Treatment and outcomes for chronic myelomonocytic leukemia compared to myelodysplastic syndromes in older adults.

Prior studies have investigated patients' characteristics, treatments, and outcomes for older adults with myelodysplastic syndromes, but most failed to distinguish chronic myelomonocytic leukemia. Recognizing potentially important differences between the diseases, we undertook a population-based comparison of baseline characteristics, treatments, and outcomes between older adults with chronic myelomonocytic leukemia and myelodysplastic syndromes. The patients' data were obtained from Surveillance Epidemiology and End Results registry data from 2001-2005, linked to Medicare claims. Baseline characteristics, treatment (red blood cell transfusions, hematopoietic growth factors, hypomethylating agents, chemotherapy or transplantation), progression to acute myeloid leukemia, and overall survival were compared using bivariate techniques. Multivariate logistic regression estimated differences in treatments received. Cox proportional hazard models estimated the effects of chronic myelomonocytic leukemia relative to myelodysplastic syndromes on progression-free survival. A larger proportion of patients with chronic myelomonocytic leukemia (n=792), compared to patients with myelodysplastic syndromes (n=7,385), failed to receive any treatment (25% versus 15%; P<0.0001), or only received red blood cell transfusions (19.8% versus 16.7%; P=0.037). A larger percentage of patients with chronic myelomonocytic leukemia progressed to acute myeloid leukemia (42.6% versus 15.5%, respectively; P<0.0001), with shorter time to progression. Chronic myelomonocytic leukemia patients had a shorter median survival (13.3 versus 23.3 months; P<0.0001) and lower 3-year survival rate (19% versus 36%; P<0.0001). Adjusted estimates, controlling for baseline characteristics and selected treatments, indicate that chronic myelomonocytic leukemia was associated with an increased risk of progression to acute myeloid leukemia or death (HR 2.22; P<0.0001), compared to myelodysplastic syndromes. In conclusion, chronic myelomonocytic leukemia is less frequently treated in older adults and is associated with worse outcomes, even after controlling for the patients' baseline characteristics and selected treatments. Our data suggest the need for continued evaluation of the biological differences between these diseases and clinical trials targeting chronic myelomonocytic leukemia.

[State of medical care of patients with hematological malignancies in Kyïv].

In the article data are presented about morbidity by oncogematologic pathology - one of the most meaningful of social-economic problems. In Ukraine annually diagnose the to 8 thousand new cases of haemoblastosis. Indexes of morbidity on a 100 thousand population are 5,2; at illness of Hodgkin's lymphoma - 2,5, at plural myeloma - 1,6; at leukemia - 8,1. Morbidity by haematological pathology in Kyiv long time remains high: annually 250 expose patients with malignant lymphnoma, 57 - with myeloma, 190 - with leukemia, from them at 55 % is a sharp form and at 40 % - chronic. The anxiety of doctors causes circumstance that the special treatment is overcome 58,1 % patients by leukemia, 68,6 % - plural myeloma and 77,8 % patients with malignant lymphoma. World experience shows that application of complex methods of therapy allows to prolong life-span 80-90 % patients with Hodgkin's malignant lymphoma on 10, and at 95 % patients by a lymphogranulomatosis - to attain nonrecurrence survival to 5 years.

Clinical pathological and epidemiological assessment of morphologically and immunologically confirmed canine leukaemia.

Traditionally, classification of leukaemia in dogs has relied on morphological examination and cytochemical staining patterns, but aberrant cellular morphology and stain uptake often curtails accurate categorization, and historical data based on this classification may be unreliable. Immunophenotyping is now the gold standard for classification of leukaemias. The purpose of this prospective study was to assess the clinical pathological and epidemiological features of a population of dogs with morphologically and immunologically confirmed leukaemia and to compare them within categories: acute and chronic lymphoid leukaemia (ALL and CLL), and acute and chronic myeloid leukaemia (AML and CML). There were 64 cases of morphologically and immunologically confirmed leukaemia: 25 cases of ALL, 17 cases of CLL and 22 cases of AML. Prevalence of B and T immunophenotypes in ALL and CLL was not statistically different. Dogs with AML were significantly younger than those with ALL at presentation (P = 0.04). Golden Retriever dogs in the study population were overrepresented in comparison with a control population of dogs (6/25 ALL cases, 8/64 leukaemia cases). No sex was overrepresented. Dogs with ALL had significantly more severe neutropenia (P = 0.001) and thrombocytopenia (P = 0.002) than those with CLL and had significantly more cytopenias. The severity and numbers of cytopenias seen in ALL and AML were not significantly different. Twenty-one of the leukaemia cases showed one cytopenia, fourteen had two cytopenias and twenty-one cases had pancytopenia. Anaemia was the most common cytopenia seen in isolation (17/21). No dogs had neutropenia without anaemia and/or thrombocytopenia. Total white blood cell counts were not different between the groups. The atypical cell counts within the peripheral blood were significantly higher in ALL than AML; both in isolation and as a percentage of the total white blood cell count (P = 0.03). This study strengthens the hypothesis that acute leukaemias give rise to more profound cytopenias, affecting more cell lines, than chronic leukaemias.

Genomic assessment of pediatric acute leukemia.

Advances in molecular genetics have revolutionized our understanding of acute myeloid and lymphoblastic leukemia. Structural and numerical chromosomal aberrations are common, and their detection is vital for leukemia diagnosis, risk stratification, and monitoring of response to therapy. Fusion proteins resulting from chromosomal translocations are necessary but not sufficient for leukemogenesis, and there is intense research activity to elucidate the cooperating molecular abnormalities that may be suitable targets for novel therapeutic approaches. Candidate gene approaches have identified mutations in kinases and transcription factors in a proportion of patients, but more comprehensive genomic approaches are required. Gene expression profiling accurately classifies known subtypes of acute leukemia and has highlighted potentially leukemogenic abnormalities in gene expression. Newer techniques, such as single-nucleotide polymorphism arrays to analyze changes in gene copy number and zygosity, cancer genome sequencing, and RNA interference, are promising tools to identify mutations, although at present, data from these approaches are limited. This review provides an overview of these techniques in clinical practice and as research tools to develop new therapeutic approaches in pediatric leukemia.

Risk assessment in patients with acute myeloid leukemia and a normal karyotype.

PURPOSE: The recognition of a number of leukemia-specific cytogenetic abnormalities and their role as independent prognostic factors have provided considerable insights into leukemia pathogenesis and have paved the way to adopt risk-adapted treatment. However, approximately 50% of newly diagnosed acute myeloid leukemia (AML) have a normal karyotype. There has therefore been much interest in identifying molecular markers that could help to improve the prognostic stratification of patients with normal-karyotype AML. EXPERIMENTAL DESIGN: Consecutive untreated AML patients (n = 67) from a single institution all with normal karyotype were analyzed for the presence of mutations in the myeloid transcription factor gene CEBPA (for CCAAT/enhancer binding protein-alpha), for internal tandem duplications (ITD) of the tyrosine kinase receptor gene FLT3 (for fms-like tyrosine kinase 3), and for expression of the BAALC gene (for brain and acute leukemia, cytoplasmic). RESULTS: 17.9% of normal-karyotype AML had mutations in the CEBPA gene, and 28.4% had FLT3-ITD; 65.7% (44 of 67) had high BAALC expression and 34.3% (23 of 67) had low BAALC expression. Patients with CEBPA mutations had a very favorable course of their disease. Median disease-free survival (DFS) and overall survival (OS) were 33.5 and 45.5 months, respectively, compared with 10 (e.g., 12 months in patients without CEBPA mutations; P = 0.0017; P = 0.0007). AML patients with FLT3-ITD had significantly shorter median DFS (P = 0.0328) and OS (P = 0.0148) than patients without FLT3-ITD. High BAALC expression predicted for a shorter DFS (P = 0.0152) and OS (P = 0.0210) compared with AML with low BAALC expression; 53.7% of normal-karyotype AML had neither FLT3-ITD nor CEBPA mutations. We found that high BAALC expression in normal-karyotype AML with neither FLT3-ITD nor CEBPA mutations (18 of 67) indicates adverse prognosis for both DFS and OS (P = 0.0001; e.g., P = 0.0001) compared with the group with low BAALC expression and absent FLT3-ITD and CEBPA mutations (18 of 67). Thus, BAALC expression represents a novel prognostic marker particularly for normal-karyotype AML patients with neither FLT3-ITD nor CEBPA mutations. CONCLUSIONS: Assessment of CEBPA mutations, FLT3-ITD, and BAALC expression permits to split normal-karyotype AML into clinically distinct subgroups.

Four-fold staining including CD45 gating improves the sensitivity of multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia.

Multiparameter flow cytometry (MFC) is capable of quantifying minimal residual disease (MRD) in acute myeloid leukemia (AML). Its broad application, however, is limited by a lack of sensitivity in about 20% of patients. CD45 gating may improve sensitivity. A broad panel of four-fold combinations of monoclonal antibodies including CD45 in each was used to define leukemia-associated aberrant immunophenotypes (LAIP), to define their sensitivities in normal bone marrow samples, and to compare results to data obtained without CD45 gating using triple staining. In 45 patients, a LAIP was defined, 11 normal bone marrow samples were analyzed as controls. The median percentage of LAIP-positive AML cells with and without CD45 gating was 21.95% (range, 3.31-82.52%) and 20.52% (range, 3.22-81.94%). The median percentage of LAIP-positive normal bone marrow cells ranged from 0.01 to 0.42% (median, 0.02%) and 0.02 to 0.58% (median, 0.15%) with and without CD45 gating. The difference of LAIP-positive cells between AML and normal bone marrow samples amounted to a median of 3.08 log (range, 1.22-4.01) and 2.28 log (range, 1.12-3.34) with and without CD45 gating. CD45 gating improves the sensitivity of MFC-based MRD monitoring in AML by 1 log.

Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

BACKGROUND AND OBJECTIVES: The percentages of CD34+ cells in the bone marrow of patients with acute myeloid leukemia (AML) vary widely. Especially in the low range (<5% CD34+ cells), the nature (normal or malignant) of the CD34+ cells is uncertain. Since only in a minority of cases are molecular techniques applicable, in this study we explored a multiparameter approach using phenotypic and functional characteristics to discriminate normal CD34+ cells from malignant ones. DESIGN AND METHODS: CD34+ cells from 24 AML patients with <5% CD34+ cells and from 3 patients with >50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by interphase fluorescent in situ hybridization on sorted CD34+ cells in 3 cases, which had concomitant presence of cytogenetic abnormalities in the CD34- blasts. In contrast, in the intermediate (1.6-3.5%) CD34+ range, the CD34+ cells appeared as normal in only 1/5 cases. In the high (51-67%) CD34+ range, as expected the majority of CD34+ cells were malignant, although in 2/3 cases a small subpopulation (i.e. 0.15% and 0.20%) of CD34+ cells were of normal origin. INTERPRETATION AND CONCLUSIONS: Our multiparameter approach enabled us to define the nature of CD34+ cells in AML. This has implications for studies dealing with the characterization of primitive malignant cells. Moreover, it enabled identification of truly CD34 negative AML, which would be eligible for CD34-based immunological purging of autologous stem cell transplants.

Assessment of monocytic component in acute myelomonocytic and monocytic/monoblastic leukemias by a chemiluminescent assay.

INTRODUCTION: Classically, the monocytic component of acute myelomonocytic (FAB-M4) and acute monocytic/monoblastic (FAB-M5) leukemias is demonstrated by nonspecific esterase positivity in cytochemical stainings. We have previously demonstrated that non-specific esterases from normal monocytes can be determined by a chemiluminescent method. In the present study, we investigated whether this assay can also determine the monocytic component of FAB-M4 and FAB-M5 and distinguish these acute myeloid leukemia (AML) categories. MATERIALS AND METHODS: Bone marrow samples were obtained from 66 patients with AML (M0, two cases; M1, 12 cases; M2, 13 cases; M3, 10 cases; M4, 11 cases; M5, 12 cases; M6, two cases; M7, four cases). Cells were incubated with a standard reaction mixture and chemiluminescence was measured for 10 min. Two parameters were assessed, the peak (PLE) and the integrated light emission (ILE). RESULTS: Both PLE and ILE were higher in FAB-M4 and FAB-M5 subtypes compared to other AML subtypes (P<0.001). In addition, the classification of AML cases into FAB-M4, FAB-M5 and nonmonocytic subtypes based on ILE analysis was concordant with alpha-naphthyl acetate esterase (ANAE) in 97% of cases (kappa coefficient 0.94, P<0.001). CONCLUSIONS: These findings indicate that this chemiluminescent assay was able to determine the monocytic component of FAB-M4 and FAB-M5 cells, and the classification of AML subtypes based on chemiluminescent analysis strongly agreed with the cytochemical ANAE-staining. In conclusion, this chemiluminescent assay is a simple, fast and objective method, which may be useful as an alternative tool in the differential diagnosis of AML subtypes.

Assessment of growth and differentiation processes in myelodysplastic syndromes by PCR analysis of Galpha16 and 5'-lipoxygenase.

Development of further diagnostic and prognostic tools in myelodysplastic syndromes (MDS) is warranted. In this study we tested two molecular markers in bone marrow of MDS patients: Galpha16, a hematopoiesis-specific G protein alpha subunit serving as an intracellular marker of hematopoietic activity, and 5'-lipoxygenase (5-LO), a putative differentiation marker. The results were correlated with clinical and laboratory features and outcome. Bone marrow mononuclear cells were evaluated by block cycler PCR in 32 patients for Galpha16 and in 25 patients for 5-LO. In 12 patients cDNA analyzed by the block cycler method was quantified by real-time quantitative (RTQ) PCR for both Galpha16 and 5-LO. The results confirmed concordance of the two methods. All FAB and WHO subtypes were positive for at least one of the two genes. Strikingly, only 1 of 11 patients with refractory anemia with ringed sideroblasts was positive for Galpha16. No correlation between Galpha16 or 5-LO and bone marrow cellularity or cytogenetic risk factors (IPSS) was detected. Only combined evaluation of Galpha16 and 5-LO expression showed a correlation with extent of anemia and thrombocytopenia. A relationship between the two markers and preleukemic duration was found. Our findings show that both Galpha16 and 5-LO can be expressed in MDS, reflecting proliferation and differentiation processes in this disease. Different expression patterns of the two molecules indicate that proliferation and differentiation of hematopoietic cells may occur independently. These parameters could constitute a new class of risk factors in MDS. Determination of Galpha16 and 5-LO expression may provide a tool for observing growth and maturation in these diseases.

Assessment of caspase activity as a possible prognostic factor in acute myeloid leukaemia.

Caspases are one of the key effector molecules in apoptosis. Caspase-3 activity (identified by cleavage of the peptide DEVD) was analysed in bone marrow blasts (minimum 70%) from 45 acute myeloid leukaemia (AML) patients. 12 patients (25%) exhibited high levels of specific DEVDase activity. These blast cells, despite having activated caspase-3, displayed none of the classical caspase-dependent morphological characteristics of apoptosis, such as degradation of DNA fragmentation factor 45, DNA fragmentation, and appeared to be more resistant to drug-induced apoptosis. Our results suggest that in these AML cells, resistance to apoptosis occurred downstream of caspase-3 activation.

A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells.

In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis.

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

Assessment of distribution of CD34 epitope classes in fresh and cryopreserved peripheral blood progenitor cells and acute myeloid leukemic blasts.

BACKGROUND AND OBJECTIVE: So far several reports have described changes in the expression of surface antigens in progenitor cells and blasts following cryopreservation. However, there are no data on the effects of cryopreservation on the expression of the three CD34 epitope classes, and on their relationship with the clonogenic capacity of PBPC collected by leukapheresis. DESIGN AND METHODS: In order to analyze the effects of freezing/thawing procedures (Eth 80C storage for 3 months) and use of dimethylsulfoxide (DMSO) on the immunophenotype profile and colony production of peripheral blood progenitor cells (PBPC) in apheresis products derived from 20 patients with stage 0-III non-Hodgkin's lymphoma (nHL), a flow cytometry study was undertaken using different CD34 monoclonal antibodies (MoAbs) capable of recognizing the 3 epitope classes of CD34 molecule (class III: HPCA-2/FITC, HPCA-2/PE, 581/FITC, 581/PE; class II: Q-Bend 10/PE; class I: ICH3/PE, BI3C5-PE, Immu-133-PE). CD34 epitope expression was also analyzed in thawed CD34+ blasts obtained from 14 patients with acute myeloid leukemia (AML), who were analyzed using a larger number (#17) of CD34 epitope class I, II, and III reactive MoAbs. RESULTS: Under our experimental conditions it was found that class III and class II CD34 epitopes (differentially resistant to enzymatic cleavage with neuraminidase, chymopapain and glycoprotease) are better preserved than class I epitope Eth sensitive to degradation Eth after cell exposure to cryoprotectant DMSO and the freezing- thawing procedures. Results further showed a concomitant decrease in class I CD34+ counts and in BFU-E colony production. A significant increase in CD34 antigen expression levels (i.e. antibody binding capacity, ABC) by cryopreserved cells stained with CD34 epitope class III, and class II reactive MoAbs was also documented, while no changes after cryopreservation were noted using class I-reactive MoAbs. The slight increase in the percentage of CD34+ cells detected after frozen storage was correlated to a concomitant decrease in the number of more mature myeloid cells (CD15+, CD13+, CD33+). Compared to pre-cryopreservation values, a slight reduction in class I CD34 epitope expression was also found in thawed CD34+ AML blasts. INTERPRETATION AND CONCLUSIONS: As far as the reduction of class I CD34 epitope is concerned, it may be hypothesized that the freezing procedure, use of DMSO, and/or lysis methodology may either damage a CD34 subset, or induce distinct alterations of the CD34 glycoprotein, possibly determining a reduction in their immunoreactivity with some CD34 MoAbs. In conclusion, this study has shown that exposure to the cryoprotectant DMSO and the freezing/thawing procedures modifies the distribution of CD34 epitopes as well as the clonogenic capacity of PBPCs from nHL patients, and CD34+ blasts from AML. These findings need to considered when selecting CD34 MoAbs for enumeration and positive selection of stem/progenitor cells for research and clinical purposes.

Cytogenetic findings in 303 Mexican patients with de novo acute myeloblastic leukemia.

In this report we show the chromosomal changes seen in a group of 303 Mexican patients with de novo Acute Myeloblastic Leukemia (AML). Two hundred forty-two patients were diagnosed and treated at two hospitals affiliated with the Instituto Mexicano del Seguro Social (IMSS). These are the Centro Medico Nacional Siglo XXI and Centro Medico La Raza Hospitals; the remaining 61 patients were diagnosed and treated at the Hospital General de Mexico (HGM). Clonal abnormalities were detected in 75.6% of the patients; this result agrees with what has been reported in other large series of AML studies. The incidence of changes per hospital was similar in patients from the IMSS hospitals (72-75%), while an increase was seen in patients from the HGM (85.2%). The chromosomal changes seen in this study in order of frequency were: t(15;17)[18.8%], t(9;22)[9.2%], miscellaneous chromosomal changes (mainly rearrangements of chromosomes 1,2,3,12y17)[8.2%], abnormalities of 16q22 [7.3%], t(8;21)[6.3%], -7/del(7q)[5.6%], t(6;9)[5.3%], and abnormalities of 11q23 [4.6%]. We reported an increase in the incidence of certain types of chromosomal changes seen in cases of AML, in comparison with reports from other countries. These differences could be due to methodological variations, although ethnic, socioeconomic and nutritional differences must not be disregarded. We support this finding when comparing distribution of changes in the population of patients seen in the IMSS hospitals with those from the HGM; the main difference lies in the socioeconomic level.

In vitro assessment of acetic-acid-soluble proteins (glutenin) toxicity in celiac disease.

Acetic-acid-soluble storage proteins from gluten of the bread wheat cv. Sprint 3 were fractionated by adsorption chromatography on 2000 A controlled-pore glass (CPG) beads, and glutenin polymers with molecular mass higher than 10(7) Da and free from monomeric gliadins were recovered. The glutenin polymers were found to consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits. Peptic-tryptic (PT) digests of glutenins were examined for their agglutination activity on human myelogenous leukemia K 562(S) cells, agglutination being strongly correlated with toxicity for the celiac intestine. The peptide fraction at a concentration of 1 g/L of culture medium was able to agglutinate 30% of K 562(S) cells, suggesting a moderate toxic effect. This toxicity may be accounted for by homologies in amino acid sequences between glutenin subunits and alpha/beta- and gamma-gliadins.

Simultaneous assessment of cell kinetics and programmed cell death in bone marrow biopsies of myelodysplastics reveals extensive apoptosis as the probable basis for ineffective hematopoiesis.

Despite hypercellular bone marrows (BM), peripheral cytopenias are the rule in patients with myelodysplastic syndromes (MDS). This study examined the roles played by cell birth and cell death rates in generating this paradox. Cell kinetics from BM biopsies of 35 MDS patients were measured using intravenous infusions of either iododeoxyuridine or bromodeoxyuridine, or both. Degree of apoptosis or programmed cell death (PCD) was estimated using in situ end-labeling of DNA directly from BM biopsies, which were simultaneously double-labeled from proliferation/PCD. MDS were found to be highly proliferative disorders with large numbers of myeloid, erythroid, and megakaryocytic cells synthesizing DNA. Median cycling time (Tc) of myeloblasts was more rapid than that of patients with acute myeloid leukemia (44.1 hr vs. 56.0 hr). Interestingly, most marrow cells of all three lineages in 32 of 34 evaluable cases were undergoing PCD. In 19 of 32 patients, greater than 75% cells were apoptotic. Surprisingly, large numbers of S-phase cells were found to be simultaneously undergoing PCD, as were stromal cells of the BM microenvironment. We conclude that the extensive apoptosis in hematopoietic cells effectively cancels the high birth rate resulting in ineffective hematopoiesis and accounting for deficient bone marrow function.

Assessment of proliferative activity in leukaemic bone marrow using the monoclonal antibody Ki-67.

AIMS: To investigate proliferative activity in leukaemic and lymphomatous bone marrow infiltrates and to assess the feasibility of transport of specimens among institutions. METHODS: Proliferative activity in bone marrow trephine cryosections from 99 patients with non-Hodgkin's lymphoma (NHL), 23 patients with acute myeloid leukaemia (AML), 11 with acute lymphoblastic leukaemia (ALL), and two with acute undifferentiated leukaemia (AUL) was investigated. Infiltration was seen in 52 out of 99 cases of NHL on bone marrow cryosections. A score was devised to assess pathological infiltrates in bone marrow trephine cryosections using the monoclonal antibody Ki-67. This method of scoring gave a measure of non-erythroid proliferative activity. RESULTS: Mean Ki-67 positivity in bone marrow infiltrates in 31 low grade B cell lymphomas (Kiel classification) was 0.3% before and 4.7% after treatment, 16.4% in seven high grade B cell lymphomas, and 17.8% in 12 peripheral T cell lymphomas. In 48 cases of NHL, bone marrow cryosections had not been infiltrated, and in all but one case the percentage of Ki-67 positive cells in normal marrow was less than 3%; the remaining case showed coexistent myelodysplasia and 8% bone marrow Ki-67 positivity. In eight cases of common ALL at diagnosis, the mean Ki-67 positivity in marrow cryosections was 24.9%, significantly higher than the 2.4% Ki-67 positivity seen in AML (p < 0.05). One of the two cases of common ALL with less than 1% Ki-67 positivity was refractory to treatment. CONCLUSIONS: Proliferative activity of erythroid elements in the bone marrow varies greatly. Immunostaining of bone marrow cryosections using Ki-67 permits accurate assessment of non-erythroid proliferative activity in lymphomas and leukaemia. High grade B cell lymphomas and peripheral T cell lymphomas invading the marrow have very similar mean proliferative activities. Such levels of proliferation are of the same order as those seen in common ALL, but much higher than those seen in AML.

Assessment of proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF) in acute myeloid leukaemia using a fluorescent ligand for the nucleoside transporter.

Nucleoside transporter expression has been linked to proliferation in a variety of haemopoietic cell types. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was given for 72 h before commencing chemotherapy in 15 patients with relapsed or refractory acute myeloid leukaemia (AML) and in 11 patients serial bone marrows were taken for measurement of [3H]thymidine labelling index, Ki-67 positivity and maximal binding of 5-(SAENTA-x8)-fluorescein, a flow cytometry ligand which enumerates nucleoside transporter sites. GM-CSF caused proliferation of marrow myeloblasts in eight of 11 patients, while in three patients there was no change in proliferative indices. The expression of nucleoside transporters increased up to 4-fold in the myeloblasts from the patients showing a proliferative response to GM-CSF but there was no increase in transporters on the myeloblasts from the three non-responding patients. A close correlation was found between the fold increase in nucleoside transporter expression and the fold increase in labelling index of marrow myeloblasts (r = 0.86, n = 9, p < 0.01). In one patient with acute megakaryoblastic leukemia, GM-CSF caused parallel increases in labelling index, Ki-67 positivity and numbers of nucleoside transporters on peripheral blood blast cells. Thus induction of proliferation by cytokine increases the expression of nucleoside transporters on leukaemic myeloblasts studied in serial samples from the same source (bone marrow or blood). The suitability of 5-(SAENTA-x8)-fluorescein for two colour flow cytometric analysis allows the rapid enumeration of nucleoside transporters in the myeloblast compartment of heterogeneous marrow samples.