Genome-wide single-nucleotide polymorphism analysis in juvenile myelomonocytic leukemia identifies uniparental disomy surrounding the NF1 locus in cases associated with neurofibromatosis but not in cases with mutant RAS or PTPN11.

Juvenile myelomonocytic leukemia (JMML) is a malignant hematopoietic disorder whose proliferative component is a result of RAS pathway deregulation caused by somatic mutation in the RAS or PTPN11 oncogenes or in patients with underlying neurofibromatosis type 1 (NF-1), by loss of NF1 gene function. To search for potential collaborating genetic abnormalities, we used oligonucleotide arrays to analyse over 116 000 single-nucleotide polymorphisms across the genome in 16 JMML samples with normal karyotype. Evaluation of the SNP genotypes identified large regions of homozygosity on chromosome 17q, including the NF1 locus, in four of the five samples from patients with JMML and NF-1. The homozygous region was at least 55 million base pairs in each case. The genomic copy number was normal within the homozygous region, indicating uniparental disomy (UPD). In contrast, the array data provided no evidence for 17q UPD in any of the 11 JMML cases without NF-1. We used array-based comparative genomic hybridization to confirm 17q disomy, and microsatellite analysis was performed to verify homozygosity. Mutational analysis demonstrated that the inactivating NF1 lesion was present on both alleles in each case. In summary, our data indicate that a mitotic recombination event in a JMML-initiating cell led to 17q UPD with homozygous loss of normal NF1, provide confirmatory evidence that the NF1 gene is crucial for the increased incidence of JMML in NF-1 patients, and corroborate the concept that RAS pathway deregulation is central to JMML pathogenesis.

Genome scan implicates adhesion biological pathways in secondary leukemia.

The genetic risk factors for etoposide-induced leukemia with MLL translocations remain largely unknown. To identify genetic risk factors for and novel characteristics of secondary leukemia, we profiled 116,204 single nucleotide polymorphisms (SNPs) in germline and paired leukemic cell DNA from 13 secondary leukemia/myelodysplasia cases and germline DNA from 13 matched and 156 unmatched controls, all with acute lymphoblastic leukemia treated with etoposide. We analyzed global gene expression from a partially overlapping cohort. No single locus was altered in most cases. We discovered 81 regions of loss of heterozygosity (LOH) in leukemic blasts and 309 SNPs whose allele frequencies differed in cases vs controls. Candidate genes were prioritized on the basis of genes whose SNPs or expression differentiated cases from controls or showed LOH or copy number change in germline vs paired blast DNA from the 13 cases. Three biological pathways were altered: adhesion, Wnt signaling and regulation of actin. Validation experiments using a genome scan for etoposide-induced leukemogenic MLL chimeric fusions in 15 HapMap cell lines also implicated genes involved in adhesion, a process linked to de novo leukemogenesis. Independent clinical epidemiologic and in vitro genome-wide approaches converged to identify novel pathways that may contribute to therapy-induced leukemia.

Comparison of genome sequencing and clinical genotyping for pharmacogenes.

We compared whole exome sequencing (WES, n = 176 patients) and whole genome sequencing (WGS, n = 68) and clinical genotyping (DMET array-based approach) for interrogating 13 genes with Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. We focused on 127 CPIC important variants: 103 single nucleotide variations (SNV), 21 insertion/deletions (Indel), HLA-B alleles, and two CYP2D6 structural variations. WES and WGS provided interrogation of nonoverlapping sets of 115 SNV/Indels with call rate >98%. Among 68 loci interrogated by both WES and DMET, 64 loci (94.1%, confidence interval [CI]: 85.6-98.4%) showed no discrepant genotyping calls. Among 66 loci interrogated by both WGS and DMET, 63 loci (95.5%, CI: 87.2-99.0%) showed no discrepant genotyping calls. In conclusion, even without optimization to interrogate pharmacogenetic variants, WES and WGS displayed potential to provide reliable interrogation of most pharmacogenes and further validation of genome sequencing in a clinical lab setting is warranted.

T-cell oncogene rhombotin-2 interacts with retinoblastoma-binding protein 2.

The LIM domain protein rhombotin-2 (RBTN-2/TTG-2/Lmo2) has distinct functions in erythropoiesis and in T-cell leukemogenesis. Additional functions for RBTN2 are indicated by its expression in non-hematopoietic tissues. These diverse functions of RBTN2 are presumed to be accomplished through physical interaction with different protein partners that bind the LIM domains of RBTN2. To identify these proteins which may modulate the activity of RBTN2, a human cDNA library was screened using the yeast two-hybrid assay. Using the RBTN2 LIM domain region as 'bait', the retinoblastoma-binding protein 2 (RBP2) was identified as a partner for RBTN2. The interaction between RBTN2 and RBP2 was confirmed using in vitro binding assays, and by co-immunoprecipitation of the two proteins. Deletion analysis showed the second LIM domain of RBTN2 was necessary and sufficient for binding to the last 69 amino acids of RBP2. The interaction between RBTN2 and RBP2 had a functional consequence: the combination of RBP2 and RBTN2 gave higher transcription in vitro, than RBTN2 alone. The interaction with RBP2 suggests two additional functions for RBTN2: (i) RBTN2 may directly affect the activity of RBP2, and/or (ii) RBTN2 may indirectly modulate the functions of the retinoblastoma protein by binding to RBP2.

Characterization of immunoglobulin heavy chain genes from an acute lymphocytic leukemia with four rearrangements.

Approximately 25% of acute leukemias of the B-cell lineage demonstrate more than two rearranged immunoglobulin heavy chain genes when examined by Southern blot analysis. The origin of the extra bands was investigated by molecular cloning and sequencing of four rearranged genes from one patient's leukemic cells. All four rearrangements were apparently derived independently. Two of the rearrangements used the VH6 variable region, attached to different diversity and joining regions. One of the two rearrangements contained a mutation in the coding sequence leading to the generation of a nonsense codon. This rearranged gene also differed from the other VH6 containing gene starting at about 330 bp upstream of the ATG initiation codon. The third rearranged gene used a member of the VH2 variable gene family. A DH-JH rearrangement was found in the fourth rearranged gene. The data indicate that the leukemia probably arose as a result of the transformation of an early B-cell progenitor that lacked rearranged immunoglobulin genes but retained some differentiation potential.

Identification of genes potentially involved in LMO2-induced leukemogenesis.

The most common translocations in childhood T cell acute lymphoblastic leukemias involve the LMO2 locus on chromosome 11p13 and cause ectopic expression of the LMO2 gene in thymocytes. Transgenic mice with enforced expression of LMO2 in their thymocytes develop T cell leukemias thus demonstrating the role of LMO2 in leukemogenesis. The physiologic and leukemogenic functions of LMO2 are mediated through its transcriptional regulatory activities, but the identity of the target genes is completely unknown. In this report, we have used cDNA representational difference analysis (cDNA-RDA) to identify genes that are over-expressed and are likely to play a role in the LMO2 induced leukemias. cDNA-RDA was performed using very small amounts of mRNA pool (from 1 microg of total RNA) to reverse transcribe the cDNAs from leukemic cells or normal thymocytes. The cDNA-RDA led to the isolation of nine distinct clones that were specifically overexpressed in the leukemic cells. Sequence analysis revealed that five of the nine clones had identity or homology to known genes that are known to play a role in the pathogenesis of leukemias or other cancers. Three clones had no significant homology to any known genes and thus represent novel candidate genes. Our study demonstrates that cDNA-RDA using very small amounts of total RNA is a highly efficient method to identify novel genes that may play a role in leukemogenesis.

Disruption of T-cell differentiation precedes T-cell tumor formation in LMO-2 (rhombotin-2) transgenic mice.

Transgenic mice expressing LMO-2 (rhombotin-2) were constructed by placing the LMO-2 gene under control of the metallothionein promoter. Thymic tumors developed in approximately 15% of the transgenic mice between 37 and 71 weeks. Only T-cell tumors were found in the transgenic mice despite high expression of LMO-2 in all tissues. The thymic tumors were aggressive and were invariably associated with metastasis to non-lymphoid organs. In approximately 50% of apparently healthy transgenic mice there was up to a 10-fold expansion of CD4-CD8- double negative (DN) cells. Expansion of the DN cells was accompanied by the compensatory decrease in CD4+CD8+ double positive (DP) cells, indicating that breach of homeostasis within the thymus had not occurred in these animals. The increase in DN cells was associated with a clonal expansion of thymocytes, and increased proliferation within the thymus. Our data indicate that the ectopic expression of LMO-2 in T-cells disrupts normal T-cell differentiation by selectively expanding the DN thymocyte population prior to breach of homeostasis and overt leukemia/lymphoma.

Elf-2, a rhombotin-2 binding ets transcription factor: discovery and potential role in T cell leukemia.

Rhombotin-2 (RBTN-2) is a proto-oncogene only in the context of T lymphocytes. We postulated that the oncogenic effect of RBTN-2 in T cells is likely mediated by binding protein(s) with T cell-specific expression. By screening a T cell cDNA library, we identified a novel ets transcription factor that binds RBTN-2. This protein was named elf-2 because its DNA-binding domain is virtually identical to that of ets family member elf-1. Northern analyses showed similar levels of two elf-2 transcripts (3.5 kb and 3.8 kb) in all tissues except thymus. Thymocytes expressed four- to 10-fold greater amounts of the 3.5 kb transcript than other tissues. Sequence analyses of cDNA clones indicated that these transcripts encode proteins differing only at their amino termini, and likely represent alternatively spliced isoforms. These isoforms (elf-2a and elf-2b) contain identical RBTN-2 binding regions and DNA-binding domains. Elf-2b lacks a putative transactivation domain. The expression patterns suggest that RBTN-2 normally interacts equally with elf-2a and elf-2b. In contrast, when RBTN-2 is inappropriately expressed in T cells, RBTN-2 would interact predominantly with elf-2b; this interaction may lead to T cell proliferation.

Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Minimal residual disease (MRD) is an independent prognostic factor in childhood acute lymphoblastic leukemia (ALL). The most widely applied MRD assays in ALL are flow cytometric identification of leukemia immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. We measured MRD by both assays in 227 patients with childhood B-lineage ALL. Of 1375 samples (736 bone marrow and 639 peripheral blood) examined, MRD was <0.01% in 1200, and > or =0.01% in 129 by both assays; MRD levels measured by the two methods correlated well. Of the remaining 46 samples, 28 had MRD > or =0.01% by flow cytometry but <0.01% by PCR. However, PCR (which had a consistent sensitivity of 0.001%) detected leukemic gene rearrangements in 26 of these 28 samples. Conversely, in 18 samples, MRD was > or =0.01% by PCR but <0.01% by flow cytometry. In nine of these samples, flow cytometry had a sensitivity of 0.001%, and detected aberrant immunophenotypes in eight samples. Therefore, the two most widely used methods for MRD detection in ALL yield concordant results in the vast majority of cases, although the estimated levels of MRD may vary in some. The use of the two methods in tandem ensures MRD monitoring in all patients.

Quantification of minimal residual disease in T-lineage acute lymphoblastic leukemia with the TAL-1 deletion using a standardized real-time PCR assay.

Hematologic relapse remains the greatest obstacle to the cure of children with acute lymphoblastic leukemia (ALL). Recent studies have shown that patients with increased risk of relapse can be identified by measuring residual leukemic cells, called minimal residual disease (MRD), during clinical remission. Current PCR methods, however, for measuring MRD are cumbersome and time-consuming. To improve and simplify MRD assessment, we developed a real-time quantitative PCR (RQ-PCR) assay for detection of leukemic cells that harbor the TAL-1 deletion. We studied serial dilutions of leukemic DNA and found the assay had a sensitivity of detection of one leukemic cell among 100,000 normal cells. We then investigated 23 samples from eight children with ALL in clinical remission. We quantified residual leukemic cells by using the TAL-1 RQ-PCR assay and by using limiting dilution analysis. In 17 samples, both methods detected MRD levels > or =0.001%. The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.926). In the remaining six samples, both methods detected fewer than 0.001% leukemic cells. We conclude the TAL-1 RQ-PCR assay can be used for rapid, sensitive and accurate assessment of MRD in T-lineage ALL with the TAL-1 deletion.

Molecular evidence for minimal residual bone marrow disease in children with 'isolated' extra-medullary relapse of T-cell acute lymphoblastic leukemia.

Central nervous system (CNS) relapse confers a poor prognosis in children with acute lymphoblastic leukemia (ALL). It is uncertain whether morphologically undetectable leukemia is present in the bone marrow at the time of CNS relapse, or whether the CNS acts as a 'sanctuary' site to allow reseeding of the marrow at a later time. We examined DNA from bone marrow samples from six patients with T-cell ALL with isolated CNS relapse using sensitive polymerase chain reaction (PCR) assays to detect minimal residual disease. One of these PCR assays was based on amplification of leukemia-specific TCR-delta gene rearrangements, while the other assay relied upon detection of the c-tal deletion. In four patients, where bone marrow samples were taken at the time of CNS relapse, residual disease was detectable in every sample at a level below morphological detection. In addition, three patients had residual disease detected in their subsequent bone marrow when CNS disease was not evident. Our findings, although preliminary, suggest that relapse of leukemia in the CNS reflects resurgence of the disease in the bone marrow that is first detected clinically in the CNS. The concomitant molecular detection of bone marrow leukemia at time of 'isolated' CNS relapse in children with T-cell ALL explains subsequent bone marrow relapse in some of these children, and argues for intensive systemic therapy of these patients.

Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Children with acute lymphoblastic leukemia (ALL) with > or = 0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 10(4) or more normal cells), accurate (r2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels > or = 1 in 10(4). The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.978). Of the remaining 50 samples, 48 had MRD levels < 1 in 10(4). In only two samples results were discordant: 2 in 10(4) and 5 in 10(4) leukemic cells by PCR but < 1 in 10(4) by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.

Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome.

We investigated the level of minimal residual disease (MRD) in 26 children with B-lineage acute lymphoblastic leukemia (ALL) after intensive induction therapy. A quantitative semi-nested polymerase chain reaction (PCR) detecting the clone-specific rearranged immunoglobulin heavy chain genes was developed to improve sensitivity and specificity of amplification. In all patients, one leukemic cell could be detected in a background of 10(5) normal blood mononuclear cells. All patients investigated were in complete remission at the end of induction therapy as evaluated by morphologic criteria. Nineteen patients (73%) had no detectable residual leukemic cells using the sensitive semi-nested PCR. Seven patients (27%) were PCR positive. Three had a low level (<2 x 10(-5) leukemic cells per bone marrow cell), while four patients had a high level (>2 x 10(5)) of detectable residual leukemic cells. All patients with low or undetectable levels of residual leukemia remained in complete remission at a median of 63 months from diagnosis (range 40-80 months), while all four patients with a high level of residual leukemia subsequently relapsed at a median of 21 months from diagnosis (range 13-37 months). The patient groups with undetectable or low, and high level of MRD did not differ significantly in other clinical or genetic features with prognostic significance. We conclude that the level of MRD at the end of the intensive induction therapy period is predictive of outcome in childhood B lineage ALL. If confirmed by large prospective studies, the level of MRD might be useful in stratifying patients into high and low risk categories.

Expression of the V(D)J recombinase gene RAG-1 is tightly regulated and involves both transcriptional and post-transcriptional controls.

The V(D)J recombinase activating genes, RAG-1 and RAG-2, are coexpressed only in immature lymphocytes, and are sufficient and necessary for V(D)J recombination to occur in non-lymphoid cells. In order to examine control mechanisms operative in the regulation of RAG-1 and RAG-2, we have studied the pattern of expression of these genes in human pre-T cells, pre-B cells, and thymocytes treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA); an agent which mimics some of the lymphocyte maturation changes seen in vivo. The expression of RAG-1 and RAG-2 was tightly controlled in a rapid, yet very complex, manner with both positive and negative control elements operating. Treatment of immature lymphocytes with TPA caused the specific and rapid elimination of steady-state RAG-1 and RAG-2 RNA. Nuclear run-on assays showed that TPA completely repressed the transcription of RAG-1 within 30 min. In addition to repressing the transcription of RAG-1, TPA treatment caused the rapid and specific degradation of RAG-1 transcripts by decreasing the apparent half-life of RAG-1 mRNA more than two-fold. As judged by cycloheximide treatment of cells, the effects of TPA were not dependent on new protein synthesis. A labile transcriptional repressor, separate from the TPA-associated repression of transcription, was also active in cells transcribing RAG-1 and RAG-2 RNA. After depletion of this labile repressor by cycloheximide treatment, steady-state RAG-1 and RAG-2 RNA levels, and their transcription rates, were elevated four- to six-fold; but were still susceptible to elimination by TPA treatment. Treatment of pre-T CEM cells with interleukin-2, or theophylline (an agent that increases intracellular cAMP) resulted in a two-fold increase in RAG-1 RNA suggesting that lymphokines, either independently or through second messengers, may modulate RAG-1 and RAG-2 expression. The complex, rapid and precise regulation of RAG-1 and RAG-2 expression is consistent with the view that it is necessary for the cell to tightly regulate V(D)J recombinase levels; lower expression may result in inefficient recombination of Ig/TCR genes, whereas increased expression may lead to recombination errors that are deleterious to the cell.

Factors affecting the integrity of the intestinal mucosa of Gambian children.

The interrelationship between diarrhea, malnutrition, and small bowel integrity was investigated prospectively in 68 Gambian infants aged 0-18 mo. Profiles of growth and morbidity were recorded for 8 mo. Each month intestinal permeability was measured by the differential uptake of orally administered lactulose (L) and mannitol (M). In well infants the mean L:M ratio was 0.42 (range 0.11-1.42). This ratio was increased slightly for underweight (60-80% wt for age) infants (mean 0.52) but considerably for those with marasmus (less than 60% wt for age) (mean 1.3, p less than 0.001), for those with acute or chronic diarrhea (mean 1.0 and 2.85, respectively; p less than 0.001), or with measles (mean 1.4, p less than 0.001). Sequential studies of ward patients with malnutrition and diarrhea showed a rapid fall in L:M ratios with resolution of diarrhea. These studies suggest that damage to the small intestine may play an important part in the development of infant malnutrition in The Gambia.

Radionuclide studies in intestinal transplantation. Diagnosis of rejection and assessment of permeability.

Three patients who received intestinal allografts were studied using two distinct radionuclide investigations. In the first, 111In or 99mTc-labeled leukocyte scanning was performed to assist in the diagnosis of rejection. It was able to demonstrate the occurrence of rejection in the transplanted intestine, and the response to antirejection therapy. In 1 case, the abnormality on the scan preceded the histological confirmation of rejection. The second technique studied mucosal integrity by serial 51Cr-EDTA/14C-mannitol permeability tests. These studies demonstrated the initial marked impairment and the slow return to normal function of the intestinal mucosal barrier. In 1 patient, this occurred by 91 days; in another, it took 232 days. A single assay performed in the third patient at the time of allograft rejection was also abnormal. Both radionuclide tests were helpful in the care of these complicated cases.

Rectal organ culture as a model for the investigation of bacterial adhesion and invasion.

A system was developed for the in vitro culture of human rectal mucosa. Its viability was proved by histological appearances and by metabolic studies. Biopsy samples were cultured in the presence of appropriate bacteria isolated from the faeces of patients with ulcerative colitis or with dysenteric illnesses. Attempts to show adhesion of bacteria to the mucosa or invasion of the cultured tissue failed. Problems with the use of this model are discussed.

The energy cost of triglyceride-fatty acid recycling in nonobese subjects after an overnight fast and four days of starvation.

The basal blood glycerol concentration was determined and the rate of glycerol turnover was assessed by a nonradioactive infusion technique in six healthy nonobese adults after an overnight fast and again after four days of total starvation. Simultaneously, estimates of total energy expenditure and net fat oxidation were made from measurements of oxygen consumption, carbon dioxide production, and urinary nitrogen excretion. The data were combined to provide quantitative estimates of the activity of the triglyceride/fatty acid cycle. The basal concentration of glycerol in venous blood rose from a mean value of 54 +/- 8 mumol/L (SEM) before starvation to 154 +/- 5 mumol/L on day 4 of starvation. Glycerol turnover rates correlated well with the basal blood glycerol concentration (r = .95) and increased from a mean value of 115 +/- 17 mumol/min before starvation (equivalent to mobilization of about 3.95 kJ triglyceride/min) to 304 +/- 20 mumol/min (equivalent to mobilization of about 18.41 kJ/min). The estimated rate of net fat oxidation was 3.00 +/- 0.47 kJ/min before starvation and 4.00 +/- 0.14 kJ/min on day +4 of starvation. The rate of triglyceride energy recycling or rate of deposition of triglyceride energy into fat stores was calculated from the difference in the rate of fat energy mobilization and the rate of energy released during net fat oxidation. The values were found to be 0.94 +/- 0.26 kJ/min before starvation and 6.29 +/- 0.54 kJ/min on day +4 of starvation.(ABSTRACT TRUNCATED AT 250 WORDS)

Case record review of adverse events: a new approach.

OBJECTIVES: To redesign the existing clinical review form (RF2) used in previous retrospective case record review studies in order to clarify the review process and provide a more powerful analysis of adverse events; and then to ask clinicians to pilot and evaluate the new modular review form (MRF2). The review form is divided into five sections, each with a defined purpose, providing a modular structure. DESIGN: Design and testing of the MRF2 on a sample of medical and nursing records, and evaluation of the reviewers' responses regarding the new review form. SETTING: Hospital based teams from eight countries. RESULTS: The modular review form was reported to be comprehensive, well structured, and clear. Most of the reviewers agreed with the positive statements regarding the review form. Overall, the modular structure was thought to be helpful. Several modifications have been made to the final version to take account of criticisms and suggestions. CONCLUSIONS: The full potential of case record review has yet to be explored. The benefits of this review form include a modular format which enables reviewers or project leaders to select the focus of their review based on resources and the purpose of the review, and to identify contributory factors which indicate areas for improvement and prevention. The training of reviewers is of vital importance for record review. Record review remains one of the primary methods for assessing the incidence of adverse events and the new format is suitable for both prospective and retrospective review.

Evaluation of specific serum anti-Giardia IgM antibody response in diagnosis of giardiasis in children.

Diagnosis of Giardia intestinalis infection is usually made by examination of stool specimens and/or by more invasive methods such as microscopy of duodenal juice or small bowel mucosal biopsies. Serological diagnostic methods have been developed but have not been evaluated in children. In this study specific anti-Giardia immunoglobulin (Ig) M, IgG and IgA antibody titres were measured by enzyme-linked immunosorbent assay. Giardia parasites were sought in jejunal mucosal biopsies and in faeces from 72 children in The Gambia, West Africa; 50 jejunal biopsies, 271 stool samples and 95 serum samples were examined for evidence of Giardia infection. As a diagnostic test, a raised specific anti-Giardia IgM antibody titre (greater than or equal to 1:800) had a sensitivity of 63% and specificity of 93%, with a positive predictive value of 85% and a negative predictive value of 81%. There was poor correlation between positive microscopical identification of Giardia and elevated specific anti-Giardia IgG or IgA antibody titres in children on admission to the study. Elevated serum anti-Giardia IgM, however, was correlated well with active Giardia infection and may prove useful in epidemiological studies of giardiasis in developing countries.