Ching-Hon Pui

Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling

Treatment of pediatric acute lymphoblastic leukemia (ALL) is based on the concept of tailoring the intensity of therapy to a patients risk of relapse. To determine whether gene expression profiling could enhance risk assignment, we used oligonucleotide microarrays to analyze the pattern of genes expressed in leukemic blasts from 360 pediatric ALL patients. Distinct expression profiles identified each of the prognostically important leukemia subtypes, including T-ALL, E2A-PBX1, BCR-ABL, TEL-AML1, MLL rearrangement, and hyperdiploid >50 chromosomes. In addition, another ALL subgroup was identified based on its unique expression profile. Examination of the genes comprising the expression signatures provided important insights into the biology of these leukemia subgroups. Further, within some genetic subgroups, expression profiles identified those patients that would eventually fail therapy. Thus, the single platform of expression profiling should enhance the accurate risk stratification of pediatric ALL patients.

Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia.

Chromosomal aberrations are a hallmark of acute lymphoblastic leukaemia (ALL) but alone fail to induce leukaemia. To identify cooperating oncogenic lesions, we performed a genome-wide analysis of leukaemic cells from 242 paediatric ALL patients using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing. Our analyses revealed deletion, amplification, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A), EBF1, LEF1, IKZF1 (IKAROS) and IKZF3 (AIOLOS). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer.

Acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia, a malignant disorder of lymphoid progenitor cells, affects both children and adults, with peak prevalence between the ages of 2 and 5 years. Steady progress in development of effective treatments has led to a cure rate of more than 80% in children, creating opportunities for innovative approaches that would preserve past gains in leukaemia-free survival while reducing the toxic side-effects of current intensive regimens. Advances in our understanding of the pathobiology of acute lymphoblastic leukaemia, fuelled by emerging molecular technologies, suggest that drugs specifically targeting the genetic defects of leukaemic cells could revolutionise management of this disease. Meanwhile, studies are underway to ascertain the precise events that take place in the genesis of acute lymphoblastic leukaemia, to enhance the clinical application of known risk factors and antileukaemic agents, and to identify treatment regimens that might boost the generally low cure rates in adults and subgroups of children with high-risk leukaemia.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia

Human T cell leukemias can arise from oncogenes activated by specific chromosomal translocations involving the T cell receptor genes. Here we show that five different T cell oncogenes (HOX11, TAL1, LYL1, LMO1, and LMO2) are often aberrantly expressed in the absence of chromosomal abnormalities. Using oligonucleotide microarrays, we identified several gene expression signatures that were indicative of leukemic arrest at specific stages of normal thymocyte development: LYL1+ signature (pro-T), HOX11+ (early cortical thymocyte), and TAL1+ (late cortical thymocyte). Hierarchical clustering analysis of gene expression signatures grouped samples according to their shared oncogenic pathways and identified HOX11L2 activation as a novel event in T cell leukemogenesis. These findings have clinical importance, since HOX11 activation is significantly associated with a favorable prognosis, while expression of TAL1, LYL1, or, surprisingly, HOX11L2 confers a much worse response to treatment. Our results illustrate the power of gene expression profiles to elucidate transformation pathways relevant to human leukemia.

Deletion of IKZF1 and Prognosis in Acute Lymphoblastic Leukemia

BACKGROUND Despite best current therapy, up to 20% of pediatric patients with acute lymphoblastic leukemia (ALL) have a relapse. Recent genomewide analyses have identified a high frequency of DNA copy-number abnormalities in ALL, but the prognostic implications of these abnormalities have not been defined. METHODS We studied a cohort of 221 children with high-risk B-cell-progenitor ALL with the use of single-nucleotide-polymorphism microarrays, transcriptional profiling, and resequencing of samples obtained at diagnosis. Children with known very-high-risk ALL subtypes (i.e., BCR-ABL1-positive ALL, hypodiploid ALL, and ALL in infants) were excluded from this cohort. A copy-number abnormality was identified as a predictor of poor outcome, and it was then tested in an independent validation cohort of 258 patients with B-cell-progenitor ALL. RESULTS More than 50 recurring copy-number abnormalities were identified, most commonly involving genes that encode regulators of B-cell development (in 66.8% of patients in the original cohort); PAX5 was involved in 31.7% and IKZF1 in 28.6% of patients. Using copy-number abnormalities, we identified a predictor of poor outcome that was validated in the independent validation cohort. This predictor was strongly associated with alteration of IKZF1, a gene that encodes the lymphoid transcription factor IKAROS. The gene-expression signature of the group of patients with a poor outcome revealed increased expression of hematopoietic stem-cell genes and reduced expression of B-cell-lineage genes, and it was similar to the signature of BCR-ABL1-positive ALL, another high-risk subtype of ALL with a high frequency of IKZF1 deletion. CONCLUSIONS Genetic alteration of IKZF1 is associated with a very poor outcome in B-cell-progenitor ALL.

BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros.

The Philadelphia chromosome, a chromosomal abnormality that encodes BCR–ABL1, is the defining lesion of chronic myelogenous leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). To define oncogenic lesions that cooperate with BCR–ABL1 to induce ALL, we performed a genome-wide analysis of diagnostic leukaemia samples from 304 individuals with ALL, including 43 BCR–ABL1 B-progenitor ALLs and 23 CML cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR–ABL1 ALL, but not in chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR–ABL1 ALL.

Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukemia.

PURPOSE To define more uniform criteria for risk-based treatment assignment for children with acute lymphoblastic leukemia (ALL), the Cancer Therapy Evaluation Program (CTEP) of the National Cancer Institute (NCI) sponsored a workshop in September 1993. Participants included representatives from the Childrens Cancer Group (CCG), Pediatric Oncology Group (POG), Dana-Farber Cancer Institute (DFCI), St Jude Childrens Research Hospital (SJCRH), and the CTEP. METHODS Workshop participants presented and reviewed data from ALL clinical trials, using weighted averages to combine outcome data from different groups. RESULTS For patients with B-precursor (ie, non-T, non-B) ALL, the standard-risk category (4-year event-free survival [EFS] rate, approximately 80%) will include patients 1 to 9 years of age with a WBC count at diagnosis less than 50,000/microL. The remaining patients will be classified as having high-risk ALL (4-year EFS rate, approximately 65%). For patients with T-cell ALL, different treatment strategies have yielded different conclusions concerning the prognostic significance of T-cell immunophenotype. Therefore, some groups/institutions will classify patients with T-cell ALL as high risk, while others will assign risk for patients with T-cell ALL based on the uniform age/WBC count criteria. Workshop participants agreed that the risk category of a patient may be modified by prognostic factors in addition to age and WBC count criteria, and that a common set of prognostic factors should be uniformly obtained, including DNA index (DI), cytogenetics, early response to treatment (eg, day-14 bone marrow), immunophenotype, and CNS status. CONCLUSIONS The more uniform approach to risk-based treatment assignment and to collection of specific prognostic factors should increase the efficiency of future ALL clinical research.

Molecular Diagnosis of Thiopurine S-Methyltransferase Deficiency: Genetic Basis for Azathioprine and Mercaptopurine Intolerance

Thiopurine S-methyltransferase (TPM) is a cytosolic enzyme that preferentially catalyzes the S-methylation (that is, inactivation) of such therapeutic agents as mercaptopurine, azathioprine, and thioguanine [1]. These thiopurine medications are currently used to treat many diseases, including cancer [2], autoimmune hepatitis [3], inflammatory bowel disease [4, 5], rheumatoid arthritis [6], multiple sclerosis [7], and autoimmune myasthenia gravis [8]; they are also used as immunosuppressants after organ transplantation [9, 10]. Several clinical studies have shown that patients with low TPM activity are at high risk for severe and potentially fatal hematopoietic toxicity if they are treated with conventional doses of mercaptopurine (for example, 75 mg/m2 body surface area per day) or azathioprine [9, 11-13]. Thiopurine S-methyltransferase activity shows codominant genetic polymorphism [14, 15]. About 90% of white and black persons have high TPM activity, and 10% have intermediate activity caused by heterozygosity at the TPM locus. About 1 in 300 persons inherits TPM deficiency as an autosomal recessive trait. Clinical studies have established an inverse correlation between TPM activity and accumulation of the active thioguanine nucleotide metabolites of mercaptopurine and azathioprine in erythrocytes. Patients with less efficient methylation of these thiopurine medications have more extensive conversion to active thioguanine nucleotides [2, 16]. Patients who have TPM deficiency accumulate higher levels of thioguanine nucleotides in erythrocytes if they receive standard doses of mercaptopurine or azathioprine. This accumulation of nucleotides usually leads to severe hematopoietic toxicity and possibly death [9], but this outcome can be averted if the thiopurine dose is decreased substantially (an 8- to 15-fold reduction) [17-19]. Patients who have intermediate TPM activity that is caused by heterozygosity at the TPM locus accumulate about 50% more thioguanine nucleotides than do patients who have high TPM activity [2]; this places patients with intermediate TPM activity at an intermediate risk for toxicity. Most of these patients are identified only after an episode of severe toxicity occurs. Although prospective measurement of erythrocyte TPM activity has been advocated by some investigators [4, 16], TPM assays are not widely available. Moreover, organ transplant recipients and patients who have recently received a diagnosis of cancer are frequently given transfusions of red blood cells; this precludes measurement of constitutive TPM activity before thiopurine therapy is started. Because thiopurine toxicity can be life threatening in TPM-deficient patients [9] and because of the intermediate risk for toxicity in heterozygous patients, a reliable method to identify patients who have inherited this trait is needed. If the genetic basis for TPM deficiency can be defined and polymerase chain reaction (PCR)-based methods can be developed to detect these inactivating mutations in genomic DNA, it should be possible to diagnose TPM deficiency and heterozygosity on the basis of genotype (as is now possible for other polymorphic enzymes) [17, 18]. To this end, we isolated and characterized two mutant alleles that are associated with TPM deficiency, TPM*2 and TPM*3A [19, 20]. The structures of these alleles are depicted in Figure 1. The molecular defect in TPM*2 is a G238C transversion mutation that leads to an amino acid substitution at codon 80 (Ala80Pro). Heterologous expression of this mutant allele in yeast showed a 100-fold decrease in S-methylation activity. The TPM*3A allele contains two nucleotide transition mutations (G460A and A719G) that lead to the amino acid substitutions Ala154Thr and Tyr240Cys. Heterologous expression of TPM*3A complementary DNA (cDNA) in yeast showed a greater than 200-fold reduction in TPM protein and undetectable activity. Moreover, marked instability of catalytic activity was evident for TPM proteins that were encoded by mutant cDNA containing either of these point mutations alone [20, 21]. We report the development, validation, and application of PCR-based methods for detection of these TPM mutations in the genomic DNA of patients and the elucidation of the polymorphic nature of the TPM gene locus in white persons. We also report a reliable method for the molecular diagnosis of TPM deficiency and heterozygosity that has excellent concordance between genotype and phenotype. Figure 1. Allelic variants at the human thiopurine S-methyltransferase (TPM) locus. Methods Human Patients and Determination of Phenotype Through methods described elsewhere [15], erythrocytes and leukocytes were isolated from the peripheral blood of healthy volunteers and children who had acute lymphoblastic leukemia. The volunteers were unselected blood donors who had been identified during a 2-month period, as described elsewhere [15]. The children were being treated at St. Jude Childrens Research Hospital or had been referred for evaluation because they could not tolerate chemotherapy. Genotype was determined for all unrelated white patients who had TPM activity that indicated heterozygous or deficient genotypes and for the same number of unrelated persons who had high activity that indicated a homozygous wild-type genotype. We focused our initial studies on white patients because they belong to the ethnic group in which we have identified the largest number of TPM-deficient and heterozygous persons. The activity of TPM in erythrocytes was determined by the radiochemical assay of Weinshilboum and colleagues [22], whose methods we modified, as described elsewhere [15]. The TPM phenotype was assigned on the basis of TPM activity in erythrocytes and according to the criteria of Weinshilboum and Sladek (that is, patients who had <5.0 U/mL of packed red blood cells were considered TPM deficient, those who had 5 to 10 U/mL were considered heterozygous, and those who had >10 U/mL were considered homozygous wild-type) [14]. We used the lowest value of TPM activity in erythrocytes that was measured in each person. We extracted RNA from leukocytes by using the method of Chomczynski and Sacchi [23], and genomic DNA was isolated by chloroform-phenol extractions. The studies were approved by the institutional review board for clinical trials at St. Jude Childrens Research Hospital, and informed consent was obtained from the patients or their guardians. Determination of Intronic Sequences The presence of a TPM-processed pseudogene [24] that could confound PCR-based genotyping methods and the absence of data on the genomic structure of the human TPM gene led us to initially use PCR primers that were complementary to TPM exon sequences to amplify genomic DNA by Expand PCR (Boehringer Mannheim, Indianapolis, Indiana) and thereby identify intronic sequences in the human TPM gene. The final volume for all PCR assays was 50 micro L. Through use of 1 g of placental genomic DNA (Clontech Laboratories, Inc., Palo Alto, California) as a template, PCR was done with primers A (5-GAGTTCTTCGGGGAACATTTCATTG-3) and B (5-CACCTGGATTAATGGCAAC TAATGC-3) in buffer D (Invitrogen, San Diego, California). The buffer contained Tris hydrochloride (pH 8.5), 60 mmol/L; ammonium sulfate, 15 mmol/L; and magnesium chloride, 3.5 mmol/L. The primers had been developed to amplify a fragment of genomic DNA (which included nucleotide 460) for detection of the G460A mutation. The concentration of each oligonucleotide was 0.1 OU/mL (about 0.5 mol/L), and 0.2 L Taq polymerase (Perkin Elmer Cetus, Norwalk, Connecticut) was used. With a Hybaid OmniGene thermocycler (Woodbridge, New Jersey), amplification was done for 30 cycles consisting of denaturation at 94 C for 1 minute, annealing at 55 C for 2 minutes, and extension at 72 C for 1 minute. A final extension step at 72 C for 7 minutes was also done. For the initial cycle, 5 L of deoxynucleoside triphosphates (dNTP, 10 mmol/L) was added after the temperature reached 80 C (following the hot start protocol). An amplified fragment of 138 base pairs was anticipated in the absence of intron sequences; the resulting fragment of 746 base pairs showed the presence of an intervening intron. This fragment was directly cloned into the plasmid pCR-II (Invitrogen). The recombinant plasmid was purified with Qiagen plasmid kits (Chatsworth, California) and sequenced with an automated sequencer using the cycle sequencing reaction and fluorescence-tagged dye terminators (Prism, Applied Biosystems, Foster City, California). The resulting intron sequence and the intron-exon boundary was then used to develop intron-specific primer P460F. Through a similar strategy, Expand PCR was used to amplify intron sequences that flanked the exons containing the G238C mutation (intron 4) and the A719G mutation (intron 9). The resulting intron-containing fragments were directly cloned into the plasmid pCR-II; the plasmid was purified and sequenced as described above. These sequences permitted the development of intron-specific PCR primers P2C and P719F for the detection of G238C and A719G mutations. Detection of TPM Mutations by Polymerase Chain Reaction Detection of G238C We used PCR amplification to determine whether the G238C transversion was present at the TPM locus. Genomic DNA, 400 ng, was amplified under conditions similar to those discussed for the intronic sequence except that 2 L of primer P2W (5-GTATGATTTTAT GCAGGTTTG-3) or P2M (5-GTATGATTTTATGCAGGTTTC-3) was used with primer P2C (5-TAAATAGGAACCATCGGACAC-3) (0.1 OU/mL) in each amplification. Unpurified PCR products were analyzed by electrophoresis in 2.5% MetaPhor gels (MetaPhor Agarose, FMC Bioproducts, Rockland, Maine) stained with ethidium bromide. A DNA fragment was amplified with P2M and P2C primers when C238 (mutant) was present, whereas a DNA fragment was amplified with P2W and P2C primers when G238 (wild-type) was present (Figure 2). Figure 2. Schematic of polymerase chain reac

Treating Childhood Acute Lymphoblastic Leukemia without Cranial Irradiation

BACKGROUND Prophylactic cranial irradiation has been a standard treatment in children with acute lymphoblastic leukemia (ALL) who are at high risk for central nervous system (CNS) relapse. METHODS We conducted a clinical trial to test whether prophylactic cranial irradiation could be omitted from treatment in all children with newly diagnosed ALL. A total of 498 patients who could be evaluated were enrolled. Treatment intensity was based on presenting features and the level of minimal residual disease after remission-induction treatment. The duration of continuous complete remission in the 71 patients who previously would have received prophylactic cranial irradiation was compared with that of 56 historical controls who received it. RESULTS The 5-year event-free and overall survival probabilities for all 498 patients were 85.6% (95% confidence interval [CI], 79.9 to 91.3) and 93.5% (95% CI, 89.8 to 97.2), respectively. The 5-year cumulative risk of isolated CNS relapse was 2.7% (95% CI, 1.1 to 4.3), and that of any CNS relapse (including isolated relapse and combined relapse) was 3.9% (95% CI, 1.9 to 5.9). The 71 patients had significantly longer continuous complete remission than the 56 historical controls (P=0.04). All 11 patients with isolated CNS relapse remained in second remission for 0.4 to 5.5 years. CNS leukemia (CNS-3 status) or a traumatic lumbar puncture with blast cells at diagnosis and a high level of minimal residual disease (> or = 1%) after 6 weeks of remission induction were significantly associated with poorer event-free survival. Risk factors for CNS relapse included the genetic abnormality t(1;19)(TCF3-PBX1), any CNS involvement at diagnosis, and T-cell immunophenotype. Common adverse effects included allergic reactions to asparaginase, osteonecrosis, thrombosis, and disseminated fungal infection. CONCLUSIONS With effective risk-adjusted chemotherapy, prophylactic cranial irradiation can be safely omitted from the treatment of childhood ALL. (ClinicalTrials.gov number, NCT00137111.)