Childhood acute lymphoblastic leukemia: Integrating genomics into therapy

Acute lymphoblastic leukemia (ALL), the most common malignancy of childhood, is a genetically complex entity that remains a major cause of childhood cancer‐related mortality. Major advances in genomic and epigenomic profiling during the past decade have appreciably enhanced knowledge of the biology of de novo and relapsed ALL and have facilitated more precise risk stratification of patients. These achievements have also provided critical insights regarding potentially targetable lesions for the development of new therapeutic approaches in the era of precision medicine. In this review, the authors delineate the current genetic landscape of childhood ALL, emphasizing patient outcomes with contemporary treatment regimens as well as therapeutic implications of newly identified genomic alterations in specific subsets of ALL. Cancer 2015;121:3577–3590 . © 2015 American Cancer Society.     

Correlation of P-glycoprotein expression and function in childhood acute leukemia: a children's cancer group study

Clinical drug resistance may be attributed to the simultaneous selection and expression of genes modulating the uptake and metabolism of chemotherapeutic agents. P-glycoprotein (P-gp) functions as a membrane-associated drug efflux pump whose increased expression results in resistance to anthracyclines, epipodophyllotoxins, vinca alkaloids, and some alkylating agents. This type of resistance occurs as both de novo and acquired resistance to therapy for leukemia. We have studied P- gp expression and function in childhood acute leukemias by developing a series of doxorubicin- and vincristine-selected CEM, T-cell lymphoblastoid cell lines that recapitulate the low levels of expression and resistance seen clinically. These cell lines have been used to develop flow cytometric assays for the semiquantitative measurements of P-gp expression with the MRK16 monoclonal antibody and P-gp function using the enhanced retention of rhodamine 123 in the presence of verapamil, a resistance modulator. Kolmogorov-Smirnov statistics, represented by the D measurement, are used to determine the difference in level of P-gp expression by comparing MRK16 staining to an IgG2a isotype control. When D is > 0.09, there is an excellent correlation (R = 0.82) between P-gp expression and function. The evaluation of 107 bone marrow specimens from 84 children with lymphoblastic or myelogenous leukemia showed a statistically significant (P = .004) increase in P-gp function at relapse. P-gp expression at relapse, however, approached but did not reach a significant level (P = .097). Using this methodology, we can identify patients with levels of P-gp expression and function that we can define clinically, as well as children with discordant multidrug resistance phenotypes. This study supports the role of P-gp-mediated drug resistance in childhood leukemia and confirms that P-gp expression and function are measurable in their leukemic blasts. These assays provide the means for the in vitro testing of resistance modulators and the monitoring of in vivo response to treatment with these agents.     

Characterization of the IgG-Fc receptor on human platelets

To determine quantitatively the number and avidity of receptors for the Fc portion of IgG on human platelets, we have measured the binding to platelets of human monomeric monoclonal IgG, and of small covalently crosslinked polymers of IgG1 labeled with 125I. The binding of labeled IgG1 monomers to platelets is too weak to permit quantitation. The binding of dimers or larger polymers of IgG1 is much more avid (greater at 4 degrees C than 37 degrees C), is readily reversible, and is saturable. The number of receptor sites ranges from 400 to 2000 per platelet and the mean equilibrium association constant (Ka) for the binding of dimers at 4 degrees C is 2.2 x 10(7) M-1 +/- 0.9 x 10(7) M- 1. The binding is specific for the Fc portion of IgG, and IgG1 and IgG3 bind to the receptors much more avidly than IgG2 or IgG4. Unlabeled IgG1 dimers are about 7--8-fold more potent in inhibiting binding than are IgG1 monomers, and larger polymers are even more potent than dimers. Thus, the Fc receptors on platelets bind human IgG1 with the same specificity and similar avidity as Fc receptors on polymorphonuclear leukocytes (PMNs), but PMNs have about 300-fold more receptors per unit of surface area than platelets.     

Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria

The purpose of these studies was to determine the molecular basis of the phenotypic mosaicism that is a defining feature of paroxysmal nocturnal hemoglobinuria (PNH). Analysis of T cell clones from a female patient revealed four distinct phenotypes based on surface expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). When PIG-A (the gene that is mutant in PNH) from these clones was analyzed, four discrete somatic mutations were identified. Analysis of X chromosomal inactivation among the abnormal T cell clones was consistent with polyclonality. Together, these studies demonstrate that the phenotypic mosaicism that is characteristic of PNH is a consequence of genotypic mosaicism and that, at least in this case, PNH is a polyclonal rather than a monoclonal disease. That four distinct somatic mutations were present in a single patient suggests that in conditions that predispose to PNH PIG-A may be hypermutable.     

The response of red cell hexose monophosphate shunt after sulfhydryl inhibition

In this investigation, we studied the importance of cellular glutathione (GSH) in the hexose monophosphate shunt (HMPS) activity of unstimulated human erythrocytes and the mechanism by which pyruvate stimulates the HMPS. The rate of HMPS activity was measured by the production of radioactive CO2 from 14C-1-glucose or 14C-1-ribose using a vibrating reed electrometer and ionization chamber. HMPS activity was not significantly impaired by N-ethylmaleimide (NEM) in concentrations which bound all red cell GSH. Red cells incubated under carbon monoxide (CO), an experimental condition which eliminates peroxide production, still had HMPS activity which was 44% of the value under air. Pyruvate stimulation of the HMPS was unaffected by doses of NEM which bound all cellular GSH or by incubation under CO. These data indicated that pyruvate stimulation of the HMPS occurs by pathways which do not involve peroxide formation, GSH, or oxygen. This study indicates that sulfhydryl blockade of GSH does not necessarily inhibit HMPS activity and that HMPS activity in red cells may respond to reactions not linked directly to glutathione reduction.     

Monosomy 7 associated with pediatric acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS): successful management by allogeneic hematopoietic stem cell transplant (HSCT)

Pediatric acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) with monosomy 7 is associated with poor disease-free survival when treated by conventional chemotherapy, immunosuppression or supportive measures. Hematopoietic stem cell transplant (HSCT) may improve outcomes; however, data to support this are limited. To better understand the curative potential of HSCT in these patients, all cases of AML and MDS with monosomy 7 treated by two transplant programs (1992 to present) were reviewed. A total of 16 patients were treated, all by allogeneic HSCT. Primary diagnoses were MDS (N = 5), therapy-related MDS (N = 3), AML (N = 5) and therapy-related AML (N = 3). In all, 11 patients (69%) survive event-free at 2 years with median follow-up of 986 days (range 330-2011 days). Toxicity caused deaths of the five nonsurviving patients, four of whom were transplanted with active leukemia. Allogeneic HSCT is effective therapy for childhood AML and MDS associated with monosomy 7, particularly for patients with AML in complete remission and MDS.     

Genome-wide copy number profiling reveals molecular evolution from diagnosis to relapse in childhood acute lymphoblastic leukemia

The underlying pathways that lead to relapse in childhood acute lymphoblastic leukemia (ALL) are unknown. To comprehensively characterize the molecular evolution of relapsed childhood B-precursor ALL, we used human 500K single-nucleotide polymorphism arrays to identify somatic copy number alterations (CNAs) in 20 diagnosis/relapse pairs relative to germ line. We identified 758 CNAs, 66.4% of which were less than 1 Mb, and deletions outnumbered amplifications by approximately 2.5:1. Although CNAs persisting from diagnosis to relapse were observed in all 20 cases, 17 patients exhibited differential CNA patterns from diagnosis to relapse. Of the 396 CNAs observed in 20 relapse samples, only 69 (17.4%) were novel (absent in the matched diagnosis samples). EBF1 and IKZF1 deletions were particularly frequent in this relapsed ALL cohort (25.0% and 35.0%, respectively), suggesting their role in disease recurrence. In addition, we noted concordance in global gene expression and DNA copy number changes (P = 2.2 x 10(-16)). Finally, relapse-specific focal deletion of MSH6 and, consequently, reduced gene expression were found in 2 of 20 cases. In an independent cohort of children with ALL, reduced expression of MSH6 was associated with resistance to mercaptopurine and prednisone, thereby providing a plausible mechanism by which this acquired deletion contributes to drug resistance at relapse.     

The biochemical and clinical consequences of 2'-deoxycoformycin in refractory lymphoproliferative malignancy

A deficiency of adenosine deaminase, an enzyme important in purine nucleoside catabolism, is associated with a severe combined immunodeficiency disease in children. Inhibition of this enzyme in vitro and in vivo results in an impairment in lymphoblast proliferation. We have investigated the pharmacologic inhibition of this enzyme by 2'-deoxycoformycin in 15 patients with hematologic malignancies. Biochemical consequences of the administration of this agent were closely monitored in erythrocytes, nucleated peripheral blood and bone marrow cells, serum, and urine. A marked rise in erythrocyte dATP was accompanied by a depletion of ATP in those patients exhibiting toxicity. Most patients excreted large amounts of deoxyadenosine but not adenosine in the urine. Serum deoxyadenosine rose in patients demonstrating a marked decrease in cell mass. The biochemical disturbances and clinical toxicity, including hepatic, renal, and conjunctival abnormalities, were usually reversible. Central nervous system toxicity, which potentially was the most serious consequence, was associated with high erythrocyte dATP/ATP ratios and high levels of cerebrospinal fluid deoxyadenosine. In patients with lymphoma and leukemia, objective responses were observed but were short- lived. Patients with chronic lymphocytic leukemia receiving weekly low doses of the drug demonstrated minimal toxicity and some efficacy. The chemotherapeutic potential o 2'-deoxycoformycin, as either a single agent or in combination with Ara-A, merits further exploration.