D. Robert Sutherland

Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors. While flow cytometry is the method of choice for identifying cells deficient in GPI‐linked proteins and is, therefore, necessary for the diagnosis of PNH, to date there has not been an attempt to standardize the methodology used to identify these cells.

Peripheral blood progenitor cell collections in multiple myeloma: predictors and management of inadequate collections

Thirty‐seven patients with previously treated multiple myeloma (MM) underwent peripheral blood progenitor cell (PBPC) collection following high‐dose cyclophosphamide and GM‐CSF or sequential IL‐3 and GM‐CSF. Patients with an inadequate collection were considered for a second or third collection. 25 patients underwent subsequent autotransplant. The only variable predictive of CFU‐GM yield was the extent of prior melphalan therapy. All repeat collections were unsuccessful and patients infused with an autograft obtained from multiple sets of collections had a high incidence of delayed engraftment. We conclude that melphalan should be avoided or PBPC collection performed early in the disease course in patients who are potential transplant candidates.

Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Activated Protein Kinase C Directly Phosphorylates the CD34 Antigen in Acute Lymphoblastic Leukemia Cells

The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.

Role of Aberrant Sialylation of Chronic Myeloid Leukemia Granulocytes on Binding and Signal Transduction by Chemotactic Peptides and Colony Stimulating Factors

Chronic myelogenous leukemia (CML) granulocytes exhibit a number of characteristics attributable to immature granulocytes, including marked increases in cell surface sialylation of glycoproteins which may be due, at least in part, to an increased activity of cytidine 5-monophosphate-N-acetylneuraminic acid:Ga1 beta 1-3Ga1NAc alpha(2-3)-sialyltransferase (EC 2.4.99.4), and perhaps to altered activity of other glycosyltransferases and sialidases. This aberrant sialylation of CML granulocytes contributes to the decreased binding of the synthetic chemotactic peptide, formyl Met Leu Phe (fMLP), to the surface of CML granulocytes which leads to a rapid, transient increase in cytosolic free calcium ([Ca2+]i), an integral step in the biochemical cascade leading to cell activation. To determine if the decrease in binding of fMLP to CML granulocytes translates into a functional deficit, we measured fMLP-induced increases in [Ca2+]i. Compared to normal granulocytes, fMLP-induced increases in [Ca2+]i were markedly decreased in CML granulocytes. After sialidase treatment, a significant augmentation in fMLP-induced increases in [Ca2+]i was noted in CML granulocytes, indicating that the decreased signalling may be a consequence of aberrant sialylation. To determine if the effects of aberrant sialylation also alters the binding of endogenous polypeptide mediators, we determined the effect of desialylation of CML and normal granulocytes on binding of the colony stimulating factor for granulocytes and monocytes (GM-CSF), which plays a role in differentiation and proliferation of myeloid-lineage cells. As with fMLP binding, we also showed that the binding of GM-CSF to CML granulocytes, but not normal granulocytes, was markedly increased after sialidase treatment. Similarly, binding of GM-CSF to undifferentiated HL-60 cells was markedly increased after sialidase treatment. Therefore, we have demonstrated that aberrant sialylation of CML granulocytes not only alters the binding of fMLP and GM-CSF to their receptor(s), but may also alter signal transduction. Thus, aberrant glycosylation of CML granulocytes may reduce the binding of hematopoietic growth factors, which in turn may be responsible for the immature phenotype of CML granulocytes.

How we treat paroxysmal nocturnal hemoglobinuria: A consensus statement of the Canadian PNH Network and review of the national registry

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease characterized by intravascular hemolysis, thrombophilia, and marrow failure. Its phenotype is due to absent or reduced expression of GPI‐linked complement regulators and subsequent sensitivity of hematopoietic cells to complement‐mediated damage and lysis. Introduction of the terminal complement inhibitor eculizumab drastically improved outcomes in PNH patients; however, despite this improvement, there remain several challenges faced by PNH patients and physicians who care for them. One of the most important is increasing awareness of the heterogeneity with which patients can present, which can lead to significant delays in recognition. Data from the Canadian PNH Registry are presented to demonstrate the variety of presenting symptoms. In Canada, geography precludes consolidation of care to just a few centers, so management is distributed across academic hospitals, linked together as the Canadian PNH Network. The Network over the last several years has developed educational programs and clinical checklists and has worked to standardize access to diagnostics across the country. Herein, we address some of the common diagnostic and therapeutic challenges faced by PNH physicians and give our recommendations. Gaps in knowledge are also addressed, and where appropriate, consensus opinion is provided.