Jeffrey J. Pu

Natural history of paroxysmal nocturnal hemoglobinuria clones in patients presenting as aplastic anemia

Objective: To investigate the natural history of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with acquired aplastic anemia (AA). Patients and Methods: Twenty‐seven patients with AA and a detectable PNH clone were monitored for a median of 5.7 years (range1.5–11.5 years). Twenty‐two patients received high‐dose cyclophosphamide (HiCy) therapy. The erythrocyte and granulocyte PNH clone sizes were measured using flow cytometry and analyzed via CellQuest software. PE‐conjugated anti‐glycophorin A, anti‐CD15, FITC‐conjugated anti‐CD59, and FLAER staining were used to define glycosylphosphatidylinositol‐AP‐deficient cells. Results: We found a linear relationship between PNH clone size and the development of intravascular hemolysis, assessed by lactate dehydrogenase (LDH) values (Pearson correlation coefficient = 0.80, P < 0.001 for erythrocyte PNH clones; and Pearson correlation coefficient = 0.73, P < 0.0001 for granulocyte PNH clones). An erythrocyte PNH size of 3–5% and granulocyte PNH size of 23% were the thresholds to predict hemolysis as measured by an elevated LDH (receiver operating characteristic analyses with AUC = 0.96 for erythrocyte PNH clone sizes and AUC = 0.88 for granulocyte PNH clone sizes). Patients with small (≤15%) initial PNH clone sizes were less likely to develop an elevated LDH (mean ± SD: 236.9 ± 109.9 vs. 423.1 ± 248.8; P = 0.02). Over time, the PNH clone sizes remained stable in 25.9% of patients; 48.1% experienced a rise in the PNH clone size; and 25.9% experienced a decrease. Conclusion: The risk of developing clinically significant PNH after HiCy therapy appears to be low in AA patients with PNH clones, especially for those with small initial PNH clones and for those who respond to HiCy therapy.

Optimal Sequencing of Ibrutinib, Idelalisib, and Venetoclax in Chronic Lymphocytic Leukemia: Results from a Multi-Center Study of 683 Patients.

Background Ibrutinib, idelalisib, and venetoclax are approved for treating CLL patients in the United States. However, there is no guidance as to their optimal sequence. Patients and methods We conducted a multicenter, retrospective analysis of CLL patients treated with kinase inhibitors (KIs) or venetoclax. We examined demographics, discontinuation reasons, overall response rates (ORR), survival, and post-KI salvage strategies. Primary endpoint was progression-free survival (PFS). Results A total of 683 patients were identified. Baseline characteristics were similar in the ibrutinib and idelalisib groups. ORR to ibrutinib and idelalisib as first KI was 69% and 81%, respectively. With a median follow-up of 17 months (range 1-60), median PFS and OS for the entire cohort were 35 months and not reached. Patients treated with ibrutinib (versus idelalisib) as first KI had a significantly better PFS in all settings; front-line [hazard ratios (HR) 2.8, CI 1.3-6.3, P = 0.01], relapsed-refractory (HR 2.8, CI 1.9-4.1, P < 0.001), del17p (HR 2.0, CI 1.2-3.4, P = 0.008), and complex karyotype (HR 2.5, CI 1.2-5.2, P = 0.02). At the time of initial KI failure, use of an alternate KI or venetoclax had a superior PFS when compared with chemoimmunotherapy. Furthermore, patients who discontinued ibrutinib due to progression or toxicity had marginally improved outcomes if they received venetoclax (ORR 79%) versus idelalisib (ORR 46%) (PFS HR .6, CI.3-1.0, P = 0.06). Conclusions In the largest real-world experience of novel agents in CLL, ibrutinib appears superior to idelalisib as first KI. Furthermore, in the setting of KI failure, alternate KI or venetoclax therapy appear superior to chemoimmunotherapy combinations. The use of venetoclax upon ibrutinib failure might be superior to idelalisib. These data support the need for trials testing sequencing strategies to optimize treatment algorithms.BACKGROUND Ibrutinib, idelalisib, and venetoclax are approved for treating CLL patients in the US. However, there is no guidance as to their optimal sequence. PATIENTS AND METHODS We conducted a multicenter, retrospective analysis of CLL patients treated with kinase inhibitors (KIs) or venetoclax. We examined demographics, discontinuation reasons, overall response rates (ORR), survival, and post-KI salvage strategies. Primary endpoint was progression-free survival (PFS). RESULTS A total of 683 patients were identified. Baseline characteristics were similar in the ibrutinib and idelalisib groups. ORR to ibrutinib and idelalisib as first KI was 69% and 81% respectively. With a median follow up of 17 months (range 1-60), median PFS and OS for the entire cohort were 35 months and not reached. Patients treated with ibrutinib (vs. idelalisib) as first KI had a significantly better PFS in all settings; front-line (HR 2.8, CI1.3-6.3 p=.01), relapsed-refractory (HR 2.8, CI 1.9-4.1 p<.001), del17p (HR 2.0, CI 1.2-3.4 p=.008), and complex karyotype (HR 2.5, CI 1.2-5.2 p=.02). At the time of initial KI failure, use of an alternate KI or venetoclax had a superior PFS as compared to chemoimmunotherapy (CIT). Furthermore, patients who discontinued ibrutinib due to progression or toxicity had marginally improved outcomes if they received venetoclax (ORR 79%) versus idelalisib (ORR 46%) (PFS HR .6, CI.3-1.0, p=.06). CONCLUSIONS In the largest real-world experience of novel agents in CLL, ibrutinib appears superior to idelalisib as first KI. Further, in the setting of KI failure, alternate KI or venetoclax therapy appear superior to CIT combinations. The use of venetoclax upon ibrutinib failure might be superior to idelalisib. These data support the need for trials testing sequencing strategies to optimize treatment algorithms.

Paroxysmal Nocturnal Hemoglobinuria from Bench to Bedside

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease that presents with protean manifestations. Clinical and laboratory investigation over the past 25 years has uncovered most of the basic science underpinnings of PNH and has led to the development of a highly effective targeted therapy. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a somatic mutation in a gene called phosphatidylinositol glycan anchor biosynthesis, class A (PIG‐A). The PIG‐A gene is required for the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Failure to synthesize GPI anchors leads to an absence of all proteins that utilize GPI to attach to the plasma membrane. Two GPI‐anchor proteins, CD55 and CD59, are complement regulatory proteins; their absence on the surface of PNH cells leads to complement‐mediated hemolysis. The release of free hemoglobin leads to scavenging of nitric oxide and contributes to many clinical manifestations, including esophageal spasm, fatigue, and possibly thrombosis. Aerolysin is a pore‐forming toxin that binds GPI‐anchored proteins and kills normal cells, but not PNH cells. A fluorescinated aerolysin variant (FLAER) binds GPI‐anchor and serves as a novel reagent diagnosing PNH. Eculizumab, a humanized monoclonal antibody against C5, is the first effective drug therapy for PNH. Clin Trans Sci 2011; Volume 4: 219–224

Successful discontinuation of eculizumab therapy in a patient with aHUS

Dear Editor, Atypical hemolytic uremic syndrome (aHUS) is a lifethreatening complement-mediated thrombotic microangiopathy that leads to progressive thrombocytopenia, kidney injury, mental status changes, and even death if without proper treatment [1]. Eculizumab is so far the only medication that effectively resolves the clinical symptoms and significantly improves end-organ function [2]. Current practice suggests a long-term eculizumab therapy to maintain terminal complement inhibition. We describe here an aHUS patient who achieved remission with eculizumab treatment and remains in clinical remission 1 year after discontinuation of eculizumab. The patient, an 85-year-old female, initially presented with fever, abdominal cramping pain, diarrhea, and hematochezia. One blood culture was positive for Pseudomonas aeruginosa . She was aggressively treated with systemic antibiotics but developed mental status changes with seizure activity requiring protective intubation and acute renal failure needing hemodialysis (HD). Her peripheral blood smear showed 30 % of schistocytes. Her laboratory data showed a hemoglobin of 6.9 g/dL (reference range, 13–17 g/dL), a platelet count of 16× 10/L (reference range, 150–350×10/L), an LDH of 1,974 U/L (reference range, 313—618 U/L), a serum creatinine of 4.6 mg/dL (reference range, 0.7–1.3 mg/ dL), reduced eGFR of 14 mL/min/1.73 m, and ADAMTS13 activity of 71 % (reference range, >67 %). She started on therapeutic plasma exchange (TPE) with platelet count improvement. However, her renal function deteriorated to oliguria, and she lost consciousness while on TPE/HD. Further laboratory workup revealed a low C3 of 57 mg/dL (reference range, 88–185 mg/dL), a normal CH50 of 35 U/mL (reference range, 31–60 U/mL), and negative tests for complement factor H (CFH) mutation and antibody. A test for shiga toxin-producing Escherichia coli was negative. Eculizumab then was administered 2 weeks after receiving meningococcal vaccine. Her mental status, seizure activity, and kidney function dramatically improved after several doses of eculizumab. She was extubated and stopped hemodialysis in 2 and 4 weeks of starting eculizumab therapy, respectively (Fig. 1). Twelve weeks after receiving eculizumab therapy, she developed sepsis originating from a urinary tract infection. She was treated with intravenous antibiotics, and her eculizumab was discontinued for suspicion of meningitis. Her lumbar puncture showed a clear CSF, and she eventually recovered from sepsis. She has been of eculizumab for 12 months with a normal CBC profile, mental status, and kidney function. aHUS is characterized by an uncontrolled activation of the alternative complement pathway and is typically caused by a deficit in complement regulatory proteins or other factors. This patient’s clinical presentation and laboratory results were consistent with a diagnosis of aHUS. Though tests for both CFH mutation and Statement: This “Letter to the editor” reports and discusses an interesting aHUS patient case. This retrospective patient case study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All procedures followed this study were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. J. J. Pu :A. Sido Penn State Hershey Cancer Institute, Penn State Hershey Medical Center, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA

Onset of expression of the components of the kell blood group complex

BACKGROUND: Kell and XK, two distinct red blood cell membrane proteins, are linked by a disulfide bond and form the Kell blood group complex. Kell surface antigens are expressed early during erythropoiesis but the onset of expression of XK which carries the Kx antigen is unknown.

Complement blockade with a C1 esterase inhibitor in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure, and a propensity for thrombosis. These patients experience both intra- and extravascular hemolysis in the context of underlying complement activation. Currently eculizumab effectively blocks the intravascular hemolysis PNH. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade to block complement at the classical and alternative pathways. C1 esterase inhibitor (C1INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway through inhibition of the classical pathway by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. In this study, we show that commercially available plasma derived C1INH prevents lysis induced by the alternative complement pathway of PNH erythrocytes in human serum. Importantly, C1INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patient on eculizumab therapy. This could suggest a role for inhibition of earlier phases of the complement cascade than that currently inhibited by eculizumab for incomplete or nonresponders to that therapy.

PD-1 pathway and its clinical application: A 20 year journey after discovery of the complete human PD-1 gene

Anti-PD-1 therapy is a novel immune-checkpoint inhibition therapy with tremendous potential in treating refractory/relapsed cancers. The 20year journey of human PD-1 research went through 3 phases: 1) discovering PD-1 gene structure and genomic organization, 2) understanding the mechanism of PD-1 mediated immune-checkpoint regulatory effects in coordination with its ligands (PD-L1 and L2), 3) and translating our knowledge of PD-1 gene into a robust clinical anticancer approach by targeting the PD-1 immune-checkpoint pathway. The success of human PD-1 gene study reflects the advancement and trends of modern biomedical research from the laboratory to the bedside. However, our journey of understanding the PD-1 gene is not yet complete. Clinical investigation data show a high variety of response rates among different types of cancers to PD-1 immune-checkpoint inhibition therapy, with a range of 18% to 87%. There is no reliable biomarker to predict an individual patients response to PD-1 inhibitory immunotherapy. Patients can present with primary, adaptive, or even acquired resistance to PD-1 immune-checkpoint inhibition therapy. Furthermore, the emerging data demonstrates that certain patients experience hyperprogressive disease status after receiving PD-1 immune-checkpoint inhibition therapy. In conclusion, PD-1 immune-checkpoint inhibition therapy has opened up a new venue of advanced cancer immunotherapy. Meanwhile, further efforts are still warranted in both basic scientific mechanism studies and clinical investigation using the principles of personalized and precision medicine.

The small population of PIG-A mutant cells in myelodysplastic syndromes do not arise from multipotent hematopoietic stem cells

Background Patients with paroxysmal nocturnal hemoglobinuria harbor clonal glycosylphosphatidylinositol-anchor deficient cells arising from a multipotent hematopoietic stem cell acquiring a PIG-A mutation. Many patients with aplastic anemia and myelodysplastic syndromes also harbor small populations of glycosylphosphatidylinositol-anchor deficient cells. Patients with aplastic anemia often evolve into paroxysmal nocturnal hemoglobinuria; however, myelodysplastic syndromes seldom evolve into paroxysmal nocturnal hemoglobinuria. Here, we investigate the origin and clonality of small glycosylphosphatidylinositol-anchor deficient cell populations in aplastic anemia and myelodysplastic syndromes. Design and Methods We used peripheral blood flow cytometry to identify glycosylphosphatidylinositol-anchor deficient blood cells, a proaerolysin-resistant colony forming cell assay to select glycosylphosphatidylinositol-anchor deficient progenitor cells, a novel T-lymphocyte enrichment culture assay with proaerolysin selection to expand glycosylphosphatidylinositol-anchor deficient T lymphocytes, and PIG-A gene sequencing assays to identify and analyze PIG-A mutations in patients with aplastic anemia and myelodysplastic syndromes. Results Twelve of 15 aplastic anemia patients were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient granulocytes; 11 of them were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient erythrocytes, 10 patients were detected to harbor glycosylphosphatidylinositol-anchor deficient T lymphocytes, and 3 of them were detected only after T-lymphocyte enrichment in proaerolysin selection. PIG-A mutation analyses on 3 patients showed that all of them harbored a matching PIG-A mutation between CFU-GM and enriched T lymphocytes. Two of 26 myelodysplastic syndromes were found to harbor small populations of glycosylphosphatidylinositol-anchor deficient granulocytes and erythrocytes transiently. Bone marrow derived CD34+ cells from 4 patients grew proaerolysin-resistant colony forming cells bearing PIG-A mutations. No glycosylphosphatidylinositol-anchor deficient T lymphocytes were detected in myelodysplastic syndrome patients. Conclusions In contrast to aplastic anemia and paroxysmal nocturnal hemoglobinuria, where PIG-A mutations arise from multipotent hematopoietic stem cells, glycosylphosphatidylinositol-anchor deficient cells in myelodysplastic syndromes appear to arise from more committed progenitors.

Burst-forming unit-erythroid assays to distinguish cellular bone marrow failure disorders.

Patients with cytopenias and a cellular bone marrow can be a diagnostic and therapeutic challenge. Previous reports suggested a role for progenitor assays for diagnosis and predicting response to therapy. We report the results of Burst-forming unit-erythroid (BFU-E) assays in 48 consultative cases of single or multilineage cytopenias with cellular marrows. The final diagnoses included 17 patients with myelodysplastic syndrome, 9 patients with pure red cell aplasia (non-large granular lymphocytosis [LGL] in etiology], 15 patients with LGL (eight of whom had a single-lineage cytopenia only, whereas the other seven had multilineage cytopenias), and 7 patients with cytopenias associated with systemic inflammation from autoimmune conditions. In this cohort, nonmalignant diseases were well-distinguished from myelodysplastic syndrome by BFU-E growth. Our data suggest that low BFU-E growth (less than 10 BFU-E per 10(5) marrow mononuclear cells) helps to exclude LGL, pure red cell aplasia, or cytopenias associated with systemic inflammation as a cause of pancytopenia with a sensitivity of 96.8%, specificity of 76.5%, and a predictive value of 88.2% (p = 0.0001). BFU-E growth also was examined to predict treatment response. Of the 29 patients in this cohort treated with immunosuppressive therapy, there was an 86% response rate with 25 responders (11 partial responses and 14 complete responses) and 4 nonresponders. This result correlated with higher BFU-E growth. Our results suggest that BFU-E assays are a useful adjunct in the diagnosis and management of cytopenias in the setting of a normocellular or hypercellular marrows.

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.