, and liver volume decreased by 33.4 ± 2.2%. Lipid profiles and elevated liver transaminase levels improved or normalized by 1 year and remained stable through 2 years of treatment. Overall, 99% of treatment-emergent adverse events were mild or moderate, with one treatment-related serious adverse event (extrasystoles; previously documented cardiomyopathy). No individual discontinued due to an adverse event. CONCLUSION: Treatment with olipudase alfa is well tolerated and reduces manifestations of chronic ASMD with sustained efficacy. Trial registration NCT02004691 registered 9 December 2013, https://clinicaltrials.gov/ct2/show/NCT02004691.

Secondary haemophagocytic lymphohistiocytosis in COVID-19: correlation of the autopsy findings of bone marrow haemophagocytosis with HScore.

BACKGROUND: Secondary haemophagocytic lymphohistiocytosis (sHLH) is characterised by a hyper activation of immune system that leads to multiorgan failure. It is suggested that excessive immune response in patients with COVID-19 could mimic this syndrome. Some COVID-19 autopsy studies have revealed the presence of haemophagocytosis images in bone marrow, raising the possibility, along with HScore parameters, of sHLH. AIM: Our objective is to ascertain the existence of sHLH in some patients with severe COVID-19. METHODS: We report the autopsy histological findings of 16 patients with COVID-19, focusing on the presence of haemophagocytosis in bone marrow, obtained from rib squeeze and integrating these findings with HScore parameters. CD68 immunohistochemical stains were used to highlight histiocytes and haemophagocytic cells. Clinical evolution and laboratory parameters of patients were collected from electronic clinical records. RESULTS: Eleven patients (68.7%) displayed moderate histiocytic hyperplasia with haemophagocytosis (HHH) in bone marrow, three patients (18.7%) displayed severe HHH and the remainder were mild. All HScore parameters were collected in 10 patients (62.5%). Among the patients in which all parameters were evaluable, eight patients (80%) had an HScore >169. sHLH was not clinically suspected in any case. CONCLUSIONS: Our results support the recommendation of some authors to use the HScore in patients with severe COVID-19 in order to identify those who could benefit from immunosuppressive therapies. The presence of haemophagocytosis in bone marrow tissue, despite not being a specific finding, has proved to be a very useful tool in our study to identify these patients.

A reappraisal of Gaucher disease-diagnosis and disease management algorithms.

Type 1 (non-neuronopathic) Gaucher disease was the first lysosomal storage disorder for which an effective enzyme replacement therapy was developed and it has become a prototype for treatments for related orphan diseases. There are currently four treatment options available to patients with Gaucher disease, nevertheless, almost 25% of Type 1 Gaucher patients do not gain timely access to therapy because of delays in diagnosis after the onset of symptoms. Diagnosis of Gaucher disease by enzyme testing is unequivocal, but the rarity of the disease and nonspecific and heterogeneous nature of Gaucher disease symptoms may impede consideration of this disease in the differential diagnosis. To help promote timely diagnosis and optimal management of the protean presentations of Gaucher disease, a consensus meeting was convened to develop algorithms for diagnosis and disease management for Gaucher disease.

Development and validation of a sequential two-step algorithm for the screening of individuals with potential polycythaemia vera.

In 2016, the WHO included haemoglobin values within normal ranges as a diagnostic criterion for Polycythaemia Vera (PV). Since then, concerns have arisen that a large number of patients are undergoing unnecessary screening for PV. To address this issue, we estimated the prevalence of JAK2 p.V617F in individuals with elevated haemoglobin or haematocrit and developed and validated a screening algorithm for PV. A total of 15,366 blood counts performed in seven non-consecutive days were reviewed, of which 1001 were selected for subsequent JAK2 p.V617F mutation screening. Eight (0.8%) new JAK2 p.V617F-mutated cases were detected. From ROC curves, a two-step algorithm was developed based on the optimal cut-off for the detection of the JAK2 p.V617F mutation. The algorithm was prospectively validated in an independent cohort of 15,298 blood counts. A total of 1595 (10.4%) cases met the criterion for haemoglobin or haematocrit, of whom 581 passed to step 2 (3.8% of the total). The JAK2 p.V617F mutation was detected in 7 of the 501 patients tested, which accounts for 0.04% of the total cohort and 0.4% of patients with erythrocytosis. In conclusion, this data show that our two-step algorithm improves the selection of candidates for JAK2 p.V617F testing.

Early detection of lysosomal diseases by screening of cases of idiopathic splenomegaly and/or thrombocytopenia with a next-generation sequencing gene panel.

Lysosomal diseases (LD) are a group of about 70 rare hereditary disorders (combined incidence 1:5000) in which diverse lysosomal functions are impaired, impacting multiple organs and systems. The first clinical signs and symptoms are usually unspecific and shared by hundreds of other disorders. Diagnosis of LD traditionally relies on performing specific enzymatic assays, if available, upon clinical suspicion of the disorder. However, the combination of the insidious onset of LD and the lack of awareness on these rare diseases among medical personnel results in undesirable diagnostic delays, with unchecked disease progression, appearance of complications and a worsened prognosis. We tested the usefulness of a next-generation sequencing-based gene panel for quick, early detection of LD among cases of idiopathic splenomegaly and/or thrombocytopenia, two of the earliest clinical signs observed in most LD. Our 73-gene panel interrogated 28 genes for LD, 1 biomarker and 44 genes underlying non-LD differential diagnoses. Among 38 unrelated patients, we elucidated eight cases (21%), five with LD (GM1 gangliosidosis, Sanfilippo disease A and B, Niemann-Pick disease B, Gaucher disease) and three with non-LD conditions. Interestingly, we identified three LD patients harboring pathogenic mutations in two LD genes each, which may result in unusual disease presentations and impact treatment. Turnaround time for panel screening and genetic validation was 1 month. Our results underline the usefulness of resequencing gene panels for quick and cost-effective screening of LDs and disorders sharing with them early clinical signs.

Bone marrow infiltration by flow cytometry at diffuse large B-cell lymphoma NOS diagnosis implies worse prognosis without considering bone marrow histology.

BACKGROUND: The significance of discrepant findings between histology (BMB) and flow cytometry (FC) in bone marrow (BM) examination at diffuse large B-cell lymphoma (DLBCL) diagnosis is uncertain. METHODS: We performed a 5-year retrospective single-center study of patients diagnosed by DLBCL not otherwise specified (n = 82), divided into three groups according to BM infiltration at diagnosis: BMB-/FC- (75.6%), BMB+/FC+ (13.4%), and BMB-/FC+ (11%). RESULTS: Median infiltration by FC analysis of the BMB-/FC+ group was 0.8% and if we considered BM infiltration as positive in all cases, 4/9 would be upstaged. Median follow was 33 months. Event-free survival (EFS) after 18 months was 82, 23, and 27% for BMB-/FC-, BMB-/FC+, and BMB+/FC+, respectively (p < .001). After 18 months of observation, OS was 87, 46, and 55% for BMB-/FC-, BMB-/FC+, and BMB+/FC+, respectively (p = .001). In multivariate analysis (BM infiltration vs. cell-of-origin according to Hans algorithm and standard IPI), BM infiltration was independently associated with EFS (HR: 1.94, 95% CI: 1.3-2.9) and overall survival (HR: 1.69, 95% CI: 1.1-2.7). CONCLUSION: In summary, minimal BM infiltration, detected by FC but not by BMB, has same prognostic implications than overt BM infiltration and should be considered as extranodal involvement regardless the infiltration quantity.

The utility of multiparametric seven-color flow cytometry in the detection of double hit lymphoma in ascitic fluid samples.

Double-hit lymphoma (DHL) is a rare type of lymphoma with concurrent chromosomal translocations of C-MYC with BCL2 or BCL6, associated with unfavorable prognosis. We describe a case of DHL in a 79-year-old female patient previously diagnosed with diffuse large B-cell lymphoma (DLBCL) with an early relapse in the ascitic fluid. A seven-color multiparametric flow cytometry immunophenotyping study of the ascitic fluid was carried out, and revealed 99.78% of large in size and high cellular complexity B-cells positive for CD19, CD10 (64.27%), CD45 dim, CD22 dim, CD25 (60%), CD43 bright, CD38 bright, and IgM (18.53%); and negative for CD20, CD5, CD23, CD79b, CD103, CD200, CD11c, and FMC7, and 78.99% without light chain expression and 21% with Lambda chain restriction. Due to the expression of CD19 and CD10 with overexpression of BCL-2 protein and due to CD43 and CD38 positivity detected, those cells showed features between DLBCL and Burkitt lymphoma. Fluorescence in situ hybridization (FISH) confirmed both c-MYC/IGH and BCL2/IGH rearrangement. Our findings may help to identify cases requiring additional cytogenetic analysis. © 2015 International Clinical Cytometry Society.

Reticulocyte parameters of delta beta thalassaemia trait, beta thalassaemia trait and iron deficiency anaemia.

AIMS: To analyse the differences in reticulocyte indices between delta beta thalassaemia trait (δß-TT), beta thalassaemia trait (ß-TT) and iron deficiency anaemia (IDA), and to correlate those differences with the physiopathological features of these three types of microcytoses. METHODS: We performed a descriptive study of 428 samples (43 δß-TT, 179 ß-TT and 206 IDA) that were run on Advia 2120 analyser (Siemens). The following reticulocyte indices were assessed: absolute reticulocyte count (ARC), percentage of reticulocytes, mean corpuscular volume of reticulocytes (MCVr), haemoglobin content of reticulocytes (CHr), mean corpuscular haemoglobin concentration of reticulocytes, red blood cell distribution width of reticulocytes (RDWr), haemoglobin distribution width of reticulocytes (HDWr) and reticulocyte subpopulations based on their fluorescence according to mRNA (low (L-R), medium (M-R) and high (H-R)), MCV ratio and MCHC ratio. Correlation between fetal haemoglobin (HbF) and RDWr in patients with thalassaemia was evaluated. RESULTS: RDWr was significantly higher in δß-TT compared with ß-TT (15.03% vs 13.82%, p<0.001), and so were HDWr (3.65% vs 3.27%, p<0.001), CHr (23.68 vs 22.66 pg, p<0.001) and MCVr (88.3 vs 85.5 fL, p<0.001). A good correlation was observed between HbF and RDWr (r=0.551, p<0.001). IDA subjects have more immature reticulocytes, but less ARC than ß-TT, suggesting a certain degree of inefficient erythropoiesis in IDA in comparison with ß-TT. CONCLUSIONS: Previously described differences between δß-TT, ß-TT and IDA in the corpuscular indices of mature red blood cell can also be observed in reticulocytes. The degree of anisocytosis in reticulocytes from patients with thalassaemia is correlated with HbF.

Laboratory parameters provided by Advia 2120 analyser identify structural haemoglobinopathy carriers and discriminate between Hb S trait and Hb C trait.

BACKGROUND: Haemoglobinopathies have spread owing to human migration, and the number of people needing diagnosis and management of these conditions is increasing. Clinicians need to accurately identify carriers and provide adequate genetic counselling in order to prevent the occurrence of homozygous or compound heterozygous offspring. OBJECTIVES: To identify red blood cell (RBC) laboratory parameters that discriminate between structural haemoglobinopathy carriers and healthy subjects, and to compare RBC laboratory indices between HbAS and HbAC individuals. METHODS: Samples of 500 variant Hb carriers (355 HbAS, 104 HbAC, 19 HbAD, 7 HbAE, 7 HbAO-Arab, 4 α-chain variants and 4 Hb Lepore) and 251 normal controls were run on an Advia 2120 analyser (Siemens). Classic haematological parameters and RBC populations were assessed in all subjects. A multivariable binary logistic regression model was created to predict the probability of a subject carrying any structural haemoglobinopathy. HbAS (n=355, 71%) and HbAC (n=104, 20.8%) subjects were compared. RESULTS: A clinical prediction rule was developed by assigning one point to each of the most efficient variables: mean corpuscular volume (MCV) <88.4 fL, RBC distribution width >13.4%, percentage of microcytic RBCs (%MICRO) >0.7% and the ratio of microcytic RBCs to hypochromic RBCs >0.8. A score of 0, 1, 2, 3 or 4, resulted in a probability of 9.6%, 36.3%, 66.7%, 85.2% or 98.3%, respectively. Among the most frequent variant Hb, HbAC subjects had lower values of parameters related to cell size (MCV, %MICRO) and higher values of parameters related to haemoglobin concentration (MCHC, %HYPER) than HbAS subjects. Coexistence of α-thalassaemia in both HbAS and HbAC individuals resulted in decreased Hb, MCV, MCH and MCHC. CONCLUSIONS: Structural haemoglobinopathy should be investigated in subjects belonging to ethnic groups with high prevalence of variant Hb and with a score of 3 or 4. Erythrocytes of HbAC subjects are smaller and denser than those of HbAS subjects.