Study of inheritance of diabetes mellitus in Western Indian population by pedigree analysis.

OBJECTIVES: To study the inheritance pattern of diabetes mellitus in Western Indian population by analysing the pedigree of diabetes patients. METHODS: 3,921 individuals from 300 families were interviewed for family history in this study, out of which 770 were diabetic individuals. Statistical analysis of the data was carried out using T-test and Chi-square test. RESULTS: 37% cases of Type 1 DM and 58% cases of Type 2 DM showed family history of the disease. Of the cases showing family history for diabetes, 92% in case of Type 1 DM and 59% in case of Type 2 DM showed family history of Type 2 DM with a decrease in age of onset in the successive generations. Both the parents, when diabetic conferred equal risk of inheriting diabetes in offspring. The sex ratio of offspring suffering from diabetes was not influenced when only one of the parents was diabetic. However it was observed that the male offspring were highly susceptible when both parents were diabetic (Chi-square value=4.55 with 1 d.f.). The age of onset of diabetes did not show significant correlation with whether one or both the parents were diabetic. However, it was noteworthy that in case of familial history of diabetes there was a decrease in the age of onset in successive generations. CONCLUSION: This study suggests that family history of diabetes results in predisposition to early onset of the disease in successive generations and a cluster of genes involved in Type 2 DM may show a parental effect for predisposition to Type 1 DM in the offspring in this set of Indian population.

Efficacy and safety of extended dosing schedules of CC-486 (oral azacitidine) in patients with lower-risk myelodysplastic syndromes.

CC-486, the oral formulation of azacitidine (AZA), is an epigenetic modifier and DNA methyltransferase inhibitor in clinical development for treatment of hematologic malignancies. CC-486 administered for 7 days per 28-day treatment cycle was evaluated in a phase 1 dose-finding study. AZA has a short plasma half-life and DNA incorporation is S-phase-restricted; extending CC-486 exposure may increase the number of AZA-affected diseased target cells and maximize therapeutic effects. Patients with lower-risk myelodysplastic syndromes (MDS) received 300 mg CC-486 once daily for 14 days (n=28) or 21 days (n=27) of repeated 28-day cycles. Median patient age was 72 years (range 31-87) and 75% of patients had International Prognostic Scoring System Intermediate-1 risk MDS. Median number of CC-486 treatment cycles was 7 (range 2-24) for the 14-day dosing schedule and 6 (1-24) for the 21-day schedule. Overall response (complete or partial remission, red blood cell (RBC) or platelet transfusion independence (TI), or hematologic improvement) (International Working Group 2006) was attained by 36% of patients receiving 14-day dosing and 41% receiving 21-day dosing. RBC TI rates were similar with both dosing schedules (31% and 38%, respectively). CC-486 was generally well-tolerated. Extended dosing schedules of oral CC-486 may provide effective long-term treatment for patients with lower-risk MDS.

Comparison of risk stratification tools in predicting outcomes of patients with higher-risk myelodysplastic syndromes treated with azanucleosides.

Established prognostic tools in patients with myelodysplastic syndromes (MDS) were largely derived from untreated patient cohorts. Although azanucleosides are standard therapies for higher-risk (HR)-MDS, the relative prognostic performance of existing prognostic tools among patients with HR-MDS receiving azanucleoside therapy is unknown. In the MDS Clinical Research Consortium database, we compared the prognostic utility of the International Prognostic Scoring System (IPSS), revised IPSS (IPSS-R), MD Anderson Prognostic Scoring System (MDAPSS), World Health Organization-based Prognostic Scoring System (WPSS) and the French Prognostic Scoring System (FPSS) among 632 patients who presented with HR-MDS and were treated with azanucleosides as the first-line therapy. Median follow-up from diagnosis was 15.7 months. No prognostic tool predicted the probability of achieving an objective response. Nonetheless, all five tools were associated with overall survival (OS, P=0.025 for the IPSS, P=0.011 for WPSS and P<0.001 for the other three tools). The corrected Akaike Information Criteria, which were used to compare OS with the different prognostic scoring systems as covariates (lower is better) were 4138 (MDAPSS), 4156 (FPSS), 4196 (IPSS-R), 4186 (WPSS) and 4196 (IPSS). Patients in the highest-risk groups of the prognostic tools had a median OS from diagnosis of 11-16 months and should be considered for up-front transplantation or experimental approaches.

Phase I and pharmacodynamic study of the topoisomerase I-inhibitor topotecan in patients with refractory acute leukemia.

PURPOSE: To determine the feasibility of escalating the hydrophilic topoisomerase I (topo I)-inhibitor topotecan (TPT) above myelosuppressive doses in adults with refractory or relapsed acute leukemias and to assess pharmacodynamic determinants of TPT action. PATIENTS AND METHODS: Seventeen patients received 33 courses of TPT as a 5-day infusion at doses ranging from 0.70 to 2.7 mg/m2/d. Pharmacologic studies were performed to determine the TPT concentrations at steady-state (Css) and to examine parameters in the patients' leukemic blasts ex vivo that may be related to TPT sensitivity, eg, topo I content, p-glycoprotein (Pgp) expression, and the inhibitory effects of relevant TPT concentrations on the growth of blast colonies in clonogenic assays relative to the range of TPT Css values achieved. RESULTS: Severe mucositis of the oropharynx and perianal tissues was intolerable at TPT doses greater than 2.1 mg/m2/d, the recommended dose for phase II studies in leukemia. One complete response (CR) in a patient with chronic myelogenous leukemia in blast crisis (CML-B) and one partial response (PR) in a patient with acute myelogenous leukemia (AML) were noted. Significant reductions in circulating blast-cell numbers occurred in all courses, and complete leukemia clearance from the peripheral blood, albeit transient, was noted in 11 courses. TPT Css values ranged from 4.8 to 72.5 nmol/L. Colony-forming assays showed that the TPT LD90 (dose that inhibits the growth of leukemia blast colonies by 90%) values for blasts varied from 6 to 22 nmol/L, a range that overlapped with TPT Css values. In view of these variations in TPT sensitivity, several aspects of topo I-mediated drug action were also studied. In 10 of 11 samples, the multi-drug resistance (Mdr) modulator quinidine altered nuclear daunorubicin (DNR) accumulation and whole-cell TPT accumulation by less than 15%, which suggests that Pgp-mediated effects on drug efflux are insufficient to explain the fourfold range of TPT sensitivities in the colony-forming assays. Immunohistochemistry showed that topo I was expressed in all of the blasts from individual patients without detectable cell-to-cell heterogeneity in each marrow. Western blots indicated that topo I content varied over a 10-fold range. Although the sample size was small, topo I content appeared to be higher in acute lymphoblastic leukemia (ALL), intermediate in AML, and lower in CML-B. Topo I content did not appear to be related to the proliferative status of the blasts. CONCLUSION: These results indicate that substantial dose escalation of TPT above myelosuppressive doses reached in solid-tumor patients is feasible in patients with refractory leukemia, that biologically relevant TPT Css values are achievable, and that further developmental trials are warranted.

Identification of growth factor-responsive acute myelogenous leukemia based on factor-dependence for survival and proliferation.

To determine whether a requirement for exogenous growth factors (GFs) for survival and proliferation could identify cases of AML which would respond best to GFs, singly and in combination, primary AML bone marrow samples were grown in suspension. Samples were classified as GF-dependent (61%), or GF-independent (39%) based on maintenance of Ki67+ cell number (proliferating cells) in the absence of exogenous GFs. GF-dependent samples had significant proliferative responses to steel factor (SF), GM-CSF and IL-3; mean Ki67+ cell number increased by 4.1-, 3.3-, and 5.2-fold, respectively. Significant stimulation was not seen for GF-independent cases; several were inhibited by exogenous GFs. SF was additive or synergistic with GM-CSF or IL-3 among GF-dependent cases; however, combinations were no more effective than single cytokines among the GF-independent group. GM-CSF plus IL-3 or the hybrid protein PIXY 321 did not increase the mean Ki67 ratio compared to the individual cytokines for any population. Flow cytometric determination of GF receptor expression was less predictive of GF response than was survival and proliferation in the absence of GFs. Suspension cultures in the absence of GFs can select patients most likely to benefit from therapeutic strategies using GFs for cell cycle recruitment.

Flow cytometric detection of rare normal human marrow cells with immunophenotypes characteristic of acute lymphoblastic leukemia cells.

Rare subpopulations of normal marrow B lymphoid cells expressing immunophenotypes typically found in B-lineage acute lymphoblastic leukaemias (ALL) were sought by multiparameter flow cytometry. First, CD34+ marrow leukocytes were isolated by immune adherence using immunomagnetic microspheres, and analyzed for coexpression of the following pairs of membrane antigens: CD34 CD22; CD34 CD20; and CD10 CD22. Terminal deoxynucleotidyl transferase expression was not assessed. All three antigen combinations were found on small percentages of the CD34-enriched cell population. Second, unseparated normal low density marrow leukocytes were examined by 'gating' on cells with the right-angle light scatter of lymphoid cells, plus either CD34+ or CD10+ immunofluorescence. This independent approach confirmed that rare subsets of normal cells coexpress 'immature' and 'mature' differentiation antigens. In addition, remission marrow cells were examined from two children who had completed therapy for ALL two and four months earlier. Both specimens had a more than threefold increase in CD34+ cells over normal marrow, and cells coexpressing immature and mature cell surface antigens were easily detected. These findings demonstrate that immunophenotypes characteristic of B-lineage ALL, previously labeled 'asynchronous' with respect to the developmental sequence of the majority of normal B lymphoid cells, exist at low frequency in normal human bone marrow.

Augmentation of phenylbutyrate-induced differentiation of myeloid leukemia cells using all-trans retinoic acid.

Despite preliminary evidence of clinical activity of the putative differentiating agent sodium phenylbutyrate (PB) in the treatment of myeloid neoplasms, it has proven difficult to maintain therapeutic levels of PB above 0.5 mM, well below the ED50 of 1-2 mM. We have studied the impact of combining PB with all-trans retinoic acid (ATRA) on the ML-1 myeloid leukemia cell line. ATRA augmented PB-induced differentiation, cell-cycle arrest, and apoptosis. ATRA augmented PB induction of the myelomonocytic marker CD11b at all doses of ATRA tested (0.0025-1 microM). Although ATRA did not significantly affect the ED50 of PB, the combination of ATRA (1 microM) and PB (0.5 mM) augmented PB-induced CD11b expression eight-fold. Compared to PB alone, this combination of ATRA and PB induced greater cell cycle arrest (S-phase 14% vs 38%; G0/G1-phase cells 72% vs 52%) and greater apoptosis (24% vs 16% by TUNEL assay). Treatment with ATRA (0.5 microM) in combination with PB (0.5 mM) led to significantly greater inhibition of colony formation (4.8% vs 48% inhibition). ATRA combined synergistically with PB to augment CD11b expression and inhibit colony formation. This combination also showed significant interaction in terms of S-phase inhibition. However, this interaction varied as a function of ATRA concentration: antagonistic at low concentrations of ATRA, synergistic at higher concentrations of ATRA. These data suggest that retinoids may significantly augment the cytostatic and differentiating activity of PB, leading to increased potency of the latter drug at clinically achievable doses.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), given concurrently with induction therapy for acute myelogenous leukemia (AML), augments the syndrome of T-lymphocyte recovery.

A transient lymphocytosis precedes myeloid recovery in many patients with AML treated with intensive chemotherapy. We describe the kinetics, clinical features, and immunology of lymphocyte recovery which is markedly augmented by the inclusion of GM-CSF in induction therapy. Lymphocyte recovery from 19 patients receiving GM-CSF as part of induction therapy was compared to a historical control of 25 patients treated with identical chemotherapy in the absence of cytokine. Kinetics and clinical features of lymphocyte recovery were analyzed. Peripheral blood was studied by flow cytometry, chromium release assays, and Southern analysis of the T-cell antigen receptor, beta chain gene. Patients treated with GM-CSF to recruit cells into cycle, exhibit markedly increased peaks of lymphocyte recovery. Recovering lymphocytes demonstrated an activated memory T-cell phenotype suggestive of a cytokine release syndrome. Lymphoid recovery was often associated with rash, fever, and lymphadenopathy. Study patients who developed peak lymphocyte counts > or = 1000 microliters were more likely to achieve remission than those with a lower peak. Recovery lymphocytes did not lyse pretreatment autologous bone marrow cells. Southern analysis demonstrated dominant potentially clonal rearrangements in the majority of patients studied. Lymphocyte recovery, which appears to include oligoclonal expansion of memory T cells is markedly augmented by administration of GM-CSF during chemotherapy. This may represent a non-specific response by a limited repertoire of T cells surviving therapy, or a specific clonal response to a powerful exogenous or endogenous antigen. Possible antileukemic activity of these cells remains to be elucidated.

Phenylbutyrate-induced G1 arrest and apoptosis in myeloid leukemia cells: structure-function analysis.

The aromatic fatty acid phenylbutyrate (PB) induces cytostasis, differentiation, and apoptosis in primary myeloid leukemic cells at clinically achievable concentrations. In the present study, we have investigated the structural and cellular basis for PB-induced cytostasis, using the ML-1 human myeloid leukemia cell line as a model system. PB induced a dose-dependent increase in cells in G1 with a corresponding decrease in cells in S-phase of the cell cycle. At comparable doses, PB induced expression of CD11b, indicating myeloid differentiation. At higher doses, the drug induced apoptosis. The antitumor activity was independent of the aromatic ring, as butyric acid (BA) was of equal or greater potency at producing these biological changes. In contrast, shortening of the fatty acid carbon chain length, as demonstrated with phenylacetate (PA), significantly diminished drug potency. Consistent with their effects on cell cycle, PB and BA, but not PA, induced the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1), and led to the appearance of hypophosphorylated Rb, suggesting a role for p21(WAF1/CIP1) in PB-induced cytostasis. Therefore, it appears that the fatty acid moiety of PB, rather than its aromatic ring, is critical for its activity in myeloid leukemic cells. These data provide a potential mechanistic basis for the increased potency of PB over PA previously demonstrated in primary leukemic samples, and support the further clinical development of PB in the treatment of hematologic malignancies.

Impact of the putative differentiating agents sodium phenylbutyrate and sodium phenylacetate on proliferation, differentiation, and apoptosis of primary neoplastic myeloid cells.

Sodium phenylacetate (PA) and sodium phenylbutyrate (PB) are aromatic fatty acids that can effect differentiation in a variety of cell lines at doses that may be clinically attainable. We have studied the impact of these two agents on lineage- and differentiation stage-specific antigen expression, proliferation, apoptosis, and clonogenic cell survival in primary cultures of bone marrow samples from patients with myeloid neoplasms at presentation and in remission and from normal volunteers. PB inhibited the proliferation of primary acute myeloid leukemia cells in suspension culture with an ID50 of 6.6 mM, similar to its ED50 in cell lines. At higher doses (>/=5 mM), PB also induced apoptosis. PB inhibited clonogenic leukemia cell growth with a median ID50 of less than 2 mM; however, colony-forming units-granulocyte/macrophage from patients with myelodysplasia and normal volunteers were inhibited with a similar ID50. In contrast to PB, its metabolite PA had no significant effect on either acute myeloid leukemia proliferation or apoptosis. Expression of the monocytic marker CD14 was increased in monocytic and myelomonocytic leukemias in response to PB, and to a lesser extent, PA. Surprisingly, both agents appeared to increase expression of the progenitor cell antigen CD34, as well as the DR locus of the human leukocyte antigen. These data indicate that PB, but not its metabolite PA, has significant cytostatic and differentiating activity against primary neoplastic myeloid cells at doses that may be achievable clinically.

Impact of in vivo administration of interleukin 3 on proliferation, differentiation, and chemosensitivity of acute myeloid leukemia.

Early clinical trials of growth factor augmentation of induction chemotherapy for acute myeloid leukemia have yielded variable results. To test the hypothesis that this heterogeneity is a consequence of the pleiotropic effects of growth factors on leukemic cell biology, we measured the effects of in vivo interleukin 3 (IL-3) administration on leukemic cell proliferation and drug sensitivity. Thirty-four patients with acute myeloid leukemia with high-risk features or advanced myelodysplasia received IL-3 as a continuous infusion beginning 3 days prior to chemotherapy and continuing for the duration of intensive induction therapy. Bone marrow cells were studied prior to and after 3 days of IL-3 administration to assess changes in overall and leukemic progenitor cell [leukemia colony-forming unit (CFU-L)] proliferation, and progenitor cell sensitivity to 1-betad-arabinofuranosylcytosine. The median fold increase in overall leukemic cell proliferation in response to IL-3, assessed as expression of the nuclear antigen Ki67 in 28 patients, was 1.2. The median fold increase in percentage of cells in S phase (assessed in 29 patients) was 1.3. Despite the increase in overall cell proliferation in 70% of cases, CFU-L number increased in only 4 of 20 patients successfully studied (median day 4:day 1 ratio of CFU-L number, 0.6). While this suggests possible terminal differentiation of leukemic progenitor cells, expression of CD34, HLA-DR, c-kit, CD15, and CD14 were not consistently affected by the cytokine. 1-betad-Arabinofuranosylcytosine sensitivity of CFU-L increased significantly in 30% of cases, decreased in 30%, and was unchanged in 40%. Changes in overall cell proliferation (Ki67 expression) and CFU-L were independent predictors of change in 1-beta-d-arabinofuranosylcytosine sensitivity; increase in percentage of cells in S phase in response to IL-3 was correlated with attainment of complete remission. While these findings support the concept of cell cycle recruitment, IL-3 has marked pleiotropic effects on proliferation, differentiation, and survival of leukemic progenitors which make the clinical impact of in vivo cytokine administration for individual patients difficult to predict.

A phase II "window" study of topotecan in untreated patients with high risk adult acute lymphoblastic leukemia.

To further evaluate the activity of topotecan (TPT) in acute leukemia, TPT was administered (2.1 mg/m2/day for 5 days by continuous i.v. infusion) to adult patients with previously untreated acute lymphoblastic leukemia (ALL) with high-risk features (13 patients) or relapsed ALL (1 patient). Patients achieving a partial response or significant hematological improvement received a second course. All patients subsequently received standard treatment for ALL. Because complete response was achieved in only 1 of 14 patients, the study was terminated prematurely. An additional patient achieved minimal response, and a third patient normalized her hemogram despite ongoing leukemia in the marrow. Overall, six patients had significant hematological improvement (normalization of platelet and/or absolute neutrophil count). Two patients expired due to infections during induction chemotherapy. The primary nonhematological toxicities were mucositis and diarrhea. Exposure to TPT did not appear to influence the response to subsequent standard chemotherapy. The mean steady-state TPT plasma concentration, 16.1+/-1 nM, overlapped the range of LD90 values of primary human leukemia specimens. Cellular topo I content varied over a 3-fold range, encompassing levels found previously in relapsed patients. No relationship was found between topo I expression and markers of cellular proliferation or response to therapy. In contrast, low expression of the apoptosis inhibitor Bcl-2 was associated with response to TPT therapy. TPT has significant, albeit modest, single-agent activity against high-risk adult ALL. This study demonstrates the feasibility of evaluating promising new therapeutic agents in untreated patients with acute leukemia at high risk for failure with conventional therapy.

A phase I and pharmacological study of topotecan infused over 30 minutes for five days in patients with refractory acute leukemia.

The principal objectives of this study were to determine the feasibility of escalating doses of the hydrophilic topoisomerase I (topo I) inhibitor topotecan (TPT) as a 30-min infusion daily for 5 days in adults with refractory or relapsed acute leukemia and to study the pharmacokinetic behavior of high doses of TPT and pharmacodynamic determinants of TPT activity. Fourteen patients received 27 courses of TPT at doses ranging from 3.5 to 5.75 mg/m2/day every 3 weeks. A constellation of unusual adverse effects, consisting of high fever, rigors, precipitous anemia, and hyperbilirubinemia, was the principal dose-limiting toxicity of high doses of TPT on this schedule. These toxicities were consistently intolerable at the 5.75 mg/m2/day dose level; however, they were neither severe nor common at lower doses. Although the precise etiology of these effects is not known, high doses of TPT may induce acute hemolytic reactions in this patient population. Severe, albeit transient, mucositis was experienced by two of eight patients in 2 of 17 courses at the next lower dose level, 4.5 mg/m2/day, which was determined to be the maximum tolerated dose and the dose recommended for further trials. The pharmacokinetic behavior of TPT at high doses was not dose dependent and resembled that at lower doses. In view of preclinical data suggesting that TPT sensitivity might correlate with topo I levels, topo I content in leukemia blasts was assessed by Western blotting. Variations in topo I content were observed. Moreover, strong correlations were evident between topo I content and two markers of proliferation, proliferating cell nuclear antigen and nuclear protein B23, raising the possibility that differences in topo I content observed among various leukemia specimens might reflect differences in the proliferating fractions of cells in various leukemia samples. Although complete clearance of circulating leukemia blasts occurred in most courses, neither sustained responses nor hematopoietic recovery were observed in the heavily pretreated, poor-risk patients enrolled in this study, and it was not possible to correlate these differences in topo I content with clinical response. These results indicate that substantial dose escalation of TPT as a 30-minute infusion for a 5-day schedule above myelosuppressive doses is feasible in adults with refractory or relapsed leukemias; however, further development of alternate high-dose schedules in leukemia may be warranted in view of the nature of the dose-limiting toxicity and the lack of sustained clinical responses in this preliminary investigation.

Impact of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia.

Sodium phenylbutyrate (PB) is an aromatic fatty acid with cytostatic and differentiating activity against malignant myeloid cells (ID(50), 1-2 mM). Higher doses induce apoptosis. Patients with myelodysplasia (n = 11) and acute myeloid leukemia (n = 16) were treated with PB as a 7-day continuous infusion repeated every 28 days in a Phase I dose escalation study. The maximum tolerated dose was 375 mg/kg/day; higher doses led to dose-limiting reversible neurocortical toxicity. At the maximum tolerated dose, PB was extremely well tolerated, with no significant toxicities; median steady-state plasma concentration at this dose was 0.29 +/- 0.16 mM. Although no patients achieved complete or partial remission, four patients achieved hematological improvement (neutrophils in three, platelet transfusion-independence in one). Other patients developed transient increases in neutrophils or platelets and decrements in circulating blasts. Monitoring of the percentage of clonal cells using centromere fluorescence in situ hybridization over the course of PB administration showed that hematopoiesis remained clonal. Hematological response was often associated with increases in both colony-forming units-granulocyte-macrophage and leukemic colony-forming units. PB administration was also associated with increases in fetal erythrocytes. These data document the safety of continuous infusion PB and provide preliminary evidence of clinical activity in patients with myeloid malignancies.

Steel factor supports the cycling of isolated human CD34+ cells in the absence of other growth factors.

Steel factor (SF) acts synergistically with other hematopoietic growth factors to support the proliferation of progenitor cells in a variety of culture systems. To determine whether SF alone could directly stimulate proliferation of early hematopoietic progenitor cells, isolated CD34+ cells from adult bone marrow and umbilical cord blood were studied in a short-term suspension culture in serum-free medium. Numbers of CD34+ and proliferating cells were quantified by flow cytometry; proliferation was assessed by simultaneous measurement of expression of the nuclear antigen Ki67 and DNA content (propidium iodide [PI]). In the absence of growth factors, the numbers of CD34+ and cycling cells declined over 2 days. Cells cultured in the presence of SF alone maintained the number of CD34+ and cycling cells at levels equal to the starting population. Withdrawal of growth factors led to a dramatic decrease in the number of cells in G1. Compared to cells grown in the absence of growth factors, cells grown in the presence of SF had significantly higher numbers of CD34+ and cycling cells (mean fold increase = 1.3 and 2; p < 0.05 and 0.01, respectively). SF increased the numbers of cells in both G1 and later phases of the cell cycle. Addition of interleukin-3 (IL-3) to SF led to further significant increases in CD34+ and cycling cells. The effects of SF could not be attributed to inhibition of apoptosis. CD34+ cells isolated from peripheral blood (PB) from patients with chronic myelogenous leukemia (CML) displayed similar characteristics. As assessed by binding of phycoerythrin (PE)-labeled ligand and flow cytometry, c-kit was expressed on 65 +/- 6% of isolated CD34+ cells and was detected on HLA-DRlow, CD38low, and Thy1+ subsets, as well as on more mature progenitor cells. Thus, while the effects of SF are most marked in combination with other growth factors, SF appears to bind to and directly maintain the active cell-cycle characteristics of isolated CD34+ cells.

Validation of flow-cytometric determination of Ki67 expression as a measure of growth factor response in acute myelogenous leukemia.

Despite its growing use as a marker of proliferation, the relationship between expression of the nuclear antigen Ki67 and other indices of proliferation in acute myelogenous leukemia (AML) has not been elucidated. In this study, short-term primary suspension cultures of human AML bone marrow cells were used to compare flow-cytometric methods of quantifying Ki67 expression and to test whether flow-cytometric determination of Ki67 expression correlates with incorporation of 3H-thymidine or bromodeoxyuridine (BrdU). BrdU incorporation was determined by staining of cells with anti-BrdU and propidium iodide (PI) followed by flow cytometry. When samples were double-labeled with Ki67 and PI, Ki67 was underestimated compared to single-color quantification of the nuclear antigen. Ki67+ cell number correlated well with incorporation of 3H-thymidine (r = 0.89, p < 0.001). Cells from 17 cases of AML were cultured for 3 days in the presence and absence of a variety of growth factors and growth factor combinations before comparison of Ki67 expression and BrdU incorporation. Ki67 expression correlated strongly with BrdU incorporation (r = 0.82, p < 0.001). Unlike the double-label with PI, containing with a phycoerythrin (PE)-labeled antibody directed against a cell surface antigen did not significantly affect Ki67 quantification. Flow cytometric determination of Ki67 is a simple and valid measurement of proliferation in AML bone marrow cells.

Alteration of transmembrane sodium and potassium gradients inhibits the action of luteinizing hormone in the luteal cell.

To evaluate the role of transmembrane ion flux on the acute response of luteal cells to LH, the effects of two drugs, ouabain and monensin, and changes in ion composition of the medium were examined. Ouabain, an inhibitor of the Na+ extrusion pump, and monensin, a selective Na+ ionophore, would be expected to increase the intracellular level of Na+. Both drugs produced a highly significant, dose-related and Na+-dependent inhibition of LH-stimulated cAMP accumulation and progesterone secretion. The IC50 values for ouabain and monensin for both responses to LH were about 50 and 0.1 microM, respectively, and inhibition of cAMP accumulation was competitive [inhibitory constant (Ki) = 20 and 0.06 microM, respectively]. Both drugs showed inhibition only in the intact cell, and no nonspecific cytotoxic effects were evident. No effect on the specific binding of gonadotropin or on LH-stimulated adenylate cyclase activity in membranes was seen, nor was inhibition reduced by coincubation of the cells with isobutyl methylxanthine, a cAMP phosphodiesterase inhibitor. Both drugs inhibited (Bu)2cAMP-stimulated progesterone accumulation. No effect of ouabain was seen on cholera toxin- or forskolin-stimulated cAMP accumulation, whereas monensin significantly inhibited this response to both agonists. Preincubation of cells with LH protected against inhibition of cAMP accumulation by ouabain and monensin. Removal of extracellular Na+ completely prevented inhibition by both drugs and slightly blunted the response to LH alone. However, reduction of extracellular Na+ from 128 to 32 mM, treatment of cells with tetrodotoxin (a sodium channel blocker), or increasing extracellular K+ from 5.4 to 66 mM had no effect on the stimulation of cAMP accumulation by LH. On the other hand, removal of extracellular Ca2+ completely blocked inhibition of LH-stimulated cAMP accumulation by ouabain or monensin. It is concluded that ouabain and monensin inhibit the acute stimulation of cAMP accumulation by LH probably as a result of an influx of Na+ into the luteal cell. The increase in intracellular Na+ does not appear to directly inhibit LH-sensitive adenylate cyclase activity, but induces a secondary influx of extracellular Ca2+ which inhibits activation of adenylate cyclase by LH at a site involved in coupling of the receptor to the enzyme. Luteal regression may be initiated by mechanisms similar to that caused by ouabain and monensin, because the characteristics of inhibition by these drugs is identical to that caused by prostaglandin F2 alpha, a physiological luteolysin.(ABSTRACT TRUNCATED AT 400 WORDS)

Stage-specific application of allogeneic and autologous marrow transplantation in the management of acute myeloid leukemia.

Allogeneic (alloBMT) and autologous bone marrow transplantation (ABMT) have become standard approaches for the management of adults with acute myeloid leukemia (AML). The indications for transplantation remain controversial as parallel improvements in intensive chemotherapy have resulted in excellent outcomes for many patients. AlloBMT is the therapy of choice for patients who fail to respond to induction chemotherapy. For those patients in first remission (CRI), a policy of intensive postremission chemotherapy with transplantation upon relapse appears to be optimal. There are no data to support transplantation in CRI, allogeneic or autologous, for those patients with leukemia characterized by favorable cytogenetic abnormalities [ie, core-binding factor type or t(15;17)], as these patients do well with nonmyeloablative strategies. Patients with relapsed disease appear to be best served with allogeneic transplantation from a human leukocyte antigen (HLA)-matched sibling or one-antigen-mismatched family member, whereas for those patients lacking a related donor, unrelated donor alloBMT or ABMT provides similar long-term overall survival. Randomized studies for the optimal management of relapsed disease are lacking but are needed. The objective of this review is to discuss the data supporting the use of alloBMT or ABMT at various points during the course of de novo adult AML.

Detection of minimal residual T cell acute lymphoblastic leukemia by flow cytometry.

We have developed a flow cytometric assay for the determination of cellular expression of terminal deoxynucleotidyl transferase (TdT) and applied this to the detection of minimal residual T cell acute lymphoblastic leukemia (T-ALL). The flow cytometric assay for TdT demonstrated requisite specificity: TdT was localized to the nucleus, and was detected in MOLT3 T lymphoblasts, clinical T-ALL samples, and normal bone marrow B lymphoid precursors, but in neither the KG1a myeloid leukemia cell line nor normal myeloid cells. Co-expression of TdT and the pan T cell marker CD5 was used to quantify T lymphoblasts. 0.25 +/- 0.13% of normal adult bone marrow CD5+ cells were TdT+; these may represent early T lymphoid precursors. When admixed with normal bone marrow, CD5+TdT+ leukemic cells could be detected above background levels at an added concentration of 0.035% (95% confidence interval 0.028-0.43%). Long term follow-up of a large number of patients will be required to determine the clinical significance of a minimal burden of leukemic cells.

Modifying histones to tame cancer: clinical development of sodium phenylbutyrate and other histone deacetylase inhibitors.

Compounds that inhibit histone deacetylase may enable the re-expression of silenced regulatory genes in neoplastic cells, reversing the malignant phenotype. Although several molecules that inhibit histone deacetylase are undergoing preclinical development, butyric acid derivatives have undergone clinical investigation for several years, initially for non-malignant indications and more recently for the treatment of cancer. Of the butyric acid derivatives, sodium phenylbutyrate has undergone the most extensive systematic investigation. Administration of phenylbutyrate by iv. and oral routes is well-tolerated clinically at concentrations which effect acetylation of histones in vitro. Higher doses lead to reversible CNS depression. The studies presented to date have been Phase I studies and do not enable assessment of efficacy. However, current development of phenylbutyrate is proceeding in combination with other agents based on rational biologically-based in vitro studies. The parallel development of combination therapy including phenylbutyrate and early clinical development of other, more potent histone deacetylase inhibitors will hopefully lead to feasible, clinically tolerable strategies for altering the malignant phenotype of cancer cells.