Standardization of Nucleic Acid Tests: the Approach of the World Health Organization.

The first World Health Organization (WHO) international standards (ISs) for nucleic acid amplification techniques were established two decades ago, with the initial focus on blood screening for three major viral targets, i.e., hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1. These reference materials have subsequently found utility in the diagnosis and monitoring of a wide range of infectious diseases in clinical microbiology laboratories worldwide. WHO collaborating centers develop ISs and coordinate international studies for their evaluation. The WHO Expert Committee on Biological Standardization is responsible for the endorsement of new standardization projects and the establishment of new and replacement ISs. Potencies of ISs are defined in international units (IU); the reporting in IU for assays calibrated with an IS (or secondary standards traceable to the IS) facilitates comparability of results for different assays and determination of assay parameters such as analytical sensitivities.

Membranes replace irradiated blast cells as growth requirement for leukemic blast progenitors in suspension culture.

The blast cells of acute myeloblastic leukemia (AML) may be considered as a renewal population, maintained by blast stem cells capable of both self-renewal and the generation of progeny with reduced or absent proliferative potential. Blast progenitor renewal is manifested in suspension culture by an exponential increase in clonogenic cells. This growth requires that two conditions be met: first, the cultures must contain growth factors in media conditioned either by phytohemagglutinin (PHA)-stimulated mononuclear leukocytes (PHA-LCM), or by cells of the continuous bladder carcinoma line HTB9 (HTB9-CM). Second, the cell density must be maintained at 10(6) blasts/ml; this may be achieved by adding irradiated cells to smaller numbers of intact blasts. We are concerned with the mechanism of the feeding function. We present evidence that (a) cell-cell contact is required. (b) Blasts are heterogeneous in respect to their capacity to support growth. (c) Fractions containing membranes from blast cells will substitute for intact cells in promoting the generation of new blast progenitors in culture. (d) This membrane function may be specific for AML blasts, since membranes from blasts of lymphoblastic leukemia or normal marrow cells were inactive.

Expression of the p53 oncogene in acute myeloblastic leukemia.

We have investigated whether the p53 oncogene is expressed in the blast cells of patients with acute myeloblastic leukemia. p53 protein was detected in the blast cells of 19 out of 34 patients, but not in normal myelopoietic cells. We find a highly significant correlation between p53 protein synthesis in leukemic blast cells and the secondary plating efficiency of these cells (p = 0.0001). The latter provides an estimate for the self renewal capacity of progenitor cells in the blast population. These data indicate that p53 may be involved in leukemic stem cell renewal.

Cytological evidence for a relationship between normal hemotopoietic colony-forming cells and cells of the lymphoid system.

The relationship between hematopoietic colony-forming stem cells and cells in the thymus and lymph nodes of unirradiated mice has been investigated using a chromosome-marker technique. It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells. It was also found that cells belonging to the same clone as colony-forming cells may reach the lymph nodes, and that nodes containing such cells can participate in an immunological response against sheep red cells. Either the precursors of cells in thymus and lymph node are identical with hematopoietic colony-forming cells, or they are both descendants of a common precursor which has not yet been identified. The results are compatible with the view that cells of the hematopoietic system and the immune system may be derived from the same stem cell.

Physical separation of hemopoietic stem cells differing in their capacity for self-renewal.

Bone marrow cells in suspension were separated into a number of fractions on the basis of cell size by sedimentation at unit gravity through gradients of fetal calf serum. The colony forming units (CFU) from the various fractions were tested for their self-renewal capacity using a double transplantation technique. The results indicate that the CFU in the fractions containing slowly sedimenting cells have an increased capacity for self-renewal in comparison with CFU in fractions containing rapidly sedimenting cells. In addition, a culture method was used to select populations containing CFU with increased self-renewal capacity, and these CFU were shown to sediment slowly in comparison with CFU of lower self-renewal capacity obtained from control cultures. It may be concluded that at least part of the heterogeneity observed in the CFU content of individual spleen colonies arises from the composition of the initial cell suspension, probably from intrinsic differences between the stem cells themselves.

Antibodies in human sera to oncorna virus-like proteins from normal or leukemia marrow cell cultures.

Some human marrows in culture release particles with oncornavirus-like properties. This study was designed to examine the immunological properties of similar particles in human marrow culture supernates. Leukemic and nonleukemic marrows were cultured for 5-7 days in the presence of [14C]uridine and [3H]leucine or [3H]glucosamine. Labeled supernatant components banding in sucrose gradient densities of 1.20-1.24 g/ml were used as antigen in a double antibody immunoprecipitation assay. The assay was validated by end point titrations and competition with unlabeled antigen; purified myeloma proteins were used as negative controls. Cross-reactivity with mammalian oncornaviruses, as judged by competitive inhibition of precipitation by these viruses, was slight and at the border of the sensitivity of the method. Precipitated antigens analyzed by SDS polyacrylamide gel electrophoresis contained three distinct polypeptides of about 70,000, 45,000 and 30,000 mol wt; these comigrated with the gp 70, pg 45, and p 30 of a murine leukemia virus. Similar polypeptides were obtained from both leukemic and nonleukemic marrow culture supernates. As determined by the radioimmunoprecipitation assay, 32 of 45 leukemic sera (71%), 36 of 45 normal sera (80%), 15 of 19 sera from family contacts of leukemic patients (79%), 14 of 21 cord blood specimens (67%), and 21 of 23 sera (91%) from patients with systemic lupus erythematosus had detectable antibody activity.

T cell receptor and immunoglobulin gene rearrangements in acute myeloblastic leukemia.

The organization and expression of the beta chain of T cell antigen receptor gene (beta-TCR) and Ig H and L chain genes were analyzed by Southern blot technique in 24 patients with a diagnosis of acute myeloblastic leukemia (AML). Rearrangements of the beta-TCR genes were seen in DNA samples from 3 of the 24 patients. One of these three patients also showed rearrangement of the Ig H chain gene. RNA samples from all three patients expressed a beta-TCR gene transcript on dot blot analysis. However, on Northern blot analysis, one patient expressed an incomplete 1.0 kb transcript and no Ig H chain mRNA, despite a rearranged configuration. The karyotypes of two of these patients showed abnormalities involving chromosome 7. Rearrangements of T cell antigen receptor genes may occur in nonlymphoid malignancy, and is consistent with the concept of lineage infidelity in AML.

Repression of colony formation reversed by antiserum to mouse thymocytes.

Marrow cells derived from C57BL/6 mice form many fewer splenic colonies in irradiated C57BL/6 x C3H F1 hybrid recipients than in irradiated C57BL/6 recipients (repression of colony formation). This effect is reversed by treatment of the hybrid recipients with active antiserum to mouse thymocytes. The repression phenomenon cannot readily be explained in immunological terms; hence the effect of the antilymphocyte serum on this phenomenon may not result from immunosuppression in the usual sense.

Ascorbic acid: a culture requirement for colony formation by mouse plasmacytoma cells.

Plasmacytoma stem cells explanted from mice formed colonies in culture only in the presence of L-ascorbic acid; this vitamin was not needed for the formation of granulocytic colonies. Isomers of L-ascorbic acid with less antiscorbutic activity also promoted plasmacytoma colony formation, but less effectively. Other redox compounds without antiscorbutic activity and an antioxidant were ineffective.

Evaluation of the modified Glasgow Prognostic Score to predict outcome in dogs with newly diagnosed lymphoma.

The modified Glasgow Prognostic Score (mGPS) assigns a numerical value (0-2) from pre-treatment serum concentrations of C-reactive protein (CRP) and albumin to predict patient outcome. CRP and albumin were evaluated in 77 untreated dogs with lymphoma to determine the relationship of mGPS to clinicopathological parameters and whether it could predict progression-free (PFS) and overall survival (OS) in treated dogs. mGPS distribution was significantly associated with clinical stage, substage b, weight loss, gastrointestinal disturbances and lethargy at presentation. On univariate analysis, mGPS was significantly associated with OS and PFS, with shorter median survival times for mGPS 2 compared to mGPS 0 and 1 combined. Hypoalbuminaemia significantly reduced OS and PFS, however increased CRP had no effect. Only clinical stage was significantly associated with OS and PFS on both univariate and multivariate analysis. mGPS has potential prognostic value for canine lymphoma , but further studies are needed.

Cytodifferentiation in the acute myeloblastic leukemias of man.

Correlation analysis of numbers of colony-forming progenitor cells was used as an approach to the quantitation of human pluripotent stem cells. Marrow specimens were obtained from 24 patients with untreated acute myeloblastic leukemia, 22 patients under treatment, and 29 patients with no hematologic malignant disease. Three classes of progenitor cells were assayed: burst-forming units dependent on erythropoietin (BFU-E), colony-forming units dependent on erythropoietin (CFU-E), and granulopoietic progenitors (CFU-C). Significant positive correlations between numbers of BFU-E, CFU-E, and CFU-C were found in all 3 groups of patients. In contrast, no such positive correlations were seen between marrow blasts and any of the classes of colony-forming progenitors. These results were compatible with a shared relationship of the colony-forming progenitors to a pluripotent cell of origin and raised the possibility that the immediate progenitors of the blasts may not be any of the myelopoietic progenitor cells monitored in these studies.

Cytotoxicity of adriamycin and daunorubicin for normal and leukemia progenitor cells of man.

A colony assay available for a subpopulation of acute myeloblastic leukemia blasts with proliferative potential was used to measure adriamycin (adria) and daunorubicin (dauno) dose-response curves following brief exposure to either drug and washing. The dose-response curves were simple negative exponentials that might be characterized by D10 (dose required to reduce survival to 10%) values. The D10 values ranged from 0.47 to 20.8 microgram/ml for adria (8 patients) and from 0.06 to 0.34 microgram/ml for dauno (3 patients). Controls consisted of committed granulopoietic and T-lymphocyte progenitors. Four measurements of granulopoietic progenitors yielded D10 values from 2.5 to 11.5 mug/ml for adria and from 0.44 to 1.2 microgram/ml for dauno. T-lymphocyte precursors from 4 normal individuals were resistant. However, following incubation of normal leukocytes with phytohemagglutinin, DNA synthesis commenced in T-lymphocyte precursors for 3 additional normal controls, which was associated with an increased data sensitivity with D10 values ranging from 4.4 to 6.2 microgram/ml.

Relationships between the mitochondrial permeability transition and oxidative stress during ara-C toxicity.

The mitochondrial permeability transition and oxidative stress seem to be critical alterations in cellular physiology that take place during programmed cell death. Failure to undergo apoptosis is associated with drug resistance in acute myeloid leukemia and other cancers. Therefore, it is important to establish causal relationships between the physiological changes that take place in apoptosis, because these are potential targets for novel treatment strategies to overcome this form of drug resistance. We describe the use of multilaser flow cytometry methods to make correlated measurements of mitochondrial membrane potential (MMP), the generation of reactive oxygen intermediates, the cellular content of reduced glutathione (GSH), intracellular calcium, and exposure of phosphatidylserine on the cell surface. Using these combined methods, we have mapped a "death sequence" that occurs after treatment of leukemic blasts with clinically relevant concentrations of 1-beta-D-arabinofuranosylcytosine (ara-C). Dual labeling of MMP and cellular glutathione content showed that loss of MMP, indicative of the permeability transition, took place in cells that were depleted of glutathione. The loss of MMP coincided with phosphatidylserine exposure and preceded a state of high reactive oxygen generation. Finally, there was an increase in intracellular calcium. These results demonstrate that the mitochondrial permeability transition takes place during ara-C toxicity but suggest that this occurs downstream of the loss of GSH. Thus, oxidative stress after ara-C-induced toxicity seems to be a biphasic phenomenon, with the permeability transition occurring after a depletion of GSH and preceding a state of high reactive oxygen generation.

Self-renewal capacity of leukemic blast progenitor cells.

A review is presented of experimental information pertaining to the characteristics of a procedure designed to quantitate the capacity for self-renewal in clonogenic cells of human acute myeloblastic leukemia. In a series of 44 previously untreated patients, a significant correlation (p less than 0.01) was seen between low capacity for self-renewal and successful remission induction. Three cytotoxic drugs (Adriamycin, 1-beta-D-arabinofuranosylcytosine, and N-[4-(19-acridinylamino)-3-methoxyphenyl]-methanesulfonamide) were tested for preferential effect against self-renewal events. Surviving clonogenic cells to these agents had, respectively, unchanged, lower, and higher capacity for self-renewal. The implications of such drug properties are discussed.

Investigating virtual reality navigation in amnestic mild cognitive impairment using fMRI.

Spatial navigation requires a well-established network of brain regions, including the hippocampus, caudate nucleus, and retrosplenial cortex. Amnestic Mild Cognitive Impairment (aMCI) is a condition with predominantly memory impairment, conferring a high predictive risk factor for dementia. aMCI is associated with hippocampal atrophy and subtle deficits in spatial navigation. We present the first use of a functional Magnetic Resonance Imaging (fMRI) navigation task in aMCI, using a virtual reality analog of the Radial Arm Maze. Compared with controls, aMCI patients showed reduced activity in the hippocampus bilaterally, retrosplenial cortex, and left dorsolateral prefrontal cortex. Reduced activation in key areas for successful navigation, as well as additional regions, was found alongside relatively normal task performance. Results also revealed increased activity in the right dorsolateral prefrontal cortex in aMCI patients, which may reflect compensation for reduced activations elsewhere. These data support suggestions that fMRI spatial navigation tasks may be useful for staging of progression in MCI.

High-dose cytosine arabinoside in the treatment of acute myelogenous leukemia: contributions to outcome of clinical and laboratory attributes.

High-dose cytosine arabinoside (HDAra-C) has been used for remission induction, and in conventional doses for maintenance in a trial of single-agent therapy in 43 previously untreated patients with acute myelogenous leukemia (AML). Rationale for the trial was provided by the observed decrease in leukemic blast cell self-renewal in culture following exposure to Ara-C. Compared with a previous trial of 57 patients treated with multidrug therapy, single-drug Ara-C was associated with a significantly improved complete remission rate (P = .010), although the survival time was not increased. All patients with low self-renewal responded to HDAra-C in contrast to the previous trial where some patients with this phenotype failed remission induction. The clinical observations are consistent with the view that the antileukemic effect of Ara-C has some specificity for cellular events required for self-renewal of blast cells. Exposure in vivo to Ara-C was associated with an increase in blast stem cell renewal at relapse, indicating that maintenance with other drugs should be tested. The study demonstrates the importance of biological attributes in design and analysis of clinical trials.

Contributions of host- and disease-related attributes to the outcome of patients with acute myelogenous leukemia.

Three sequential trials of treatment for acute myelogenous leukemia (AML) involving 173 patients were analyzed to identify clinical and myeloblast-cell progenitor properties in culture related to outcome. The latter, including self-renewal capacity expressed as plating efficiency (PE2) and drug sensitivity, were determined for a representative group of 45 patients. Despite increasingly intensive remission induction therapy, similar response rates were achieved in the three trials and no increase in the duration of survival was observed. Clinical attributes at presentation by multivariate analyses were not consistently predictable of outcome. Of the blast cell attributes, only PE2 was predictive of duration of survival (p less than 10(-6)). For patients in remission the relapse rate during the first year was 0.63 compared with 0.15 in subsequent years. The percentage marrow myeloblasts at presentation, a measure of disease activity, was significantly higher for the patients having remissions lasting less than one year. These studies demonstrate the importance of disease-related attributes on the outcome of patients with AML.

Generation of reactive oxygen intermediates after treatment of blasts of acute myeloblastic leukemia with cytosine arabinoside: role of bcl-2.

Cytosine arabinoside is usually considered to be lethal by incorporation into DNA followed by chain termination. Recently, we have reported that the radical scavenger N-acetyl-cysteine (NAC) protects cultured clonogenic AML blast cells from the lethal affects of Ara-C if given before the drug. This observation provides indirect evidence that toxic reactive oxygen intermediates (ROI) are generated in AML blast cells following Ara-C-induced damage to DNA. In the present paper we present evidence in support of this hypothesis. Using flow cytometry and multiple fluorescent probes for live cell function, we have mapped a sequence of discrete stages that occur during Ara-C cytotoxicity. An early event was the increased generation of ROI. Initially this oxidative stress was countered by an increase in the cellular content of reduced glutathione (GSH), but cells then underwent an abrupt transition to a state characterized by low GSH and very high ROI generation indicative of collapse of cellular redox balance. Next, the capacity to maintain low intracellular ionized calcium was lost, probably due to lipid peroxidation at membrane sites of calcium regulation. Finally, surface membrane integrity was lost. Concurrent measurements of clonogenic cell survival insured the relevance of these flow cytometry measurements to the stem cell population. We used OCI/AML-2 cells transfected with bcl-2 to look for the place in this sequence where bcl-2 protein protects cells against apoptosis; bcl-2 transfectants showed an increase in ROI generation similar to controls, but were able to maintain GSH levels in the face of this oxidative stress. We conclude that oxidative stress plays a major role in Ara-C toxicity, and that bcl-2 protein protects cells by maintaining cellular redox balance in a reducing state. These studies complement previous work showing how regulators of AML growth affect the sensitivity of blast cells to Ara-C by changing the concentration or stability of bcl-2 protein.

A cell culture model for the treatment of acute myeloblastic leukemia with fludarabine and cytosine arabinoside.

The purpose of this paper was to ascertain whether results obtained in cell cultures of AML clonogenic blast cells would provide a useful model for a clinical regimen that combines fludarabine (F-ara-AMP) and cytosine arabinoside (ara-C). In the cultures the nucleoside F-ara-A was used. Blast cells from the continuous lines OCI/AML-2 and OCI/AML-3 were grown, either in methylcellulose to quantify clonogenic cells, or in suspension to measure self-renewal as reflected in changes in numbers of clonogenic cells. F-ara-A, like ara-C, was found to be more toxic to blast stem cells in suspension than in the clonogenic assay, indicating that F-ara-A might, in addition to general cytotoxicity, have some specific inhibitory effects on self-renewing stem cells. F-ara-A was less cytotoxic than ara-C; but, when F-ara-A was given before ara-C, synergism was seen at some F-ara-A doses, as manifested by increased ara-C cytotoxicity. In contrast, when ara-C was given before F-ara-A, protection was observed. Control experiments make it unlikely that this effect is related to changes in the cell cycle following ara-C exposure. We conclude that the cellular studies reported here confirm previous pharmacological data indicating that F-ara-A before ara-C increases the effectiveness of ara-C by increasing the accumulation of ara-CTP. However the present experiments show that the synergism between F-ara-A and ara-C is dependent on both dose and schedule.