Synchronization and recruitment in acute leukemia.

The in vivo effects of several chemotherapeutic agents on the mitotic cycle of leukemic blasts in the bone marrow were evaluated by serial measurements of cells in mitosis and in deoxyribonucleic acid (DNA) synthesis as indicated by ability to incorporate tritiated thymidine or tritiated deoxyuridine. 28 studies were done in 23 children and 1 adult. The changes in the marrow after a single injection of L-asparaginase, hydrocortisone, cyclophosphamide, cytosine arabinoside, methotrexate, and an exchange transfusion (62% of the total blood volume) were evaluated. L-asparaginase and hydrocortisone were found to arrest the entry of cells into the S period. Cyclophosphamide appeared to inhibit DNA synthesis, arrest cells in mitosis, and inhibit the entry of cells into the S period. Cytosine arabinoside, and methotrexate inhibited DNA synthesis. During the period of time the cells were inhibited in the S phase by these two drugs, cells continued to enter the S period. Thus partial synchronization was achieved after these two drugs. An exchange transfusion had no consistent effect on the mitotic cycle, but partial synchronization in the S period was seen in one patient. To take advantage of the ability of cystosine arabinoside, to synchronize leukemic cells in the S phase, a second cycle-dependent drug was given at the time the leukemic blasts were synchronized. The second cycle-dependent drugs evaluated were vincristine, methotrexate, and cytosine arabinoside given by intravenous drip over a 12 hr period. Recruitment was found after cytosine arabinoside alone, and after prior synchronization with cytosine arabinoside and then the administration of either of these drugs. The results of these studies indicate that a greater therapeutic advantage can be achieved by a second cycle-dependent drug after synchronization than after the second drug alone.

Variation of proliferative activity in leukemic cell populations of patients with acute leukemia.

In 31 children with acute leukemia, the proliferative activity of the leukemic marrow cell population as measured by mitotic and labeling indexes varied widely from patient to patient and from one disease stage to another. Leukemic marrow had a small but statistically significant diurnal variation of proliferative activity. Changes in labeling indexes were directly related to changes in the proportion of large dividing blasts in the marrow. Generation times of dividing leukemic blast cells in 3 patients were similar at diagnosis and in relapse. Changes in proliferative activity of leukemic marrow can be explained by progressive accumulation of nondividing leukemic cells.

Drug effect in acute leukemia.

The in vivo therapeutic effect of vincristine, cytosine arabinoside, and corticosteroids on leukemic blast cells in the bone marrow was evaluated. 24 studies were done in 21 children with acute leukemia. 19 children had acute lymphoblastic leukemia, and two children had acute myeloblastic leukemia. Direct cytotoxicity or lysis of blast cells after drug administration was looked for by serial measurements of the volume of marrow buffy coat. Changes in proliferative capacity were evaluated by serial measurements of number of cells in mitosis and the per cent of cells in deoxyribonucleic acid (DNA) synthesis, as indicated by tritiated thymidine incorporation. Corticosteroid administration caused lysis of leukemic blast cells. Each drug affected the proliferative capacity of the leukemic cells by an action at a different part of the mitotic cycle. Corticosteroids suppressed the entry of cells into DNA synthesis. Vincristine arrested cells in mitosis. Cytosine arabinoside inhibited DNA synthesis. After the inhibitory effect of cytosine arabinoside, an increased number of cells began to synthesize DNA, a phenomenon indicating that partial synchronization of the mitotic cycle had been achieved in the leukemic cell population. The action of these drugs at different parts of the mitotic cycle might be important in designing treatment regimens where in two or more of these drugs are used.

Biochemical and functional abnormalities in lymphocytes from an adenosine deaminase-deficient patient during enzyme replacement therapy.

Biochemical and immunological properties of lymphocytes were measured repetitively over a period of 40 mo during enzyme replacement by transfusion in a child with adenosine deaminase (ADA) deficiency and severe combined immunodeficiency disease. Catalytically defective ADA protein is present in the child's cells. ADA activity in his lymphocytes is 7 nmol/min per 10(8) cells with 51 ng of ADA protein/10(8) cells by radioimmunoassay. ADA activities in normal cord and adult lymphocytes average 193 and 92 nmol/min per 10(8) cells, respectively, with 429 and 223 ng of ADA protein/10(8) cells. Deoxy(d)ATP accumulates in the patient's erythrocytes and lymphocytes. Transfusion of irradiated packed erythrocytes partially corrects the metabolic defects. Frank metabolic relapse occurs if transfusions are discontinued for several months. The amounts of dATP in erythrocytes and lymphocytes averaged 13 and 2 times normal, respectively, during periods when transfusions were administered every 2-4 wk. Deoxyguanosine triphosphate and deoxycytidine triphosphate in lymphocytes were normal on 11 occasions, but deoxyribosylthymine triphosphate was ninefold increased. On 11 occasions dATP was measured in lymphocytes and erythrocytes isolated simultaneously. There was a positive, but statistically insignificant, correlation between amounts of dATP in the two types of cells (r = 0.25,P > 0.1). The absolute peripheral lymphocyte count was correlated with the activity of ADA in circulating erythrocytes and with the response of lymphocytes to phytohemagglutinin (r = 0.64, P < 0.01; r = 0.49, P < 0.05). Response of lymphocytes to stimulation by phytohemagglutinin in vitro and absolute peripheral lymphocyte counts were not significantly correlated with levels of dATP in the erythrocyte or lymphocyte during periods of intensive therapy. Although there was objective improvement during enzyme replacement, the child remained immunodeficient and biochemically abnormal.

Effect of 5-fluoro-2'-deoxyuridine on deoxyribonucleotide pools in vivo.

5-Fluoro-2'-deoxyuridine (FdUrd) lowered the dTTP levels in rapidly frozen 12-day W rat embryos and in a human neuroblastoma grown in nude N:NIH(S) mice to about 20% of control values. This effect was associated with greatly increased dCTP levels and reduction of dGTP levels essentially to zero. Elimination of the dGTP pool correlated temporally with the cytotoxicity of FdUrd. Extremely rapid fixation of tissue was required to avoid artifactually high deoxyribonucleoside triphosphate values.

Phase II trial of a complex polyriboinosinic-polyribocytidylic acid with poly-L-lysine and carboxymethyl cellulose in the treatment of children with acute leukemia and neuroblastoma: a report from the Children's Cancer Study Group.

Therapeutic efficacy and toxicity were evaluated in 28 children with acute lymphoblastic leukemia, in ten with acute nonlymphoblastic leukemia (ANLL), and in 13 with metastatic neuroblastoma. All were refractory to standard chemotherapeutic agents and 25 were refractory to an investigational drug. The initial dose was 12 mg/m2/day and was based on an established maximal dose tolerated in adults. This dose was found to be intolerable in 5 of 5 children with leukemia. Similarly an initial dose of 9 mg/m2/day was intolerable in 4 of 5 patients with leukemia. The starting dose in the next 28 children with leukemia or neuroblastoma was 3 mg/m2. This drug was gradually increased to the highest tolerated dose by 3-mg/m2 increments. Fifteen children with acute lymphoblastic leukemia, 3 children with ANLL, and 2 children with neuroblastoma received the drug daily. Seven patients with ANLL and 7 patients with neuroblastoma received the drug biweekly. Seventeen patients with acute lymphoblastic leukemia, 6 patients with ANLL, and 5 patients with neuroblastoma had an adequate trial of the drug. An adequate trial was defined as a minimum of 5 weeks of therapy unless progressive disease developed. Side effects of the drug were striking and included fever, hypotension, myalgia, bone pain, arthralgia, arthritis, abdominal pain, liver toxicity, thrombocytopenia, and neurotoxicity. No complete remission occurred although interferon levels above 100 units/ml were induced in nearly 50% of the patients.

Impact of high-dose cytarabine and asparaginase intensification on childhood acute myeloid leukemia: a report from the Childrens Cancer Group.

PURPOSE: The purpose of this review was to determine the impact of high-dose cytarabine and asparaginase intensification, administered shortly after remission induction, on the outcome of childhood acute myeloid leukemia (AML). MATERIALS AND METHODS: Three consecutive Childrens Cancer Group (CCG) trials of acute myeloid leukemia, CCG 251 (1979 to 1983), CCG 213P (1983 to 1985), and CCG 213 (1985 to 1989) with a total of 1,294 patients, were reviewed and provide the basis of this report. RESULTS: CCG 213P demonstrated the importance of dose interval, in that two courses of cytarabine and asparaginase administered at 7-day intervals gave superior 5-year survival rates (58% v 41% from the end of induction, P < .04) to the same therapy administered at 28-day intervals. CCG 213 showed that there was no advantage to the maintenance therapy used for patients who received two courses of cytarabine and asparaginase at 7-day intervals (5-year survival, 68% [no maintenance] v 44% [maintenance] from the end of consolidation, P < .01). Inclusion of the 7-day interval cytarabine/asparaginase intensification was accompanied by an overall improvement in 5-year survival rates from diagnosis when compared with historical controls (CCG 213, 36% v CCG 251, 29%, P < .02) although other differences between these studies could also be responsible for the improvement seen. CONCLUSION: High-dose cytarabine and asparaginase intensification eliminated the benefit of prolonged maintenance therapy in childhood AML and was accompanied by an overall improvement in survival.

Correlation of chromosome abnormalities with patient characteristics, histologic subtype, and induction success in children with acute nonlymphocytic leukemia.

Cytogenetic analyses of bone marrow cells were performed in 195 children with acute nonlymphocytic leukemia (ANLL) at diagnosis, as part of Childrens Cancer Study Group Study No. 251. Ninety-six patients (49%) exhibited clonal abnormalities, including trisomy 8 in 18 patients, t(8;21) in 11, t(15;17) in seven, loss of a sex chromosome in seven, monosomy 7 in seven, and the Philadelphia chromosome in four. Clonal abnormalities were found significantly more often in younger patients. Furthermore, recurring cytogenetic abnormalities tended to correlate with specific ages. For example, t(8;21) was associated significantly with children over four years of age, while -7 associated with overall loss of genetic material from the long arm of chromosome 7 (7q) and 11q- were associated significantly with younger children. Recurring chromosome abnormalities also correlated with specific ANLL histologic subtypes, such as t(8;21) with acute myelogenous leukemia and t(15;17) with acute promyelocytic leukemia. Presence or absence of cytogenetic abnormalities was compared with the ability of patients to achieve remission. Individuals exhibiting clonal abnormalities in bone marrow cells had an equally likely chance of achieving remission (74%) as those individuals with normal karyotypes (75%). Nonrandom chromosome abnormalities associated with a high induction success rate included +8 with a 94% induction success rate (P = .13) and t(8;21) with a 91% success rate (P = .46). Patients exhibiting the -7 abnormality associated with overall loss of 7q had a significantly less successful induction outcome, with only 28% achieving remission (P = .02); three of seven patients with t(15;17) died during induction therapy.

Clonal remission in childhood acute myeloid leukemia is an infrequent event.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases that differ in pattern of both remission and lineage involvement. The observation that hematopoiesis remains clonal in some patients with AML in complete clinical remission suggests that the acute phase may develop from a clinically unrecognized preleukemic clone. To investigate the characteristics and significance of clonal remissions in childhood AML, we used X-chromosome-linked polymorphisms to study granulocytes obtained from pediatric female patients in complete clinical remission. Remission granulocytes from only one of 17 evaluable patients were clonally derived, suggesting that clonal remission is an infrequent event in childhood AML.

Prognostic variables in newly diagnosed children and adolescents with acute myeloid leukemia: Children's Cancer Group Study 213.

The objective of this study was to identify biologic parameters that were associated with either exceptionally good or poor outcome in childhood acute myeloid leukemia (AML). Among the children with AML who entered Children's Cancer Group trial 213, 498 patients without Down syndrome or acute promyelocytic leukemia (APL) comprise the basis for this report. Univariate comparisons of the proportion of patients attaining complete remission after induction (CR) indicate that, at diagnosis, male gender, low platelet count (< or =20 000/microl), hepatomegaly, myelodysplastic syndrome (MDS), French-American- British (FAB) category M5, high (>15%) bone marrow (BM) blasts on day 14 of the first course of induction, and +8 are associated with lower CR rates, while abnormal 16 is associated with a higher CR rate. Multivariate analysis suggests high platelet count at diagnosis (>20 000/microl), absence of hepatomegaly, < or =15% day 14 BM blast percentage, and abnormal 16 are independent prognostic factors associated with better CR. Univariate analysis demonstrated a significant favorable relationship between platelet count at diagnosis (>20 000/microl), absence of hepatomegaly, low percentage of BM blasts (< or =15%), and abnormal 16 with overall survival. Absence of hepatomegaly, < or =15% day 14 BM blast percentage, and abnormal 16 were determined to be independent prognostic factors associated with better survival.

The use of urethane sponge matrix to assess in vivo recovery of murine cellular immunity following syngeneic bone marrow transplantation.

A unique method for assessing the in vivo recovery of cellular immunity following sublethal irradiation and syngeneic bone marrow transplantation of CBA/J mice is described. This method employs the serial surgical implantation of a urethane sponge matrix followed by impregnation of the sponge matrix with alloantigen. Sponge matrices were harvested from each mouse subject at 2 weeks, 4 weeks, and 8 weeks post transplant with and without treatment with interleukin-2. Recovery of cellular immunity was assessed as a function of cytotoxic T lymphocyte activity via cell-mediated lympholysis using chromium labeled targets. Repetitive procedures of implantation and removal of the matrices from the same animal were surgically feasible and were exceptionally well tolerated by the animals with insignificant morbidity thereby permitting continuous monitoring of immunologic recovery. Although the results of treatment with interleukin-2 were inconclusive, this method provides a sensitive, technically manageable means by which to assess an antigenic response in vivo at predetermined time intervals utilizing the same mouse subject. The method additionally affords the potential of being applicable to the in vivo assessment and manipulation of other aspects of the immune response.

Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients.

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.

Acute monoblastic leukemia: a unique subtype--a review from the Childrens Cancer Study Group.

The acute non-lymphocytic leukemias (ANLL) are generally treated as a homogeneous group. However, the literature is replete with articles alluding to distinctive features of acute monoblastic leukemia (AMoL). This review addresses the unique clinical, laboratory, epidemiological, and therapeutic features of AMoL. Leukemic monoblasts are distinguished from other cells in the myelocytic series by physical properties such as greater adhesiveness, deformability, and motility. Patients with AMoL often exhibit hyperleukocytosis, disseminated intravascular coagulation, and extramedullary involvement, particularly in the skin, gingiva, and central nervous system (CNS). AMoL occurs predominantly in adults over 40 and children under 10, fifty percent of whom are under 2 years of age at diagnosis. Its relatively common occurrence in infants parallels the high rate of proliferation of monocytes in that age group. Additionally, its occurrence in young children appears to be associated with in utero exposure to marijuana and parental exposure to pesticides and solvents. Therapeutic results are generally poor due to high rates of fatal complications during induction, induction failures, and frequent extramedullary and medullary relapses. This poor outcome is particularly noted in infants. Higher remission induction rates attained with epipodophyllotoxins and incorporation of bone marrow transplantation have not yet resulted in substantial improvement of long-term outcome. Recurrence of disease in the CNS is minimized by the use of intensive CNS presymptomatic treatment, usually incorporating irradiation. Our review suggests that unique and innovative treatment strategies are needed to improve outcome for patients with AMoL.

Immunologic, morphologic and chromosomal characterization of a cell line (TC78) established from a child with acute lymphoblastic leukemia.

We characterized a cell line established from bone marrow cells from a child with acute lymphoblastic leukemia. This cell line, TC78, had lymphoblastic morphology and was cytoplasmic peroxidase and esterase negative. The cells did not have T- or B-cell properties such as E- or EAC-rosette forming ability, reactivity with monoclonal T-cell or B2 antibodies, or immunoglobulin synthesis. We concluded that TC78 was a pre-pre B-cell line based on the following monoclonal antibody staining pattern: BA-1+, BA-2+, cALLa+, Ia+, 2H7+ and OKB2+. Growth in 'Dickie' culture and reactivity with 1G10 myeloid antibody suggested coexpression of lymphoid and myeloid characteristics. However, 1G10 expression proved dependent on culture conditions, illustrating one caveat in application of monoclonal antibodies in lineage determination.

Alterations of the p15, p16,and p18 genes in osteosarcoma.

Activation of cyclin-dependent kinases (CDKs) by interaction with cyclins regulates progression through cell cycle checkpoints. This process is counterbalanced by CDK inhibitors (CDKIs), which can inhibit progression through the cell cycle. Because CDKI expression acts to inhibit cellular proliferation, CDKIs may have a role as tumor suppressors. One class of CDKIs, characterized by the presence of ankyrin repeats, has at least four members (p15INK4B), p16INK4, p18, and p19). Two of these, p15INK4B, p16INK4, have been mapped to chromosome 9p21, a region of frequent loss in a wide variety of cancers. Alterations of p16INK4 have been detected in various tumors and cell lines. We analyzed p15INK4B, p16INK4, and p18 alterations in 52 osteosarcomas (including 11 explants), and 23 other various sarcomas. Single-stranded conformation polymorphism analysis [polymerase chain reaction (PCR-SSCP)] of the coding regions of these CDKI genes detected a missense mutation of p16INK4 exon 1 in one soft tissue sarcoma. Southern blotting detected complete deletion of p15INK4B and p16INK4 genes in osteosarcomas from 2 patients and a soft tissue sarcoma from another individual. Loss of heterozygosity (LOH) at chromosome 9p21 was observed with a microsatellite probe closely linked to the INK4 genes in the latter case. Deletions of both p15INK4B and p16INK4 genes were detected in five of eight osteosarcoma cell lines. By contrast, no alterations of p18 were detected in any sample. Together these data suggest that alterations of the p15INK4B and p16INK4 genes, but not p18, may occur in approximately 5% of sarcomas. However, deletions of the p15INK4B and P16INK4 genes are frequent in osteosarcoma cell lines and probably have a role in tumor cell growth in culture. Notably, all seven detectable deletions involved both p15INK4B and p16INK4 genes, suggesting that both contribute individual tumor suppressor activity.

Congenital and neonatal leukemia.

Congenital and neonatal leukemia occur rarely, yet carry high mortality rates and pose special problems for the perinatologist and hematologist. Although the etiology is unknown, the presence of leukemia at birth suggests genetic abnormalities and possibly intrauterine exposures to drugs or other toxins as contributing factors. Specific chromosomal rearrangements that are common in congenital leukemia have recently been identified and promise to enhance our understanding of these enigmatic diseases. The differential diagnosis is broad and includes many disorders that occur frequently in the neonatal period. Infants diagnosed with congenital or neonatal leukemia require thorough investigative workup and extensive supportive care. Although the prognosis is poor, recent use of high-intensity multiagent chemotherapy regimens has produced promising results.

Effects of dimaprit on growth and differentiation of human promyelocytic cell line, HL-60.

The human promyelocytic cell line HL-60, differentiates in response to a variety of agents including dibutyryl cAMP and agents which increase intracellular cAMP concentrations (phosphodiesterase inhibitors, PGE2, and cholera toxin). HL-60 is also known to be rich in H2 -histamine sensitive adenylate cyclase activity. The present study was therefore designed to test the effects of H2-stimulation on growth and differentiation of HL-60 using the potent H2 agonist dimaprit. Dimaprit markedly increased cAMP production in a dose-dependent manner reaching maximal levels after 30-60 minutes. Intracellular cAMP levels decreased thereafter and by 24 hours were approximately 2-3 fold increased above control. Intracellular cAMP levels were not altered by dimaprit (10(-7)M to 10(-4)M) at 4 days in culture compared to either untreated HL-60 cells or dimethylsulfoxide (DMSO) (1.3%) treated cells. While exponential growth was unaltered by dimaprit (10(-7)M to 10(-4)M) as compared to control, dimaprit induced i) morphologic maturation to the myelocyte and metamyelocyte form with no differentiation seen beyond the metamyelocyte even after 6 days in culture, ii) increased NBT reductase activity and iii) dose-dependent increase in lysozyme activity which could be completely blocked by cimetidine, a specific H2 antagonist. Dimaprit-induced differentiation of HL-60 cells was associated with an initial but transient increase in intracellular cAMP production. Maturation beyond the metamyelocyte stage was not observed. Acquisition of NBT reductase and lysozyme activity correlated with morphologic maturation.

Detection of unexpected clones of monosomy 7 in childhood acute lymphoblastic leukemia using fluorescence in situ hybridization.

The feasibility of a fluorescence in situ hybridization (FISH) technique for the detection of leukemic clones with masked chromosomal aberration in interphase nuclei was tested in childhood acute lymphoblastic leukemia (ALL). Twenty-one cases of ALL previously studied by classical metaphase cytogenetics were retrospectively analysed using a centromere-specific chromosome 7 probe. Five cases with karyotypic abnormalities of chromosome 7 (2 with trisomy 7, 2 with monosomy 7 and 1 with trisomy & tetrasomy 7) showed a correlation with FISH results, whereas in five other cases monosomy 7 was found in 12-43% of cells only by FISH. The unexpected detection of monosomy 7 in these latter ALL patients suggests that either these clones are quiescent or unable to enter mitosis in vitro. This suggests that FISH and metaphase cytogenetics must be combined whenever possible to obtain comprehensive karyotypic information.

Comparison of two double labeling techniques to measure cell cycle kinetics in myeloid leukemias.

Five patients with acute myeloid leukemia (AML) received a one hour infusion of iododeoxyuridine (IUdR) 100 mg/M2 to label S-phase cells in vivo. The aspirate was labeled in vitro either with tritiated thymidine (3HTdr) or bromodeoxyuridine (BrdU) to measure the duration of S-phase (Ts). The mean Ts using 3HTdr (Ts1) was 15.9h (13.1-19.8h) and using BrdU (Ts2) was 17.1h (14.5-20.6h). Total cell cycle time (Tc) ranged between 44.7h to 158.8h using Ts1 and 54.0h to 170.5h using Ts2. Based on this close approximation between the results, we confirm the reliability of the newly developed method that relies purely on immunohistochemical reaction.

Solid malignancies in children and adolescents.

The cure rate in childhood cancer has improved markedly during the past 20 years. In the 1960s the cure rate was about 20 to 30 per cent, but today more than 50 per cent of children and adolescents with cancer are being cured. This improvement is principally due to multidisciplinary teamwork in diagnosing, staging, and treating children with cancer; newer and more chemotherapeutic agents; and a recognition that combination therapy consisting of surgery, radiotherapy, and chemotherapy is frequently indicated.