Long-term follow-up of imatinib in pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia: Children's Oncology Group study AALL0031.

We previously reported preliminary findings that post induction imatinib mesylate (340 mg/m(2)/day), in combination with intensive chemotherapy, resulted in outcomes similar to blood and marrow transplant (BMT) for pediatric patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We now report 5-year outcomes of imatinib plus intensive chemotherapy in 91 children (1-21 years) with and without allogeneic BMT (N=91). We explore the impacts of additional chromosomal abnormalities and minimal residual disease (MRD) by flow cytometry on outcomes. The 5-year disease-free survival was similar for Cohort 5 patients, treated with chemotherapy plus imatinib (70%±12%, n=28), sibling donor BMT patients (65%±11%, n=21) and unrelated donor BMT patients (59±15%; P=0.60, n=13). Patients with additional cytogenetic abnormalities had worse outcomes (P=0.05). End induction (pre-imatinib) MRD was not prognostic for Cohort 5 or allogeneic BMT patients, although limited by small numbers. The re-induction rate following relapse was similar to other higher-risk ALL groups. Longer-term follow-up confirms our initial observation of substantially good outcomes for children and adolescents with Ph+ ALL treated with imatinib plus intensive chemotherapy with no advantage for allogeneic BMT.

Using fluorescence-activated cell sorting followed by fluorescence in situ hybridization to study lineage relationships: the 8;21 translocation is present in neutrophils but not monocytes in a patient with severe congenital neutropenia and a granulocyte colony-stimulating factor-responsive clonal abnormality.

UNLABELLED: Severe congenital neutropenia (Kostmann syndrome) is a disorder that presents in the neonatal period, but predisposes to leukemia later in life. This report describes a 4-y-old female with a history of severe congenital neutropenia, who developed a clonal abnormality associated with the translocation (7;21;8) (q32;q22;q22) (AML-1/ETO). She had circulating peripheral blasts and bone marrow blast counts as high as 64% when she received recombinant granulocyte colony-stimulating factor (rG-CSF). Her marrow blasts decreased to 4-20% when rG-CSF was discontinued. Fluorescence in situ hybridization analysis was performed on bone marrow cell populations sorted by flow cytometry to determine which cell populations had the AML-1/ETO translocation. The translocation was found in mature neutrophils and blasts, but not in monocytes, lymphocytes or stem cells. CONCLUSION: These findings suggest that the translocation occurred in a neutrophil progenitor, past the point in ontogeny where monocytes and neutrophils separate. The techniques described may be useful in understanding lineage relationships and leukemogenesis in other clonal abnormalities associated with myelodysplasia and leukemia.

Observations of residual differentiation potential during lineage commitment.

We have recently described a subset of the multipotent progenitor pool that contains a common lymphoid progenitor. This subset of cells is lineage negative and expresses c-kit and Sca-1, but lacks expression of Thy 1.1 (Thyneg). Based on the observation that lethally irradiated mice transplanted with these cells die from anemia unless supported with competitor marrow, we hypothesized that these progenitors lacked erythroid potential. We analyzed the erythroid potential of these cells by transplanting them into mice allelic at the hemoglobin locus and compared their erythroid potential with the Thy-1.1low (Thylow) subset that contains hematopoietic stem cells. We also performed CFU-C assays in methylcellulose containing recombinant cytokines and determined erythroid contribution to colonies using in situ benzidine staining. Donor-derived hemoglobin was observed following transplant of Thyneg cells, even though 19 of 20 of these animals died from anemia. In contrast, recipients of Thylow cells showed complete donor-derived engraftment 30 days following transplant. While approximately 60% of day 4 colonies derived from Thyneg cells expressed hemoglobin, by day 11 less than 5% were hemoglobinized. In contrast, greater than 70% of the Thylow subset contained hemoglobinized cells at the end of the observation period. A similar transient appearance of myeloid progeny was also observed in colonies derived from c-kitlow Thyneg lymphoid progenitor cells. We conclude that these studies demonstrate commitment to the lymphoid lineage at the Thylow-to-Thyneg interface, and that the loss of erythroid and myeloid potential is gradual rather than abrupt. Hemoglobinized colonies may be undergoing apoptosis because of down-regulation of GATA-1 or because of a death signal from surrounding nonerythrocytic cells.

Phenotypic distinction and functional characterization of pro-B cells in adult mouse bone marrow.

A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1(neg)Sca-1(pos)c-Kit(low)Lin(neg). These cells were CD43(pos)CD24(pos) on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.

Congenital bone marrow failure syndromes associated with protean developmental defects and leukemia.

Congenital bone marrow failure syndromes are associated with a number of congenital abnormalities affecting a wide range of organ systems. The underlying molecular abnormalities that cause these disorders affect normal embryonic development during the critical organogenesis phase (weeks 4 to 8). These syndromes predispose patients to leukemia and other malignancies, and these genetic disorders may represent the first hit of at least two hits necessary for malignant transformation. The molecular defects underlying these diseases are just beginning to be understood; mechanisms suggested by recent research include DNA repair (FA-A, FA-G); abnormalities of the ribosomes (DBA, DC); to disorders of electron transport (FA-C, Pearson's syndrome, Barth's syndrome). Understanding these molecular mechanisms provides the knowledge necessary to develop better therapy, possibly including gene therapy, offering for the first time the potential for curing the hematologic manifestations of these illnesses.

Rapid, B lymphoid-restricted engraftment mediated by a primitive bone marrow subpopulation.

Utilizing multiparameter flow cytometry, we have defined a subset of bone marrow cells containing lymphoid-restricted differentiation potential after i.v. transplantation. Bone marrow cells characterized by expression of the Sca-1 and c-kit Ags and lacking Ags of differentiating lineages were segregated into subsets based on allele-specific Thy-1.1 Ag expression. Although hematopoietic stem cells were recovered in the Thy-1.1low subset as previously described, the Thy-1.1neg subset consisted of progenitor cells that preferentially reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy-1.1neg cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy-1.1neg cells predominantly reconstituted the bone marrow and peripheral blood of lethally irradiated recipients with B lineage cells within 2 weeks, although a low frequency of myeloid lineage cells was also detected. In contrast, myeloid progenitors outnumbered lymphoid progenitors when the Thy-1.1neg population was assayed in culture. When Thy-1. 1low stem cells were rigorously excluded from the Thy-1.1neg subset, reconstitution of T lymphocytes was rarely observed in peripheral blood after i.v. transplantation. Competitive repopulation studies showed that the B lymphoid reconstitution derived from Thy-1.1neg cells was not sustained over a 20-wk period. Therefore, the Thy-1. 1neg population defined in these studies includes transplantable, non-self-renewing B lymphocyte progenitor cells.

Hematopoiesis in the liver and marrow of human fetuses at 5 to 16 weeks postconception: quantitative assessment of macrophage and neutrophil populations.

During human development, the liver and marrow both function as hematopoietic organs, but little is known about differences in the production of macrophages and neutrophils by these two organs. We used immunohistochemical stains to quantify the ratio of neutrophils to macrophages within the liver and the marrow of 16 fetuses from 5 to 16 wk postconception. At 5 wk the liver had a ratio of one granulocyte [myeloperoxidase (MPO)-positive cell] to every 9 +/- 5 (X +/- SD) macrophages (KP-1-positive cells). Between 5 and 16 wk, the granulocyte to macrophage ratio in the liver was constant, whereas it changed markedly in the marrow. Before 8 wk no MPO-positive or KP-1-positive cells were observed in bones. At 10 wk, bones still had no MPO-positive cells, but KP-1-positive cells were abundant. At 11-12 wk the granulocyte to macrophage ratio was 1 to 1 +/- 1, but by 13-16 wk it had increased to 8 +/- 3 MPO-positive cells to one KP-1-positive cell. We hypothesized that at 13-16 wk the abundance of MPO-positive cells in the marrow and their scarcity in the liver was the result of production of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSF-R) in the marrow and their absence in the liver. However, by reverse transcriptase-PCR mRNAs for G-CSF and G-CSF-R were positive in both organs at all gestations, and G-CSF and G-CSF-R proteins (by immunohistochemistry) were also abundant in all liver and marrow specimens. We then hypothesized that progenitors in the fetal liver were intrinsically different from those in the marrow, and were unable to generate clones of neutrophils. However, progenitors from the liver produced neutrophils abundantly in culture. Thus, the explanation is likely related to as yet undescribed environmental differences between the fetal liver and marrow.

The first-appearance of neutrophils in the human fetal bone marrow cavity.

We sought to define the time of first-appearance of neutrophils within the developing human bone marrow cavity, and to compare this with the time of appearance of G-CSF and its receptor (G-CSF-R) at that site. We hypothesized that the onset of G-CSF production is an initiation signal for neutrophil production within the marrow cavity, and that therefore G-CSF mRNA and G-CSF protein in the marrow cavity would immediately precede the first-appearance of neutrophils. To test this, we determined the time of first-appearance of neutrophils in the clavicular marrow space using a monoclonal antibody against myeloperoxidase (MPOAb), and then validated these findings by flow cytometric analyses, for neutrophil cell-surface markers, of cells flushed from the marrow cavity. After thus defining the time of first-appearance of neutrophils, specific mRNA transcripts for G-CSF and G-CSF-R were sought from clavicles of varying gestational ages, using RT-PCR, and the presence of these proteins in the clavicles were sought using immunohistochemistry. We observed that; (1) MPO+ cells first appeared in the clavicular marrow cavity between the 10 to 11th weeks post-conception, (2) Flow cytometric analyses confirmed that these MPO+ marrow cells included CD11b+, CD15+ neutrophils, (3) Transcripts for G-CSF and G-CSF-R, and the specific G-CSF and G-CSF-R proteins, were present in the clavicles by 6 weeks post-conception, 4 to 5 weeks before the first-appearance of neutrophils. Thus, neutrophils first appear in the human clavicular marrow at 10-11 weeks post-conception, and G-CSF and G-CSF-R are present in the developing bone rudiment preceding the appearance of neutrophils. It is unclear whether neutrophils arise in the marrow cavity in response to the onset of production of G-CSF or to other initiation signals.

Use of alternative therapies for children with cancer.

OBJECTIVE: To compare the use of alternative therapy (AT) in families of children with cancer with its use in those with routine pediatric conditions. BACKGROUND AND RATIONALE: AT refers to healing practices such as therapeutic massage, acupuncture, and use of medicinal herbs that have become increasingly popular with the general public, but are not widely accepted by the medical profession. Although studies have investigated the use of AT in the families of both healthy children and children with cancer, no comparison of the incidence of its use between these two populations has been published. We hypothesized that AT was used more frequently among the families of children with cancer. METHODS: Using a prevalence survey design, we interviewed 81 parents of children with cancer attending a pediatric hematology/oncology clinic and 80 parents of children attending a continuity care clinic for routine check-ups and acute care. We explored the types of AT being used, the reasons for its use, and the frequency with which it was discussed with the patient's physician. RESULTS: 1) Overall, 65% of the cancer group were using AT, compared with 51% of the control group. This was not statistically significant. 2) Prayer, exercise, and spiritual healing were three AT practices most often used by the cancer group, and prayer, massage, and spiritual healing by the control group. 3) Discussion of AT with the physician varied according to group, with 53% of the cancer patients discussing its use; income level, with 59% of parents in the higher income group discussing its use; and ethnicity, with 47% of whites discussing its use. CONCLUSION: Use of AT is not limited to the families of children with life-challenging illnesses, but is commonly used by those of children with routine pediatric problems. Pediatricians need to be aware that their patients may not tell them about AT practices they are using in addition to prescribed treatment.

Tetany in a child with AIDS receiving intravenous tobramycin.

Aminoglycosides have been reported to cause tetany by inducing renal wasting of magnesium and secondary hypocalcemia. A 9-year-old girl with AIDS was brought to the emergency department because of tetany. She had just finished a 3-week course of intravenous tobramycin for bronchiectasis and had an elevated serum tobramycin trough level 1 week before the onset of tetany. Hypomagnesemia, hypokalemia, and hypocalcemia had developed. The urine revealed inappropriately high levels of magnesium and potassium despite critically low serum levels, with an appropriately low urine calcium level. This pattern is suggestive of renal toxicity due to tobramycin. Over the next several months, she continued to require supplemental calcium, magnesium, potassium, and phosphorus. She also had nephrotic range proteinuria. Aminoglycoside renal mineral wasting has not previously been described in a child.

Testosterone induced priapism in two adolescents with sickle cell disease.

Priapism is common in pubertal males with sickle cell disease, but the association between low-dose exogenous testosterone administration and priapism in such patients has not been well documented. Two adolescents with homozygous sickle cell disease (SCD) and delayed maturation with behavioral problems developed priapism about one week after receiving an intramuscular injection of testosterone enanthate. Neither had a previous history of priapism. We conclude that testosterone should not be administered to male patients with SCD because of the risk of inducing priapism and possible impotence.

The immunological response of Wistar rats to the intracranially implanted C-6 glioma cell line.

The immune response of Wistar rats to the intracranial inoculum of 1 X 10(5) C-6 glioma cells was evaluated. The growth of these cells disrupted the blood-brain barrier by day 9. The rats with the implanted tumor cells died between three to four weeks following injection. No significant cell-mediated cytotoxicity against 51Cr labelled C-6 cells was seen in the short term (4 hours) cytotoxicity assay with spleen cells obtained from glioma-bearing rats at any stage of tumor growth. In the long term (18 hours) cytotoxicity assay, significant activity was detected using whole, adherent and nonadherent spleen cells from glioma-bearing rats at every assessment point during the growth of the tumor, but this cytotoxicity was also seen in normal rat splenocytes. The lack of cell mediated cytotoxicity above normal values was not due to a generalized immunosuppression since splenocytes from glioma bearers were found to have responses to Con A comparable to normal controls. However, normal or glioma-bearer splenocytes showed augmented cytotoxicity in the presence of serum obtained from rats bearing a glioma tumor starting by day 13 of tumor growth and rising in cytotoxic activity until death. This activity was not seen with normal serum. The glioma-bearer serum, though not cytotoxic to the C-6 cells alone, became cytotoxic with the addition of rabbit complement. These data indicate that the growth of intracranially implanted glioma cells in rats elicits primarily a humoral cytotoxic immunity without a significant cell-mediated immunity. This humoral immunity develops after the breakdown of the blood-brain barrier.