Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population of platelets derived from the abnormal stem cell clone. However, identification of GPI (-) platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.

Baseline clinical characteristics and disease burden in patients with paroxysmal nocturnal hemoglobinuria (PNH): updated analysis from the International PNH Registry.

The International Paroxysmal Nocturnal Hemoglobinuria (PNH) Registry (NCT01374360) was initiated to optimize patient management by collecting data regarding disease burden, progression, and clinical outcomes. Herein, we report updated baseline demographics, clinical characteristics, disease burden data, and observed trends regarding clone size in the largest cohort of Registry patients. Patients with available data as of July 2017 were stratified by glycosylphosphatidylinositol (GPI)-deficient granulocyte clone size (< 10%, ≥ 10%-< 50%, and ≥ 50%). All patients were untreated with eculizumab at baseline, defined as date of eculizumab initiation or date of Registry enrollment (if never treated with eculizumab). Outcomes assessed in the current analysis included proportions of patients with high disease activity (HDA), history of major adverse vascular events (MAVEs; including thrombotic events [TEs]), bone marrow failure (BMF), red blood cell (RBC) transfusions, and PNH-related symptoms. A total of 4439 patients were included, of whom 2701 (60.8%) had available GPI-deficient granulocyte clone size data. Among these, median clone size was 31.8% (1002 had < 10%; 526 had ≥ 10%-< 50%; 1173 had ≥ 50%). There were high proportions of patients with HDA (51.6%), history of MAVEs (18.8%), BMF (62.6%), RBC transfusion (61.3%), and impaired renal function (42.8%). All measures except RBC transfusion history significantly correlated with GPI-deficient granulocyte clone size. A large proportion of patients with GPI-deficient granulocyte clone size < 10% had hemolysis (9.7%), MAVEs (10.2%), HDA (9.1%), and/or PNH-related symptoms. Although larger GPI-deficient granulocyte clone sizes were associated with higher disease burden, a substantial proportion of patients with smaller clone sizes had history of MAVEs/TEs.

Beneficial effects of eculizumab regardless of prior transfusions or bone marrow disease: Results of the International Paroxysmal Nocturnal Hemoglobinuria Registry.

OBJECTIVES: To evaluate the effects of eculizumab on transfusions and thrombotic events (TEs) in patients with and without prior history of transfusion in the International Paroxysmal Nocturnal Hemoglobinuria (PNH) Registry. METHODS: Registry patients enrolled on or before January 1, 2018, initiated on eculizumab no more than 12 months prior to enrollment, having known transfusion status for the 12 months before eculizumab initiation, and ≥12 months of Registry follow-up after eculizumab initiation, were included. RESULTS: Eculizumab treatment was associated with a 50% reduction in transfusions in patients with a transfusion history (10.6 units/patient-year before eculizumab vs 5.4 after; P < .0001), with greater reduction observed in those with no history of bone marrow disease vs those with bone marrow disease. Mean lactate dehydrogenase levels decreased from a mean of 6.7 to 1.4 times the upper limit of normal (ULN) in patients with transfusion history and from 5.1 to 1.2 times ULN in those with no transfusion history. TE and major adverse vascular event rates also decreased by 70% in patients with and without history of transfusion. CONCLUSIONS: The benefit of eculizumab therapy does not appear to be limited to any group defined by transfusion history or bone marrow disease history.

MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia.

Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous deletions in MSH6, a crucial part of DNA mismatch repair, are frequently detected. Our aim was to determine whether MSH6 deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down MSH6 in mismatch repair proficient cell lines (697 and UOCB1) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold) in vitro, as well as increased resistance to 6-mercaptopurine treatment in vivo No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070 vs 1722 fmol/µg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure. MSH6 knockdown cells showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant MSH6 adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.

Leukemic blasts with the paroxysmal nocturnal hemoglobinuria phenotype in children with acute lymphoblastic leukemia.

It has been proposed that genomic instability is essential to account for the multiplicity of mutations often seen in malignancies. Using the X-linked PIG-A gene as a sentinel gene for spontaneous inactivating somatic mutations, we previously showed that healthy individuals harbor granulocytes with the PIG-A mutant (paroxysmal nocturnal hemoglobinuria) phenotype at a median frequency (f) of ∼12 × 10(-6). Herein, we used a similar approach to determine f in blast cells derived from 19 individuals with acute lymphoblastic leukemia (ALL) and in immortalized Epstein-Barr virus-transformed B-cell cultures (human B-lymphoblastoid cell lines) from 19 healthy donors. The B-lymphoblastoid cell lines exhibited a unimodal distribution, with a median f value of 11 × 10(-6). In contrast, analysis of the f values for the ALL samples revealed at least two distinct populations: one population, representing approximately half of the samples (n = 10), had a median f value of 13 × 10(-6), and the remaining samples (n = 9) had a median f value of 566 × 10(-6). We conclude that in ALL, there are two distinct phenotypes with respect to hypermutability, which we hypothesize will correlate with the number of pathogenic mutations required to produce the leukemia.

The rate of spontaneous mutations in human myeloid cells.

The mutation rate (µ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of µ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median µ value was ∼9.4×10(-7) (range ∼3.6-23×10(-7)) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on µ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis.

Thrombolytic therapy is effective in paroxysmal nocturnal hemoglobinuria: a series of nine patients and a review of the literature.

BACKGROUND: Thrombosis is the major risk factor for death in patients with paroxysmal nocturnal hemoglobinuria. Previous case reports indicate that venous thrombosis in patients with paroxysmal nocturnal hemoglobinuria is amenable to thrombolysis. DESIGN AND METHODS: We reviewed the outcome of thrombolytic therapy for patients with paroxysmal nocturnal hemoglobinuria who had thromboses refractory to anticoagulation at our institutions. RESULTS: In this study of 41 patients who had at least one thrombotic event, we confirmed a very high incidence of recurrence despite anticoagulation. Nine patients with thrombosis were regarded as eligible for administration of intravenous tissue plasminogen activator, which was effective in reversing thrombi in all of 15 occasions in which it was given. Serious hemorrhagic complications developed in three cases. At last follow-up visit, of the nine patients treated, three had died, and six were in very good to excellent condition in terms of clinical outcome and radiological findings. The only patient in whom thrombolysis may have contributed to a fatal outcome also had complications of "heparin induced thrombocytopenia with thrombosis", which we diagnosed in three additional patients. In our review of the literature, nine out of 15 patients treated with thrombolysis have had a good outcome. CONCLUSIONS: Although it is associated with a significant but manageable risk of bleeding, systemic thrombolysis is a highly effective treatment for reversing venous thromboses in patients with paroxysmal nocturnal hemoglobinuria.

The use of PIG-A as a sentinel gene for the study of the somatic mutation rate and of mutagenic agents in vivo.

Mutations are an inherent risk of cell duplication. On one hand, inheritable mutations are the driving force of biological evolution; on the other hand, their accumulation in somatic cells plays a key role in the development of cancer. The frequency of mutants (f) and the rate of mutation (mu) are biological features of any cell population: their measurement could provide important information about the risk of oncogenesis and the exposure to carcinogenic agents. However, the measurement of these parameters is not trivial. To measure f and mu, a potential sentinel gene is the PIG-A gene, which encodes one of the subunits of an enzyme essential in the biosynthesis of glycosylphosphatidylinositol (GPI). Since PIG-A is X-linked, mutational inactivation of the one single copy active in somatic cells entails absence from the cell surface of all the proteins that require GPI for attachment to the membrane: thus, mutant cells display a GPI-negative surface phenotype that can be easily detected by flow cytometry. The measurement of PIG-A mutants by counting cells with the GPI-negative phenotype has proved to be effective to measure mutant frequency in peripheral blood cells of humans and of others animals. Up to now, mu has been exceedingly difficult to measure in human cells; however, by using as a sentinel the PIG-A gene in lymphoblastoid cell lines we now have a test that makes it practical to measure mu in human cells.

A quantitative analysis of genomic instability in lymphoid and plasma cell neoplasms based on the PIG-A gene.

It has been proposed that hypermutability is necessary to account for the high frequency of mutations in cancer. However, historically, the mutation rate (mu) has been difficult to measure directly, and increased cell turnover or selection could provide an alternative explanation. We recently developed an assay for mu using PIG-A as a sentinel gene and estimated that its average value is 10.6 x 10(-7) mutations per cell division in B-lymphoblastoid cell lines (BLCLs) from normal donors. Here we have measured mu in human malignancies and found that it was elevated in cell lines derived from T cell acute lymphoblastic leukemia, mantle cell lymphoma, follicular lymphoma in transformed phase, and 2 plasma cell neoplasms. In contrast, mu was much lower in a marginal zone lymphoma cell line and 5 other plasma cell neoplasms. The highest mu value that we measured, 3286 x 10(-7), is 2 orders of magnitude above the range we have observed in non-malignant human cells. We conclude that the type of genomic instability detected in this assay is a common but not universal feature of hematologic malignancies.

Mild Clinical Course of COVID-19 in 3 Patients Receiving Therapeutic Monoclonal Antibodies Targeting C5 Complement for Hematologic Disorders.

BACKGROUND Patients receiving immunosuppressive therapies might be more susceptible to COVID-19. Conversely, an exaggerated inflammatory response to the SARS-CoV-2 infection might be blunted by certain forms of immunosuppression, which could be protective. Indeed, there are data from animal models demonstrating that complement may be a part of the pathophysiology of coronavirus infections. There is also evidence from an autopsy series demonstrating complement deposition in the lungs of patients with COVID-19. This raises the question of whether patients on anti-complement therapy could be protected from COVID-19. CASE REPORT Case 1 is a 39-year-old woman with an approximately 20-year history of paroxysmal nocturnal hemoglobinuria (PNH), who had recently been switched from treatment with eculizumab to ravulizumab prior to SARS-CoV-2 infection. Case 2 is a 54-year-old woman with a cadaveric renal transplant for lupus nephritis, complicated by thrombotic microangiopathy, who was maintained on eculizumab, which she started several months before she developed the SARS-CoV-2 infection. Case 3 is a 60-year-old woman with a 14-year history of PNH, who had been treated with eculizumab since 2012, and was diagnosed with COVID-19 at the time of her scheduled infusion. All 3 patients had a relatively mild course of COVID-19. CONCLUSIONS We see no evidence of increased susceptibility to SARS-CoV-2 in these patients on anti-complement therapy, which might actually have accounted for the mild course of infection. The effect of anti-complement therapy on COVID-19 disease needs to be determined in clinical trials.

Splenic infarction and subsequent splenic rupture in a patient with paroxysmal nocturnal hemoglobinuria and heparin-induced thrombocytopenia.

We describe a patient with paroxysmal nocturnal hemoglobinuria (PNH) and no previous history of thrombosis who presented with hepatic venous thromboses and subsequently developed splenic infarction and rupture requiring splenectomy while on anticoagulation therapy for the hepatic thromboses. The patient's anticoagulation was complicated by heparin-induced thrombocytopenia (HIT) highlighting the unique management challenge presented by PNH in combination with HIT.

Spontaneously arising red cells with a McLeod-like phenotype in normal donors.

Very few human genes can be used to identify spontaneous inactivating somatic mutations. We hypothesized that because the XK gene is X-linked, it would be easy to identify spontaneously arising red cells with a phenotype resembling the McLeod syndrome, which results from inherited XK mutations. Here, by flow cytometry, we detect such phenotypic variants at a median frequency of 9 x 10(-6) in neonatal cord blood samples and 39 x 10(-6) in healthy adults (p=0.004). It may be possible to further investigate the relationship between aging, mutations, and cancer using this approach.

Analysis of TET2 mutations in paroxysmal nocturnal hemoglobinuria (PNH).

BACKGROUND: Large clonal populations of cells bearing PIG-A mutations are the sine qua non of PNH, but the PIG-A mutation itself is insufficient for clonal expansion. The association between PNH and aplastic anemia supports the immune escape model, but not all PNH patients demonstrate a history of aplasia; therefore, second genetic hits driving clonal expansion have been postulated. Based on the previous identification of JAK2 mutations in patients with a myeloproliferative/PNH overlap syndrome, we considered TET2 as a candidate gene in which mutations might be contributing to clonal expansion. METHODS: Here we sequenced the TET2 and JAK2 genes in 19 patients with large PNH clones. RESULTS: We found one patient with a novel somatic nonsense mutation in TET2 in multiple hematopoietic lineages, which was detectable upon repeat testing. This patient has had severe thromboses and has relatively higher peripheral blood counts compared with the other patients-but does not have other features of a myeloproliferative neoplasm. CONCLUSIONS: We conclude that mutations in TET2 may contribute to clonal expansion in exceptional cases of PNH.

A quantitative measurement of the human somatic mutation rate.

The mutation rate (mu) is a key biological feature of somatic cells that determines risk for malignant transformation, and it has been exceedingly difficult to measure in human cells. For this purpose, a potential sentinel is the X-linked PIG-A gene, because its inactivation causes lack of glycosylphosphatidylinositol-linked membrane proteins. We previously found that the frequency (f) of PIG-A mutant cells can be measured accurately by flow cytometry, even when f is very low. Here we measure both f and mu by culturing B-lymphoblastoid cell lines and first eliminating preexisting PIG-A mutants by flow sorting. After expansion in culture, the frequency of new mutants is determined by flow cytometry using antibodies specific for glycosylphosphatidylinositol-linked proteins (e.g., CD48, CD55, and CD59). The mutation rate is then calculated by the formula mu = f/d, where d is the number of cell divisions occurring in culture. The mean mu in cells from normal donors was 10.6 x 10(-7) mutations per cell division (range 2.4 to 29.6 x 10(-7)). The mean mu was elevated >30-fold in cells from patients with Fanconi anemia (P < 0.0001), and mu varied widely in ataxia-telangiectasia with a mean 4-fold elevation (P = 0.002). In contrast, mu was not significantly different from normal in cells from patients with Nijmegen breakage syndrome. Differences in mu could not be attributed to variations in plating efficiency. The mutation rate in man can now be measured routinely in B-lymphoblastoid cell lines, and it is elevated in cancer predisposition syndromes. This system should be useful in evaluating cancer risk and in the design of preventive strategies.

High incidence of thrombosis in African-American and Latin-American patients with Paroxysmal Nocturnal Haemoglobinuria.

Paroxysmal Nocturnal Haemoglobinuria (PNH) results in a marked thrombophilic state by unknown mechanisms. Geographic differences in thrombosis incidence in PNH have been observed. We have reviewed 64 patients with "Classic PNH" from a single institution in order to determine the rate of thrombosis in different ethnic groups. When we compared African-Americans (n=11) and Latin-Americans (n=8) with other patients (n=45), we found that African-American and Latin-American patients are at increased risk [Hazard ratio 3.66 (p=0.005) and 3.52, (p=0.035) respectively by Cox regression]. Our data also suggest that this difference in the rate of thrombosis has an impact on length of survival. These findings demonstrate that ethnicity is a risk factor for thrombosis in PNH and have implications for decision-making regarding the management of these patients, including the prevention of thrombosis.

Selective splenic artery embolization for the treatment of thrombocytopenia and hypersplenism in paroxysmal nocturnal hemoglobinuria.

BACKGROUND: PNH is associated with abdominal vein thrombosis, which can cause splenomegaly and hypersplenism. The combination of thrombosis, splenomegaly, and thrombocytopenia (TST) is challenging because anticoagulants are indicated but thrombocytopenia may increase the bleeding risk. Splenectomy could alleviate thrombocytopenia and reduce portal pressure, but it can cause post-operative thromboses and opportunistic infections. We therefore sought to determine whether selective splenic artery embolization (SSAE) is a safe and effective alternative to splenectomy for TST in patients with PNH. METHODS: Four patients with PNH and TST received successive rounds of SSAE. By targeting distal vessels for occlusion, we aimed to infarct approximately 1/3 of the spleen with each procedure. RESULTS: Three of 4 patients had an improvement in their platelet count, and 3 of 3 had major improvement in abdominal pain/discomfort. The one patient whose platelet count did not respond had developed marrow failure, and she did well with an allo-SCT. Post-procedure pain and fever were common and manageable; only one patient developed a loculated pleural effusion requiring drainage. One patient, who had had only a partial response to eculizumab, responded to SSAE not only with an improved platelet count, but also with an increase in hemoglobin level and decreased transfusion requirement. CONCLUSIONS: These data indicate that SSAE can decrease spleen size and reverse hypersplenism, without exposing the patient to the complications of splenectomy. In addition, SSAE probably reduces the uptake of opsonised red cells in patients who have had a limited response to eculizumab, resulting in an improved quality of life for selected patients.

Multiple myeloma involving skin and pulmonary parenchyma after autologous stem cell transplantation.

Pulmonary involvement and skin involvement are rare complications of plasma cell neoplasms. Here we describe what may be the first reported case of a patient with relapse in both of these sites following autologous peripheral blood stem cell transplantation.

The mutation rate in PIG-A is normal in patients with paroxysmal nocturnal hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the presence in the patient's hematopoietic system of a large cell population with a mutation in the X-linked PIG-A gene. Although this abnormal cell population is often found to be monoclonal, it is not unusual that 2 or even several PIG-A mutant clones coexist in the same patient. Therefore, it has been suggested that the PIG-A gene may be hypermutable in PNH. By a method we have recently developed for measuring the intrinsic rate of somatic mutations (mu) in humans, in which PIG-A itself is used as a sentinel gene, we have found that in 5 patients with PNH, mu ranged from 1.24 x 10(-7) to 11.2 x 10(-7), against a normal range of 2.4 x 10(-7) to 29.6 x 10(-7) mutations per cell division. We conclude that genetic instability of the PIG-A gene is not a factor in the pathogenesis of PNH.