Abstract: BACKGROUND: Residual disease (RD) is an important prognostic factor in acute lymphoblastic leukemia (ALL). Flow cytometry (FC)-based RD detection is easy to perform, but interpretation requires expert analysis due to individual differences among patients. PROCEDURE: We focused at the design of standardized and reproducible RD monitoring in ALL. RD was investigated by a uniform gating strategy, which was designed internationally and tested in one center by Ig/TCR rearrangements. RESULTS: For each gate, positivity cutoff value was assigned using quantification of non-leukemic background. Comparing to Ig/TCR at 0.1% level, 80 of 103 specimens were correctly diagnosed by FC. The predictive value of FC RD at day 15 was then analyzed. In B lineage ALL, day 15 FC significantly correlated with Ig/TCR results at day 33 and/or week 12 (P < 0.01). No significant correlation was found in T lineage ALL. CONCLUSIONS: Thus, FC with preset uniform gating at day 15 predicts PCR-detectable MRD in B precursor ALL. Presented data may be used to define new polychromatic cytometric diagnostics of MRD including semiautomatic assessment. Pediatr Blood Cancer 2010; 54:62-70. (c) 2009 Wiley-Liss, Inc.
Abstract: Children with Down syndrome (DS) have a unique form of acute megakaryocytic leukemia (AMKL) characterized by the presence of mutations in the GATA1 gene leading to increased chemosensitivity and a favorable outcome. We describe an 8-month-old male with DS who was diagnosed with AMKL without a mutation in the GATA1 gene. The patient was treated according to the DS-AML-regimen but his disease progressed and he succumbed 9 months later. This rare case of DS AMKL without a GATA1 mutation with an unfavorable outcome suggests that GATA1 testing may play a useful role in initial stratification. Pediatr Blood Cancer. (c) 2010 Wiley-Liss, Inc.
Abstract: MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose-dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic function of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT-PCR in four types of childhood acute lymphoblastic leukemia (ALL) characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly, high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies showed that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together, these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53-independent manner.
Abstract: We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.
Abstract: Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.
Abstract: PURPOSE: Brain metastases affect 25% of patients with non-small cell lung cancer (NSCLC). We hypothesized that the expression of genes in primary NSCLC tumors could predict brain metastasis and be used for identification of high-risk patients, who may benefit from prophylactic therapy. EXPERIMENTAL DESIGN: The expression of 12 genes was measured by real-time quantitative reverse transcriptase PCR in 142 frozen NSCLC tissue samples. Univariate and multivariate Cox regression analysis was used to analyze the correlation between gene expression and the occurrence of brain metastasis. Immunohistochemistry on independent samples was used to verify the findings. RESULTS: A score based on the expression levels of three genes, CDH2 (N-cadherin), KIFC1, and FALZ, was highly predictive of brain metastasis in early and advanced lung cancer. The probability of remaining brain metastasis-free at 2 years after diagnosis was 90.0+/-9.5% for patients with stage I/stage II tumors and low score compared with 62.7+/-12% for patients with high score (P<0.01). In patients with more advanced lung cancer, the brain metastasis-free survival at 24 months was 89% for patients with low score compared with only 37% in patients with high score (P<0.02). These results were confirmed by immunohistochemical detection of N-cadherin in independent cohort of primary NSCLC. CONCLUSIONS: The expression levels of three genes in primary NSCLC tumors may be used to identify patients at high risk for brain metastasis who may benefit from prophylactic therapy to the central nervous system.
Abstract: Children with Down syndrome (DS) show a spectrum of clinical anomalies, including cognitive impairment, cardiac malformations, and craniofacial dysmorphy. Moreover, hematologists have also noted that these children commonly show macrocytosis, abnormal platelet counts, and an increased incidence of transient myeloproliferative disease (TMD), acute megakaryocytic leukemia (AMKL), and acute lymphoid leukemia (ALL). In this review, we summarize the clinical manifestations and characteristics of these leukemias, provide an update on therapeutic strategies and patient outcomes, and discuss the most recent advances in DS-leukemia research. With the increased knowledge of the way in which trisomy 21 affects hematopoiesis and the specific genetic mutations that are found in DS-associated leukemias, we are well on our way toward designing improved strategies for treating both myeloid and lymphoid malignancies in this high-risk population.
Abstract: PURPOSE: Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay. PATIENTS AND METHODS: High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples. RESULTS: We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set. CONCLUSION: Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.
Abstract: Recurrent, prognostically significant chromosomal abnormalities occur in approximately 75% of paediatric acute lymphoblastic leukaemia (ALL), but only infrequently in children with Down syndrome (DS) and ALL. Recently, novel somatic activating mutations in the gene Janus kinase 2 (JAK2) were reported in 18% of DS ALL. Here we report identification and clinical correlates of JAK2 mutations in an independent cohort. JAK2 activating mutations occurred in 10/53 DS ALL cases (18.9%). Mutations were overrepresented in males (P < 0.03), occurred once in association with high hyperdiploidy and were not significantly correlated with age, initial white blood count, or event-free survival. Our results confirm the significance of JAK-STAT pathway activation in DS ALL.
Abstract: ABSTRACT : INTRODUCTION : PARP-1 (polyADP-ribose polymerase-1) is known to be activated in response to DNA damage, and activated PARP-1 promotes DNA repair. However, a recently disclosed alternative mechanism of PARP-1 activation by phosphorylated externally regulated kinase (ERK) implicates PARP-1 in a vast number of signal-transduction networks in the cell. Here, PARP-1 activation was examined for its possible effects on cell proliferation in both normal and malignant cells. METHODS : In vitro (cell cultures) and in vivo (xenotransplants) experiments were performed. RESULTS : Phenanthridine-derived PARP inhibitors interfered with cell proliferation by causing G2/M arrest in both normal (human epithelial cells MCF10A and mouse embryonic fibroblasts) and human breast cancer cells MCF-7 and MDA231. However, whereas the normal cells were only transiently arrested, G2/M arrest in the malignant breast cancer cells was permanent and was accompanied by a massive cell death. In accordance, treatment with a phenanthridine-derived PARP inhibitor prevented the development of MCF-7 and MDA231 xenotransplants in female nude mice. Quiescent cells (neurons and cardiomyocytes) are not impaired by these PARP inhibitors. CONCLUSIONS : These results outline a new therapeutic approach for a selective eradication of abundant nonhereditary human breast cancers.
Abstract: Constitutional aneuploidies are rare syndromes associated with multiple developmental abnormalities and the alterations in the risk for specific cancers. Acquired somatic chromosomal aneuploidies are the most common genetic aberrations in sporadic cancers. Thus studies of these rare constitutional aneuploidy syndromes are important not only for patient counseling and clinical management, but also for deciphering the mechanisms by which chromosomal aneuploidy affect cancer initiation and progression. Here we review the major constitutional aneuploidy syndromes and suggest some general mechanisms for the associated cancer predisposition.
Abstract: Owing to the increased central nervous system (CNS) relapse risk in T-cell acute lymphoblastic leukaemia (ALL), it is unclear whether preventive cranial radiation (pCRT) can be safely omitted. In this study, pCRT was replaced by extended triple intrathecal therapy (TIT) in prednisone good early responders - medium-risk (MR) group, accounting for 76% of T-ALL patients. From 1989 to 2003, 143 T-ALL patients aged 1-18 years were enrolled in the Israel National Studies (INS) 89 (n = 84) and INS 98 (n = 59) trials, based on ALL-Berlin-Frankfurt-Munster (BFM) 86/90 and ALL-BFM 95 protocols, respectively. Five-year event-free survival (EFS) of the MR group in the INS 89 (n = 60) was 70 +/- 5.9% and the INS 98 (n = 43), 83.7 +/- 5.6% (P = 0.12); the cumulative incidence (CI) of any CNS relapse was 5.0 +/- 2.8% and 2.3 +/- 2.3% (P = 0.50), respectively. There was no difference in outcome between MR patients with a white blood cell count (WBC) >or=100 x 10(9)/l treated with extended TIT (n = 17) or pCRT (n = 10). For all T-ALL patients, 5-year EFS was 61.9 +/- 5.3% in INS 89 and 72.9 +/- 5.8% in INS 98, (P = 0.21); the CI of any CNS relapse was 7.1 +/- 2.8% and 1.7 +/- 1.7% (P = 0.142), respectively. Outcome of T-ALL MR patients given extended TIT in the context of BFM-based protocols with long-term follow-up appeared to be comparable to studies in which a larger proportion of patients was irradiated, and was associated with low risk of CNS relapse, regardless of the WBC.
Abstract: Ets-related gene (ERG) is a member of the ETS transcription factor gene family located on Hsa21. ERG is known to have a crucial role in establishing definitive hematopoiesis and is required for normal megakaryopoiesis. Truncated forms of ERG are associated with multiple cancers such as Ewing's sarcoma, prostate cancer, and leukemia as part of oncogenic fusion translocations. Increased expression of ERG is highly indicative of poor prognosis in acute myeloid leukemia and ERG is expressed in acute megakaryoblastic leukemia (AMKL); however, it is unclear if expression of ERG per se has a leukemogenic activity. We show that ectopic expression of ERG in fetal hematopoietic progenitors promotes megakaryopoiesis and that ERG alone acts as a potent oncogene in vivo leading to rapid onset of leukemia in mice. We observe that the endogenous ERG is required for the proliferation and maintenance of AMKL cell lines. ERG also strongly cooperates with the GATA1s mutated protein, found in Down syndrome AMKL, to immortalize megakaryocyte progenitors, suggesting that the additional copy of ERG in trisomy 21 may have a role in Down syndrome AMKL. These data suggest that ERG is a hematopoietic oncogene that may play a direct role in myeloid leukemia pathogenesis.
Abstract: PURPOSE: The ETV6 gene has been reported to be fused to a multitude of partner genes in various hematologic malignancies with 12p13 aberrations. Cytogenetic analysis of six cases of childhood acute lymphoblastic leukemia revealed a novel recurrent t(8;12)(q13;p13), suggesting involvement of ETV6. EXPERIMENTAL DESIGN: Fluorescence in situ hybridization was used to confirm the involvement of ETV6 in the t(8;12)(q13;p13) and reverse transcription-PCR was used to identify the ETV6 partner gene. Detailed immunologic characterization was done, and owing to their lineage promiscuity, the leukemic blast cells were analyzed for NOTCH1 mutations. RESULTS: We have identified a novel recurrent t(8;12)(q13;p13), which results in a fusion between the transcriptional repressor ETV6 (TEL) and the transcriptional coactivator NCOA2 (TIF2) in six cases of childhood leukemia expressing both T-lymphoid and myeloid antigens. The ETV6-NCOA2 transcript encodes a chimeric protein that consists of the pointed protein interaction motif of ETV6 that is fused to the COOH terminus of NCOA2, including the cyclic AMP-responsive element binding protein-binding protein (CBP) interaction and the AD2 activation domains. The absence of the reciprocal NCOA2-ETV6 transcript in one of the cases suggests that the ETV6-NCOA2 chimeric protein and not the reciprocal NCOA2-ETV6 is responsible for leukemogenesis. In addition, ETV6-NCOA2 leukemia shows a high frequency of heterozygous activating NOTCH1 mutations, which disrupt the heterodimerization or the PEST domains. CONCLUSIONS: The ETV6-NCOA2 fusion may define a novel subgroup of acute leukemia with T-lymphoid and myeloid features, which is associated with a high prevalence of NOTCH1 mutations.
Abstract: BACKGROUND: The sharp division between curative cancer therapy and palliative care results in the late introduction of palliative care and a high incidence of suffering in children with cancer. We established a Palliative Care Unit (PCU) that is fully integrated with the Pediatric Hematology Oncology Department (PHOD). We wished to explore the impact of such integrative model on patterns of hospitalizations and exposure to palliative care of pediatric oncology patients. PROCEDURES: Retrospective search of medical records of patients admitted to the PHOD since PCU establishment in 1999, and of children who died from progressive disease between 1990 and 2005 was performed. Differences in clinical and prognostic variables between PCU and non-PCU patients, and differences in location of death before and after PCU establishment were evaluated. RESULTS: The majority (59%) of patients, who were hospitalized after the PCU establishment, were hospitalized in the PCU, including 49% of the good prognosis patients and 91% of the poor prognosis patients. Poor prognosis patients were hospitalized in the PCU earlier and with higher frequency compared to children with curable disease. After PCU opening there was a significant decline in the percentage of patients who died in the general pediatric ward, hematology-oncology ward, and at home from 40%, 26% and 28% to 4%, 8%, and 16%, respectively. CONCLUSIONS: Our integrative model results in exposure of the majority of children with cancer to palliative care. For poor prognosis patients, palliative care is introduced early enough to allow gradual transition from symptom control after diagnosis to end of life care.
Abstract: Early in mammalian development, the stem cell leukemia (SCL/TAL1) gene and its distinct 3' enhancer (SCL 3'En) specify bipotential progenitor cells that give rise to blood and endothelium, thus termed hemangioblasts. We have previously detected a minor population of SCL (+) cells in the postnatal kidney. Here, we demonstrate that cells expressing the SCL 3'En in the adult kidney are comprised of CD45+CD31- hematopoietic cells, CD45-CD31+ endothelial cells and CD45-CD31- interstitial cells. Creation of bone marrow chimeras of SCL 3'En transgenic mice into wild-type hosts shows that all three types of SCL 3'En-expressing cells in the adult kidney can originate from the bone marrow. Ischemia/reperfusion injury to the adult kidney of SCL 3'En transgenic mice results in the intrarenal elevation of SCL and FLK1 mRNA levels and of cells expressing hem-endothelial progenitor markers (CD45, CD34, c-Kit and FLK1). Furthermore, analysis of SCL 3'En in the ischemic kidneys reveals an increase in the abundance of SCL 3'En-expressing cells, predominantly within the CD45 (+) hematopoietic fraction and to a lesser extent in the CD45 (-) fraction. Our results suggest organ-injury-induced reactivation of bone marrow-derived hemangioblasts and possible local angioblastic progenitors expressing SCL and SCL 3'En.
Abstract: Children with Down syndrome (DS) have a markedly increased risk of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To identify chromosomal changes cooperating with +21 that may provide information on the pathogenesis of these leukemias, we analyzed 215 DS-ALLs and 189 DS-AMLs. Unlike previous smaller series, a significant proportion of DS-ALLs had the typical B-cell precursor ALL abnormalities high hyperdiploidy (HeH; 11%) and t(12;21)(p13;q22) (10%). The HeH DS-ALLs were characterized by gains of the same chromosomes as non-DS-HeH, suggesting the same etiology/pathogenesis. In addition, specific genetic subtypes of DS-ALL were suggested by the significant overrepresentation of cases with +X, t(8;14)(q11;q32), and del(9p). Unlike DS-ALL, the common translocations associated with non-DS-AML were rare in DS-AML, which instead were characterized by the frequent presence of dup(1q), del(6q), del(7p), dup(7q), +8, +11, del(16q), and +21. This series of DS leukemias-the largest to date-reveals that DS-ALL is a heterogeneous disorder that comprises both t(12;21) and HeH as well as DS-related abnormalities. Furthermore, this analysis confirms that DS-AML is a distinct entity, originating through other genetic pathways than do non-DS-AMLs, and suggests that unbalanced changes such as dup(1q), +8, and +21 are involved in the leukemogenic process.
Abstract: The ALL IC-BFM 2002 protocol was created as an alternative to the MRD-based AIEOP-BFM ALL 2000 study, to integrate early response criteria into risk-group stratification in countries not performing routine PCR-based MRD testing. ALL IC stratification comprises the response to prednisone, bone marrow (BM) morphology at days 15 and 33, age, WBC and BCR/ABL or MLL/AF4 presence. Here, we compared this stratification to the MRD-based criteria using MRD evaluation in 163 patients from four ALL IC member countries at days 8, 15 and 33 and week 12. MRD negativity at day 33 was associated with an age of 1-5 years, WBC<20,000 microl(-1), non-T immunophenotype, good prednisone response and non-M3 morphology at day 15. There were no significant associations with gender or hyperdiploidy in the study group, or with TEL/AML1 fusion within BCP-ALL. Patients with M1/2 BM at day 8 tended to be MRD negative at week 12. Patients stratified into the standard-risk group had a better response than intermediate-risk group patients. However, 34% of them were MRD positive at day 33 and/or week 12. Our findings revealed that morphology-based ALL IC risk-group stratification allows the identification of most MRD high-risk patients, but fails to discriminate the MRD low-risk group assigned to therapy reduction.
Abstract: Twenty-five cases of B-cell precursor acute lymphoblastic leukaemia (ALL) from Down syndrome (DS) patients were analyzed using array comparative genomic hybridization (aCGH) and compared with two other subgroups of non-DS patients with ALL; five cases with high-hyperdiploidy (HH) and nine cases with ETV6-RUNX1 positive clones. Seven cases of DS-acute megakaryoblastic leukaemia (AMKL) were also included, DS-ALL cases showed relatively stable karyotypes with cryptic losses and gains that most frequently involved chromosomes X, 1, 2, 9, 11, 16, and 17. The most consistent change involved a deletion in 2p, spanning region Chr2:88273220-91084234, which in some cases appeared to be homozygous. ALL from non-DS patients showed a similar overall karyotypic stability, although gains of chromosome 21 were infrequent in the ETV6-RUNX1 positive cases. The most consistent change in this group involved a 12p deletion, where Chr12:10383878-16017619 defined the common region of overlap. All HH-ALL karyotypes showed variable gains of chromosome 21. This overall analysis supports the suggestion that, although constitutional trisomy 21 predisposes to ALL/AMKL, the cytogenetic changes associated with DS-ALL in particular, are most similar to those found in non-DS ETV6-RUNX1 positive ALL. The HH-ALL group, however, undergoes distinct karyotypic evolution not dependent on chromosome translocation/deletion events.
Abstract: BACKGROUND: Children with Down's syndrome have a greatly increased risk of acute megakaryoblastic and acute lymphoblastic leukaemias. Acute megakaryoblastic leukaemia in Down's syndrome is characterised by a somatic mutation in GATA1. Constitutive activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) pathway occurs in several haematopoietic malignant diseases. We tested the hypothesis that mutations in JAK2 might be a common molecular event in acute lymphoblastic leukaemia associated with Down's syndrome. METHODS: JAK2 DNA mutational analysis was done on diagnostic bone marrow samples obtained from 88 patients with Down's syndrome-associated acute lymphoblastic leukaemia; and 216 patients with sporadic acute lymphoblastic leukaemia, Down's syndrome-associated acute megakaryoblastic leukaemia, and essential thrombocythaemia. Functional consequences of identified mutations were studied in mouse haematopoietic progenitor cells. FINDINGS: Somatically acquired JAK2 mutations were identified in 16 (18%) patients with Down's syndrome-associated acute lymphoblastic leukaemia. The only patient with non-Down's syndrome-associated leukaemia but with a JAK2 mutation had an isochromosome 21q. Children with a JAK2 mutation were younger (mean [SE] age 4.5 years [0.86] vs 8.6 years [0.59], p<0.0001) at diagnosis. Five mutant alleles were identified, each affecting a highly conserved arginine residue (R683). These mutations immortalised primary mouse haematopoietic progenitor cells in vitro, and caused constitutive Jak/Stat activation and cytokine-independent growth of BaF3 cells, which was sensitive to pharmacological inhibition with JAK inhibitor I. In modelling studies of the JAK2 pseudokinase domain, R683 was situated in an exposed conserved region separated from the one implicated in myeloproliferative disorders. INTERPRETATION: A specific genotype-phenotype association exists between the type of somatic mutation within the JAK2 pseudokinase domain and the development of B-lymphoid or myeloid neoplasms. Somatically acquired R683 JAK2 mutations define a distinct acute lymphoblastic leukaemia subgroup that is uniquely associated with trisomy 21. JAK2 inhibitors could be useful for treatment of this leukaemia. FUNDING: Israel Trade Ministry, Israel Science Ministry, Jewish National Fund UK, Sam Waxman Cancer Research Foundation, Israel Science Foundation, Israel Cancer Association, Curtis Katz, Constantiner Institute for Molecular Genetics, German-Israel Foundation, and European Commission FP6 Integrated Project EUROHEAR.
Abstract: The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently, E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes, we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in vivo. Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F.
Abstract: PURPOSE: Studies on musculoskeletal manifestations (MSM) of childhood acute lymphoblastic leukemia (ALL) have yielded variable findings with regard to their clinical impact. We investigated the significance for differential diagnosis, treatment and outcome of musculoskeletal complaints as presenting symptoms of ALL, and their correlation with leukemia immunophenotypes, for which data is lacking. METHODS: Data on 783 children in the national study for childhood ALL between 1984 and 2003 were reviewed retrospectively. Statistical analysis examined possible relationships between MSM at the time of diagnosis and demographic and clinical data, biological features of leukemia (peripheral blood counts, immunophenotype and main cytogenetic aberration), response to initial prednisone treatment, and outcome. RESULTS: Of 765 children with data on orthopaedic complaints, 240 presented with MSM (31.4%). Among these children, B cell precursor (BCP) was much more common (209/576, 36.3%) than T cell ALL (25/176, 14.2%). Patients with MSM had lower white blood cell counts (WBC) (median of 9 vs. 20 x 10(9)/L, P < 0.001) and percentage of blast cells in the peripheral blood at diagnosis compared to those without (median of 27 vs. 53%, P < 0.001). Hepatomegaly and splenomegaly were less common in MSM group (67 vs. 53% <3 cm, P < 0.001, and 63 vs. 50% <3 cm, P < 0.001, respectively). Poor response to initial treatment with prednisone was recorded in 7.1% of patients with MSM versus 11.5% of those without (P = 0.086). The analysis revealed no independent effect of MSM on event-free survival (EFS), after correcting for differences in EFS related to immunophenotype or initial WBC. CONCLUSIONS: MSM occur mostly in children with BCP ALL who present with less involvement of extramedullary organs, low peripheral blood blasts and white blood cells counts. These findings highlight the importance of including ALL in the differential diagnosis of MSM even in the presence of an apparently normal peripheral blood count. Our study also suggests that MSM are caused by leukemic cells with enhanced biological propensity to remain relatively confined within the intramedullary bone-marrow space.
Abstract: Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.
Abstract: Chromosomal aneuploidy is commonly observed in neoplastic diseases and is an important prognostic marker. Here we examine how gene expression profiles reflect aneuploidy and whether these profiles can be used to detect changes in chromosome copy number. We developed two methods for detecting such changes in the gene expression profile of a single sample. The first method, fold-change analysis, relies on the availability of gene expression data from a large cohort of patients with the same disease. The expression profile of the sample is compared with that of the dataset. The second method, chromosomal relative expression analysis, is more general and requires the expression data from the tested sample only. We found that the relative expression values are stable among different chromosomes and exhibit little variation between different normal tissues. We exploited this novel finding to establish the set of reference values needed to detect changes in the copy number of chromosomes in a single sample on the basis of gene expression levels. We measured the accuracy of the performance of each method by applying them to two independent leukemia datasets. The second method was also applied to two solid tumor datasets. We conclude that chromosomal aneuploidy can be detected and predicted by analysis of gene expression profiles. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.
Abstract: Although mitosis is a general physiologic process, cancer cells are unusually sensitive to mitotic inhibitors. Therefore, there is an interest in the identification of novel mitotic inhibitors. Here, we report the novel discovery of the SIL gene as a regulator of mitotic entry and cell survival. The SIL gene was cloned from leukemia-associated chromosomal translocation. It encodes a cytosolic protein with an unknown function and no homology to known proteins. Previously, we observed an increased expression of SIL in multiple cancers that correlated with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. Here, we show that SIL is important for the transition from the G(2) to the M phases of the cell cycle. Inducible knockdown of SIL in cancer cells in vitro delayed entrance into mitosis, decreased activation of the CDK1 (CDC2)-cyclin B complex, and induced apoptosis in a p53-independent manner. SIL is also essential for the growth of tumor explants in mice. Thus, SIL is required for mitotic entry and cancer cell survival. Because increased expression of SIL has been noted in multiple types of cancers and correlates with metastatic spread, it may be a suitable target for novel anticancer therapy.
Abstract: BACKGROUND: Bernard-Soulier syndrome (BSS) is a severe inherited bleeding disorder that is caused by a defect in glycoprotein (GP)Ib-IX-V complex, the platelet membrane receptor for von Willebrand factor. PATIENTS: The diagnosis of BSS was made in two members of a Bukharian Jewish family who had life-long thrombocytopenia associated with mucocutaneous bleeding manifestations. METHODS AND RESULTS: Flow cytometry and Western blot analyses showed only trace amounts of GPIb and GPIX on the patients' platelets. Sequence analysis of the GPIbalpha gene revealed a homozygous T > G transversion at nucleotide 709 predicting Trp207Gly substitution in the mature protein. Introduction of the mutation into a mammalian expression construct abolished the surface expression of GPIbalpha in transfected baby hamster kidney cells. The crystal structure of the N-terminus of GPIbalpha (PDB: 1SQ0) indicates that Trp207 is completely buried and located in a disulfide loop structure that interacts with the leucine-rich repeat (LRR) domain. CONCLUSION: A novel mutation, Trp207Gly, causes BSS and predicts disruption of the interaction between a disulfide loop and the LRR domain that is essential for the integrity of GPIbalpha structure.
Abstract: PURPOSE: Applying current diagnostic methods, overt CNS involvement is a rare event in childhood acute lymphoblastic leukemia (ALL). In contrast, CNS-directed therapy is essential for all patients with ALL because without it, the majority of patients eventually will experience relapse. To approach this discrepancy and to explore potential distinct biologic properties of leukemic cells that migrate into the CNS, we compared gene expression profiles of childhood ALL patients with initial CNS involvement with the profiles of CNS-negative patients. PATIENTS AND METHODS: We evaluated leukemic gene expression profiles from the bone marrow of 17 CNS-positive patients and 26 CNS-negative patients who were frequency matched for risk factors associated with CNS involvement. Results were confirmed by real-time quantitative polymerase chain reaction analysis and validated using independent patient samples. RESULTS: Interleukin-15 (IL-15) expression was consistently upregulated in leukemic cells of CNS-positive patients compared with CNS-negative patients. In multivariate analysis, IL-15 expression levels greater than the median were associated with CNS involvement compared with expression equal to or less than the median (odds ratio [OR] = 10.70; 95% CI, 2.95 to 38.81). Diagnostic likelihood ratios for CNS positivity were 0.09 (95% CI, 0.01 to 0.65) for the first and 6.93 (95% CI, 2.55 to 18.83) for the fourth IL-15 expression quartiles. In patients who were CNS negative at diagnosis, IL-15 levels greater than the median were associated with subsequent CNS relapse compared with expression equal to or less than the median (OR = 13.80; 95% CI, 3.38 to 56.31). CONCLUSION: Quantification of leukemic IL-15 expression at diagnosis predicts CNS status and could be a new tool to further tailor CNS-directed therapy in childhood ALL.
Abstract: MOTIVATION: Existing computational methods that identify transcription factor (TF) binding sites on a gene's promoter are plagued by significant inaccuracies. Binding of a TF to a particular sequence is assessed by comparing its similarity score, obtained from the TF's known position weight matrix (PWM), to a threshold. If the similarity score is above the threshold, the sequence is considered a putative binding site. Determining this threshold is a central part of the problem, for which no satisfactory biologically based solution exists. RESULTS: We present here a method that integrates gene expression data with sequence-based scoring of TF binding sites, for determining a global score threshold for each TF. We validate our method, STOP (Searching TFs Of Promoters), in several ways: (1) we calculate the average expression values of groups of human putative target genes of each TF, and compare them to similar averages derived for random gene groups. The groups of putative targets show significantly higher relative average expression. (2) We find high consistency between the induced lists of putative targets in human and in mouse. (3) The expression patterns associated with human and mouse genes (ordered by PWM scores for each TF) exhibit high similarity between human and mouse, indicating that our method has firm biological basis. (4) Comparison of results obtained by STOP and PRIMA (Elkon et al., 2003) suggests that determining the score threshold using gene expression, as is done in STOP, is more biologically tuned. AVAILABILITY: Software package will be available for academic users upon request. SUPPLEMENTARY INFORMATION: Supplementary data are available on Bioinformatics online.
Abstract: BACKGROUND: Gene duplication and exonization of intronic transposed elements are two mechanisms that enhance genomic diversity. We examined whether there is less selection against exonization of transposed elements in duplicated genes than in single-copy genes. RESULTS: Genome-wide analysis of exonization of transposed elements revealed a higher rate of exonization within duplicated genes relative to single-copy genes. The gene for TIF-IA, an RNA polymerase I transcription initiation factor, underwent a humanoid-specific triplication, all three copies of the gene are active transcriptionally, although only one copy retains the ability to generate the TIF-IA protein. Prior to TIF-IA triplication, an Alu element was inserted into the first intron. In one of the non-protein coding copies, this Alu is exonized. We identified a single point mutation leading to exonization in one of the gene duplicates. When this mutation was introduced into the TIF-IA coding copy, exonization was activated and the level of the protein-coding mRNA was reduced substantially. A very low level of exonization was detected in normal human cells. However, this exonization was abundant in most leukemia cell lines evaluated, although the genomic sequence is unchanged in these cancerous cells compared to normal cells. CONCLUSION: The definition of the Alu element within the TIF-IA gene as an exon is restricted to certain types of cancers; the element is not exonized in normal human cells. These results further our understanding of the delicate interplay between gene duplication and alternative splicing and of the molecular evolutionary mechanisms leading to genetic innovations. This implies the existence of purifying selection against exonization in single copy genes, with duplicate genes free from such constrains.
Abstract: Extra copies of chromosome 21 are often found in sporadic leukemias. Constitutional trisomy 21 of Down syndrome (DS) is associated with markedly increased risk for childhood leukemia. Thus the oncogenic role of trisomy 21 in the more common sporadic childhood leukemias may be revealed through the investigations of the relatively rare leukemias of DS. Recent studies of the megakaryoblastic leukemias of Down syndrome have uncovered a developmental leukemogenic mechanism characterized by a unique pre-natal collaboration between overexpressed genes from chromosome 21 and an acquired mutation in the transcription factor GATA1. The base of the markedly enhanced risk for acute lymphoblastic leukemia conferred by trisomy 21 is still unclear. Studies of the leukemias of DS are likely to contribute to the general understanding of the oncogenic mechanisms of chromosomal aneuploidies, the most common abnormalities in cancer.
Abstract: Abnormal number of chromosomes, aneuploidy, is the most common abnormality in leukemia and cancer. However, the casual relationship between aneuploidy and cancer is unclear. Additional copies of chromosome 21 are frequently found in leukemic cells. Constitutional trisomy 21 that characterizes Down Syndrome is associated with markedly increased risk for childhood leukemia. In this perspective I review recent studies that suggest that constitutional trisomy 21 promotes leukemic transformation during fetal hematopoiesis. As most of childhood leukemias arise in-utero, these studies are of general relevance to sporadic childhood leukemias.
Abstract: OBJECTIVE: The Hedgehog family of intercellular proteins has a crucial role in embryonic development. Recent experimental data suggests that the Hedgehog pathway may play a role in early hematopoiesis and angiogenesis. Stem cell leukemia (SCL), a basic helix-loop-helix (bHLH) transcription factor, is essential for the specification and function of the hemangioblast. SCL expression in early hematopoietic precursors and endothelium is directed by a 3' enhancer. We hypothesized that the SCL 3' enhancer is regulated by Hedgehog signaling during specification of mesoderm towards hemangioblastic fate. MATERIALS AND METHODS: Whole embryos derived from transgenic mouse lines carrying reporter genes under the regulation of SCL 3' enhancer were cultured in the presence of active Hedgehog peptide. Hedgehog transcriptional regulation of SCL 3' enhancer was studied by in vitro and in vivo binding and reporter assays. RESULTS: Hedgehog induced expansion of cells in which the SCL 3' enhancer was transcriptionally activated. A Gli-binding site within the 3' enhancer of SCL was identified and Gli1 was demonstrated to bind and transactivate this enhancer in a sequence-dependent manner. We further demonstrated that the core region of the SCL 3' enhancer is transcriptionally regulated by Hedgehog in-vivo and that the Gli-binding site located in this enhancer is essential for Hedgehog transcriptional regulation in vitro. CONCLUSION: These findings suggest that SCL may be a direct target of Hedgehog signaling during hemangioblast specification.
Abstract: A kindred is reported with four members affected with neurodegenerative disorder and 3-methylglutaconic aciduria. Two siblings developed thrombocytopenia heralding a myelodysplastic syndrome; in one patient it evolved into acute myeloid leukemia with monosomy 7 in the marrow. The hematologic complications have hitherto not been previously reported in other cases of 3-methylglutaconic aciduria and are thus thought to represent a new disease entity. This family adds additional evidence to the genetic heterogeneity of Mendelian disorders in which the primary mutation may have a mutator effect that could give origin to myelodysplastic syndrome and acute myeloid leukemia through acquired chromosomal changes.
Abstract: The Hedgehog proteins play a crucial role in metazoan embryo development. Constitutive activation of the pathway is associated with multiple types of cancer. Recent experimental data suggest involvement of Hedgehog signaling in vascular remodeling, germ cell migration, and axon guidance. The molecular mechanisms underlying these effects remain elusive. Here we show that yolk sac-derived endothelial cells and embryonic fibroblasts can directly respond to the Hedgehog signal by increased migration in an in vitro scratch (wound) assay. We also identify Hedgehog transcriptional target genes in these cells, many of which participate in cell migration, axon guidance, and angiogenesis processes. Inhibition of one such molecular pathway, neuropilin-flavomonooxygenase, blocks Hedgehog-induced cell migration. These findings suggest that Hedgehog signaling directly affects embryonic endothelial and fibroblast cell migration via molecules and pathways known to regulate cell migration in response to a variety of environmental cues.
Abstract: On the basis of epidemiological studies, infection was suggested to play a role in the etiology of human cancer. While for some cancers such a role was indeed demonstrated, there is no direct biological support for the role of viral pathogens in the pathogenesis of childhood leukemia. Using a novel bioinformatic tool that alternates between clustering and standard statistical methods of analysis, we performed a 'double-blind' search of published gene expression data of subjects with different childhood acute lymphoblastic leukemia (ALL) subtypes, looking for unanticipated partitions of patients, induced by unexpected groups of genes with correlated expression. We discovered a group of about 30 genes, related to the interferon response pathway, whose expression levels divide the ALL samples into two subgroups; high in 50, low in 285 patients. Leukemic subclasses prevalent in early childhood (the age most susceptible to infection) are over-represented in the high-expression subgroup. Similar partitions, induced by the same genes, were found also in breast and ovarian cancer but not in lung cancer, prostate cancer and lymphoma. About 40% of breast cancer samples expressed the 'interferon-related' signature. It is of interest that several studies demonstrated mouse mammary tumor virus-like sequences in about 40% of breast cancer samples. Our discovery of an unanticipated strong signature of an interferon-induced pathway provides molecular support for a role for either inflammation or viral infection in the pathogenesis of childhood leukemia as well as breast and ovarian cancer.
Abstract: The TEL/AML1 (ETV6/RUNX1) fusion gene is the most common genetic rearrangement in paediatric acute lymphoblastic leukaemia (ALL). Although considered to be a low-risk leukaemia, it is associated with a relapse rate of 10-20%. The coexistence of different subclones at diagnosis, based on polymerase chain reaction (PCR) studies of IG/TCR gene rearrangement, with differential response to chemotherapy, was recently reported in this subtype of ALL. We wished to demonstrate such subclones at diagnosis by a recently developed technique of quantitative multiparametric fluorescence in situ hybridization (FISH). Bone marrow cells from 80 paediatric patients with ALL at diagnosis were analysed for the presence of the TEL/AML1 fusion gene by interphase FISH. Fourteen patients were positive for the translocation. Four of them had several subclones associated with various combinations of additional chromosomal abnormalities. The most striking was an atypical and unexpected hybridization pattern consistent with a submicroscopic deletion of the 5' region of the AML1 breakpoint. Other abnormalities included TEL deletion, trisomy and tetrasomy 21 as well as double TEL-AML1 fusion. The presence of numerous subclones in about 25% of patients with TEL/AML1+ ALL suggests extensive clonal evolution by the time of diagnosis.
Abstract: SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.
Abstract: OBJECT: Pediatric low-grade gliomas (LGGs) are the largest group of central nervous system neoplasms in children. Although these tumors are generally benign, 5 to 10% of patients with pediatric LGGs present with leptomeningeal dissemination. The genetic and biological nature of these tumors is poorly understood. The authors looked for certain molecular abnormalities that may differentiate disseminated gliomas from the other pediatric LGGs. METHODS: Comparative genomic hybridization (CGH) was applied to 18 pediatric LGGs. Six cases featuring disseminated pediatric LGGs were compared with 12 control cases involving nondisseminated pediatric LGGs. Fluorescence in situ hybridization (FISH) analysis and immunohistochemical analysis were used to highlight further specific genetic targets. The CGH revealed multiple chromosomal abnormalities in five of six cases with disseminated gliomas and in six of 12 control cases. No correlation was found between the number of chromosomal abnormalities and dissemination status. Amplification of chromosome 7 was noted in four of six cases with disseminated gliomas as opposed to one of 12 control cases (p = 0.02). The FISH analysis revealed epidermal growth factor receptor (EGFR) amplification in one case negative to chromosome 7 amplification by CGH, raising the amplification cases to five of six (p = 0.0038). Immunohistochemical analysis for EGFR was positive in six of six cases and in two of 12 control cases (p = 0.0015). At the end of a mean follow-up period of 7.2 years, all patients with disseminated gliomas are alive with variable but slow disease progression. CONCLUSIONS: The high rate of EGFR gene amplification and protein expression in disseminated pediatric LGGs is intriguing and may have implications for our understanding of the role of EGFR in glioma genesis. Targeted therapies may be available for these children. Larger-scale studies are needed to establish further these findings.
Abstract: Aneuploidy is one of the hallmarks of cancer. Acquired additions of chromosome 21 are a common finding in leukemias, suggesting a contributory role to leukemogenesis. About 10% of patients with a germ line trisomy 21 (Down syndrome) are born with transient megakaryoblastic leukemia. We and others have shown acquired mutations in the X chromosome gene GATA1 in all these cases. The gene or genes on chromosome 21 whose overexpression promote the megakaryoblastic phenotype are presently unknown. We propose that ERG, an Ets transcription factor situated on chromosome 21, is one such candidate. We show that ERG is expressed in hematopoietic stem cells, megakaryoblastic cell lines, and in primary leukemic cells from Down syndrome patients. ERG expression is induced upon megakaryocytic differentiation of the erythroleukemia cell lines K562 and UT-7, and forced expression of ERG in K562 cells induces erythroid to megakaryoblastic phenotypic switch. We also show that ERG activates the gpIb megakaryocytic promoter and binds the gpIIb promoter in vivo. Furthermore, both ERG and ETS2 bind in vivo the hematopoietic enhancer of SCL/TAL1, a key regulator of hematopoietic stem cell and megakaryocytic development. We propose that trisomy 21 facilitates the occurrence of megakaryoblastic leukemias through a shift toward the megakaryoblastic lineage caused by the excess expression of ERG, and possibly by other chromosome 21 genes, such as RUNX1 and ETS2, in hematopoietic progenitor cells, coupled with a differentiation arrest caused by the acquisition of mutations in GATA1.
Abstract: Sil (SCL interrupting locus) was cloned from the most common chromosomal rearrangement in T-cell acute lymphoblastic leukemia. It is an immediate early gene whose expression is associated with cell proliferation. Sil protein levels are tightly regulated during the cell cycle, reaching peak levels in mitosis and disappearing on transition to G1. A recent study found Sil to be one of 17 genes whose overexpression in primary adenocarcinomas predicts metastatic spread. We hypothesized that Sil might have a role in carcinogenesis. To address this question, we utilized several approaches. Using a multitumor tissue array, we found that Sil protein expression was increased mostly in lung cancer, but also at lower levels, in a subset of other tumors. Microarray gene expression analysis and immunohistochemistry of lung cancer samples verified these observations. Sil gene expression in lung cancer correlated with the expression of several kinetochore check-point genes and with the histopathologic mitotic index. These observations suggest that overexpression of the Sil gene characterizes tumors with increased mitotic activity.
Abstract: BACKGROUND: In vertebrates the hematopoietic and renal tissues share a common mesodermal origin. Recently, we have analyzed global gene expression during human nephrogenesis and observed up-regulation of stem cell leukemia (SCL), a transcription factor critical for hematopoietic and endothelial lineage specification. Here we characterize the expression of SCL along with its distinct 3' hematopoietic and endothelial enhancer (SCL 3'En) during kidney development. METHODS: mRNA and protein expression of SCL were examined in developing murine and human kidneys by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The activity of SCL 3'En was examined by X-galactosidase (X-gal) staining of embryonic kidneys obtained from SCL +6E5/lacZ/3'En transgenic mice and by reporter lacZ assay in various renal cell lines. RESULTS: We found developmental regulation of SCL mRNA with highest levels of expression in embryonic day 17 (E17) mouse kidneys and lowest in postnatal and adult kidneys. Immunostaining of human fetal kidneys demonstrated the protein predominantly in the nephrogenic cortex and particularly in mesenchymal cells and developing glomeruli. Similarly, SCL +6E5/lacZ/3'En transgenic kidneys showed prominent lacZ staining in cells resembling undifferentiated mesoderm cells in close proximity to S and comma-shaped primitive nephrons and in peritubular and glomerular vessel endothelium. The SCL 3'En was activated in the human embryonic kidney cell line (HEK 293), but not in cell lines derived from adult kidney. CONCLUSION: These observations suggest a possible role for SCL in renal vasculogenesis. Undifferentiated mesenchymal cells expressing SCL during early nephrogenesis might represent putative progenitors that can simultaneously give rise to kidney, blood, and endothelium.
Abstract: Acquired resistance towards apoptosis is the hallmark of most if not all types of cancer. We have previously identified and characterized ARTS, a broadly expressed protein localized to mitochondria. ARTS was initially shown to mediate TGF-beta induced apoptosis. Recently, we have found that high levels of ARTS induce apoptosis without additional pro-apoptotic stimuli. Further, ARTS promotes apoptosis in response to a wide variety of pro-apoptotic stimuli. Here, we report that the expression of ARTS is lost in all lymphoblasts of more than 70% of childhood acute lymphoblastic leukemia (ALL) patients. The loss of ARTS is specific, as the related non-apoptotic protein H5, bearing 83% identity to ARTS, is unaffected. During remission, ARTS expression is detected again in almost all patients. Two leukemic cell lines, ALL-1 and HL-60 lacking ARTS, were resistant to apoptotic induction by ara-C. Transfection of ARTS into these cells restored their ability to undergo apoptosis in response to this chemotherapeutic agent. We found that methylation process contributes to the loss of ARTS expression. We conclude that the loss of ARTS may provide a selective advantage for cells to escape apoptosis thereby contributing to their transformation to malignant lymphoblasts. We therefore propose that ARTS can function as a tumor suppressor protein in childhood ALL.
Abstract: Leukaemia is characterized by the accumulation of malignant haematopoietic precursors. Recent studies have revealed that acquired alterations in genes that regulate normal haematopoiesis are frequently detected in leukaemia. The progression to leukaemia depends on additional mutations that promote the survival of developmentally arrested cells. This review describes three examples of this general paradigm of leukaemogenesis: RUNX1 abnormalities in acute leukaemias, GATA1 mutations in the leukaemias of Down syndrome, and SCL and LMO2 ectopic expression in T cell acute lymphoblastic leukaemia.
Abstract: The pace of disappearance of leukemic blasts in response to therapy has long been recognized as the most important prognostic factor in childhood acute lymphoblastic leukemia (ALL). Recent technological advancements enable detection of submicroscopic leukemic cells. The extent of reduction in the level of minimal residual disease (MRD) during the first phase of therapy can be exploited for improved risk classification of children with ALL. Current prospective studies test the hypothesis that tailoring treatment to the level of MRD will improve patients' outcome.
Abstract: Transcriptional regulation in eukaryotes is a multilevel hierarchical process. It is becoming clear that higher-order chromatin structure, occurring via modifications of histones in their nucleosome structure, plays a crucial role in regulating gene expression, both in normal and pathological states. Deacetylation of histones by histone deacetylases (HDACs) modifies the chromatin from an open gene active euchromatin structure to a closed gene silenced heterochromatin structure. Several cancer promoting mutations and chromosomal translocations result in repression of transcription through abnormal recruitment and activation of HDACs, leading to neoplastic transformation. This is the rationale for the evolvement of HDAC inhibitors as a new class in cancer therapy. Trials have shown anti-proliferation effect of histone deacetylase inhibitors in cell culture, animal models and in human with both hematological and solid tumors. The exact mechanism by which histone deacetylase inhibitors exert their effect is still obscure. Reversal of the alteration in gene expression by fusion transcription factors or overexpressed repressors is just one of several possible explanations. The territory of heterochromatin in the vicinity of the nuclear periphery raised the possibility of involvement of nuclear envelope proteins in the regulation of transcription. Our laboratory is interested in the transcription repression mechanism induced by the nuclear envelope lamina associated polypeptide 2 (LAP2) family of proteins through chromatin modification. Here, we will describe the structure of the nucleosome, review regulation of gene expression by acetylation of histones and give an update on the current phase I and phase II clinical trials with histone deacetylase inhibitors.
Abstract: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is frequently used to mobilize CD34+ cells in healthy donors and patient with malignant diseases prior to peripheral blood stem cell (PBSC) harvest. To analyze the effects of rhG-CSF on morphology and genotype of white blood cells, a novel multiparametric cell scanning system that combines morphologic, immune and genotypic analyses of the same cells was used. We report here that tetraploid myeloid cells are present in the peripheral blood of donors treated with rhG-CSF. The tetraploidy was detected in up to 0.6% of differentiated myeloid cells and all observed CD34+ cells were diploid. Thus, short treatment with rhG-CSF of PBSC donors induces numerfical chromosomal alterations in a small subset of mature myeloid cells.
Abstract: Patients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s. We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL.
Abstract: The association of specific congenital syndromes with leukemia provides an opportunity to study the process of leukemogenesis on the background of known genetic alterations. The role of the intracellular DNA damage response system in suppressing leukemia is demonstrated by the congenital disorders of genomic instability. Specific collaborations between survival and differentiation pathways characterize the leukemias observed in Down, Noonan and neurofibromatosis syndromes. As these syndromes clearly reveal, childhood leukemia arises when the delicate balance between growth, development and differentiation of the fetal and early post-natal hematopoietic system is disrupted.
Abstract: BACKGROUND: Medulloblastoma is a malignant, invasive embryonic tumor of the cerebellum. Sonic hedgehog (SHH) is a secreted glycoprotein that has a major role in the developing cerebellum. Activation of the SHH pathway resulting from mutations in the PATCH gene, which is an inhibitor of the pathway, are associated with hereditary and sporadic medulloblastomas. The GLI3 protein is another negative regulator of SHH signaling. The authors hypothesized that mutations in GLI3 may be associated with meduloblastomas. METHODS: The authors describe a patient with hereditary Greig syndrome, which was caused by mutations in GLI3, and medulloblastoma. Another such patient was described in the literature. They also sequenced the GLI3 gene, including all exon-intron boundaries, in an additional 12 sporadic medulloblastomas. RESULTS: The authors detected a new nonsense germline mutation in a child with Greig syndrome and medulloblastoma. This mutation generates a stop codon in position 809 of GLI3 that has been predicted to result in massive truncation of the protein. Several new polymorphisms, but no tumor-associated mutations, were found in sporadic tumors. CONCLUSIONS: Gli3 is mutated rarely in medulloblastoma.
Abstract: Advances in the fields of molecular genetics and cell biology are transforming medicine. Discoveries made today in the laboratory are translated at a rapid pace into new diagnostics and therapeutics. The aim of this series of review on different aspects of molecular medicine is to update physicians on new advances at the bench that are likely to impact bedside medicine.
Abstract: Holoprosencephaly (HPE) is the most common congenital malformation of the brain and face in humans. In this study we report the analysis of SIL (Sumacr;CL iumacr;nterrupting lumacr;ocus) as a candidate gene for HPE. Fluorescent in situ hybridization (FISH) analysis using a BAC 246e16 confirmed the assignment of SIL to 1p32. Computational analysis of SIL at the protein level revealed a 73% overall identity between the human and murine proteins. Denaturing high performance liquid chromatography (dHPLC) techniques were used to screen for mutations and these studies identified several common polymorphisms but no disease-associated mutations, suggesting that SIL is not a common factor in HPE pathogenesis in humans.
Abstract: The Sil gene encodes a cytosolic protein required for mouse embryonic midline and left/right axial development. Based on the phenotype of Sil mutant embryos, we hypothesized that Sil may be required for the activity of Sonic Hedgehog (Shh), a secreted signaling molecule also critically important for the development of the embryonic axes and found mutated in multiple types of cancer. Here we tested the genetic interaction between Sil and the Shh pathway by generating and analyzing embryos carrying mutations in both Sil and Patched (Ptch), a Shh receptor that normally inhibits the signaling pathway in the absence of ligand and when mutated leads to constitutive activation of the pathway. We find that Sil(-/-) Ptch(-/-) embryos do not activate the Shh pathway and instead have a phenotype indistinguishable from Sil(-/-) embryos, in which there is a loss of activity of Shh. These results provide genetic evidence that Sil is an essential component of the Shh response, acting downstream to Ptch.
Abstract: A physiological strain index (PSI) based on rectal temperature (Tre) and heart rate (HR) was recently suggested to evaluate exercise-heat stress. The purpose of this study was to evaluate PSI for gender differences under various combinations of exercise intensity and climate. Two groups of eight men each were formed according to maximal rate of O2 consumption (VO2 max). The first group of men (M) was matched to a group of nine women (W) with similar (P > 0.001) VO2 max (46.1 +/- 2.0 and 43.6 +/- 2.9 ml. kg-1. min-1, respectively). The second group of men (MF) was significantly (P < 0. 001) more fit than M or W with VO2 max of 59.1 +/- 1.8 ml. kg-1. min-1. Subjects completed a matrix of nine experimental combinations consisting of three different exercise intensities for 60 min [low, moderate, and high (300, 500, and 650 W, respectively)] each at three climates (comfortable, hot wet, and hot dry [20 degrees C 50% relative humidity (RH), 35 degrees C 70% RH, and 40 degrees C 35% RH, respectively]). No significant differences (P > 0.05) were found between matched genders (M and W) at the same exposure for sweat rate, relative VO2 max (%VO2 max), and PSI. However, MF had significantly (P < 0.05) lower strain than M and W as reflected by %VO2 max and PSI. In summary, PSI applicability was extended for exercise-heat stress and gender. This index continues to show potential for wide acceptance and application.
Abstract: The establishment of the main body axis and the determination of left-right asymmetry are fundamental aspects of vertebrate embryonic development. A link between these processes has been revealed by the frequent finding of midline defects in humans with left-right anomalies. This association is also seen in a number of mutations in mouse and zebrafish, and in experimentally manipulated Xenopus embryos. However, the severity of laterality defects accompanying abnormal midline development varies, and the molecular basis for this variation is unknown. Here we show that mouse embryos lacking the early-response gene SIL have axial midline defects, a block in midline Sonic hedgehog (Shh) signalling and randomized cardiac looping. Comparison with Shh mutant embryos, which have axial defects but normal cardiac looping, indicates that the consequences of abnormal midline development for left-right patterning depend on the time of onset, duration and severity of disruption of the normal asymmetric patterns of expression of nodal, lefty-2 and Pitx2.
Abstract: The SIL gene was discovered at the site of a cancer-associated interstitial deletion in which its promoter assumed the regulation of a second gene, SCL. The human SIL gene encodes a 1287-amino acid cytosolic protein that has been found to be highly conserved in the mouse. SIL is expressed in proliferating cells and is down-regulated when cellular proliferation ceases because of serum starvation, contact inhibition, or induction of terminal differentiation. SIL is induced within 1 h of stimulation by 20% serum in growth-arrested 3T3 cells. This induction is independent of protein synthesis because "superinduction" is observed in the presence of the protein synthesis inhibitor cyclohexamide. Thus, SIL is an immediate-early gene. Upon release from serum starvation of 3T3 fibroblasts, SIL mRNA and protein levels display a biphasic pattern during the first cell cycle. In contrast, in exponentially growing EL4 lymphoblasts, SIL mRNA is stable throughout the cell cycle, whereas SIL protein accumulates into G2 phase and then falls precipitously at the completion of the cell cycle. This pattern of cell cycle expression suggests that SIL may play an important role in cellular growth and proliferation.
Abstract: BACKGROUND: Pulmonary complications occur commonly during HIV infection. The aim of this study was to evaluate the clinical value of lung tissue examination in the diagnosis and treatment of pulmonary disorders in children with HIV infection. METHODS: The medical records of 347 children enrolled between January, 1990, and April, 1994, into various antiretroviral therapy protocols were reviewed to identify patients who underwent a lung biopsy. RESULTS: Fourteen patients underwent diagnostic lung biopsies on 16 separate occasions. The most common radiologic findings were nodular infiltrates which were localized in 7 patients and diffuse in 6. Eight patients presented with fever and progressive respiratory distress unresponsive to empiric therapy, whereas the rest had progressive nodular infiltrates. The pathologic diagnoses included opportunistic infection in 7 patients, lymphocytic interstitial pneumonitis in 5, non-Hodgkin's lymphoma in 3 and interstitial fibrosis in 1. The biopsy led to a major change in the treatment of 7 patients which resulted in a significant improvement of the pulmonary process in all of them. In an additional patient the excisional biopsy proved curative. CONCLUSIONS: When patients are selected appropriately, lung biopsy might have a significant impact on therapy and outcome in HIV-infected children with pulmonary infiltrates.
Abstract: The potential of low dose (30 mg t.i.d) pyridostigmine to reduce the ocular side effects of double dose transdermal controlled release hyoscine was evaluated by the study of near visual acuity, accommodation amplitude and pupil diameter in a placebo controlled, double masked study. We studied 47 healthy men (age 18-21 yr) in 3 groups: 16 assigned to placebo hyoscine and placebo pyridostigmine, 15 assigned to double dose hyoscine and placebo pyridostigmine, and 16 to double dose hyoscine and pyridostigmine. Subjects were tested during 48 h of treatment and 48 h of washout period. Blood cholinesterase inhibition level and amount of hyoscine released from the patches were used as parameters of reliability. Difference between groups was assessed using change from baseline scores. Double dose hyoscine caused decrease in near visual acuity to a mean of 14/18. Accommodation amplitude was decreased in the double dose transdermal hyoscine group from 9.19 +/- 1.04 to 4.83 +/- 1.97 diopters of accommodation. This decrease was significant when compared to the placebo group (p < 0.05) and to the pyridostigmine-protected group (p < 0.05). Pyridostigmine, however, did not significantly change the hyoscine-induced mydriasis of 1.47 + 0.15 mm change from baseline (p < 0.05). These results suggest that pyridostigmine administration may be beneficial in shortening recovery time when near vision impairment is experienced following single and double dose transdermal hyoscine administration.
Abstract: BACKGROUND. Ifosfamide has been associated with proximal renal tubular dysfunction resembling Fanconi-like syndrome and leading to rickets in young children. The characteristic manifestations of this nephrotoxicity include phosphaturia and hypophosphatemia, glycosuria, aminoaciduria, renal tubular acidosis, and urinary loss of low molecular weight serum proteins. However, the relationship between acute ifosfamide nephrotoxicity, which is frequently subclinical, and long term renal damage is unclear. In this prospective study, the laboratory features of ifosfamide-induced acute nephrotoxicity were characterized further and correlated with the development of chronic nephropathy. METHODS. The renal function of newly diagnosed children and young adults with high risk sarcomas was followed during therapy with a high dose ifosfamide-containing regimen. Serum and urine were collected regularly immediately before and after 5-day cycles of ifosfamide throughout treatment for determination of the fractional excretion of electrolytes (sodium, potassium, phosphate, magnesium, calcium) and glucose and urinary excretion of amino acids and beta 2-microglobulin. RESULTS. Significant changes in the renal threshold of phosphate excretion, the fractional excretion of calcium and glucose, and the urinary excretion of beta 2-microglobulin were observed when comparing pretreatment values with those at the end of a 5-day treatment cycle. The median renal threshold of phosphate excretion decreased from 1.22 to 0.82 mmol/L (P < 0.0001). The median fractional excretions of calcium and glucose increased from 1.05% to 1.68% (P < 0.0001) and 0.05% to 0.08% (P = 0.0006), respectively. Beta 2-microglobulin excretion increased by 70-fold from 0.02 to 1.42 mg/mmol (P < 0.0001). Except for glucose and beta 2-microglobulin excretion, renal parameters returned to baseline before the next ifosfamide treatment cycle. Acute aminoaciduria was observed in 21 of 23 patients. Chronic nephrotoxicity, as defined by the development of a Fanconi-like syndrome or chronic tubular electrolyte loss requiring oral supplementation, developed in the three patients with the highest urinary excretion of beta 2-microglobulin after ifosfamide therapy. CONCLUSIONS. Prospectively, high dose ifosfamide was associated with a 4% incidence of Fanconi-like syndrome; however, evidence of acute reversible subclinical nephrotoxicity was observed for all patients. Severe beta 2-microglobulinuria appeared to be a prognostic laboratory indicator for the development of chronic nephrotoxicity.
Abstract: Acute pancreatitis is an uncommon but serious complication of cancer chemotherapy. A 16-year-old girl with metastatic osteosarcoma experienced recurrent bouts of symptomatic pancreatitis 24 hours after treatment with ifosfamide administered as a single agent. To the authors' knowledge, this is the first report of an association between ifosfamide and pancreatitis. Because serum amylase levels are not monitored routinely during treatment with most chemotherapeutic agents, subclinical cases of pancreatitis with ifosfamide and other agents may go undetected.
Abstract: Elevated blood lactate levels that declined to normal after erythrocyte transfusion were observed in 17 of 37 otherwise healthy infants with anemia of prematurity (26.1 +/- 2.1 mg/dl vs 12.3 +/- 0.9 mg/dl; p < 0.001). Posttransfusion heart rate in this group decreased from 155 +/- 1 beats/min to 150 +/- 2 beats/min (p = 0.01). Blood lactate concentration may be a predictor of the need for transfusion in anemia of prematurity.
Abstract: The E2A/PBX1 and the BCR/ABL fusion genes result from the t(1;19)(q23;p13) and the t(9;22)(q34;q11), respectively, and encode oncoproteins which are thought to play an important role in the development of acute lymphoblastic leukemia (ALL) subtypes associated with adverse prognosis. The use of the polymerase chain reaction (PCR) for the detection of these genetic rearrangements may offer advantages over cytogenetic techniques which are often unsatisfactory in patients with ALL and, furthermore, provide a useful tool for monitoring of residual disease. However, it has not yet been evaluated whether the employment of PCR at the time of diagnosis improves the detection rate of these clinically relevant genetic anomalies. We have developed a multiprimer-PCR protocol which facilitates the detection of each of the four chimeric E2A/PBX1 and BCR/ABL mRNAs in a single reaction. This protocol was used for the evaluation of bone-marrow or blood samples from 251 children with ALL in whom cytogenetic analyses had been performed. Of the 251 patients, 221 had a B-cell precursor immunophenotype. In this group, 21 patients (9.5%) carrying the E2A/PBX1 rearrangement and three patients (1.4%) with BCR/ABL transcripts were detected by PCR. Twelve of these cases had escaped the detection by conventional cytogenetic analysis. In two of 12 patients with a typical t(1;19)(q23;p13), no E2A/PBX1 transcripts were identified by PCR, thus suggesting the presence of different molecular rearrangements. Residual leukemic cells were detected by PCR in five of eight patients who were followed during complete clinical remission. The frontline use of PCR has an important impact on the timely diagnosis, therapeutic decisions, and monitoring of high-risk patients with B-cell precursor leukemia who carry the E2A/PBX1 or BCR/ABL fusion genes.
Abstract: The translocation t(1;19)(q23;p13) in pediatric patients with acute lymphoblastic leukemia (ALL) was demonstrated in early reports to result in a consistent fusion between the E2A and PBX1 gene sequences and in the expression of a uniform, chimeric E2A/PBX1 mRNA with transforming potential. More recent observations suggested that cytogenetically identical t(1;19) translocations can result in the transcription of different mRNA species and that expression of the E2A/PBX1 message may be restricted to patients with the t(1;19) who display a pre-B phenotype of ALL. To further assess the correlation between the immunologic subtypes of the disease and specific genetic alterations, we have performed cytogenetic and molecular analyses in 221 children with B-lineage ALL. Expression of the chimeric E2A/PBX1 message was detected in 21 patients. Out of 14 patients, in whom cytoplasmic immunoglobulin (cig) analyses were available, no less than four had a cig- B-cell precursor ALL, whereas 10 displayed a cig+ B-ALL immunophenotype. These findings are at variance with a recent report in which expression of the E2A/PBX1 message was linked exclusively to a subset of patients who displayed a cig+ pre-B-cell precursor phenotype of ALL. In seven cases diagnosed before 1986, cig analyses were not available, and consequently E2A/PBX1 expression could not be correlated to a particular immunological subtype of B-cell precursor ALL. Our results have important implications for the detection of residual disease in pediatric patients where expression of the typical E2A/PBX1 mRNA may occur both in cig+ (pre-B) and cig- (early pre-B) immunologic subtypes of ALL.
Abstract: Spectral analysis of heart rate fluctuations was used to investigate the role of the autonomic nervous system in the pathogenesis of vasovagal syncope. Nine adolescents with a history of at least three episodes of vasovagal syncope and nine age-matched healthy controls were studied. All subjects were tested in supine position and at a 60 degrees inclination for 60 min or less if syncope developed. Blood pressure and heart rate were measured, while the ECG and respiration traces were recorded on magnetic tape for later spectral analysis. Baseline heart rate was lower in control subjects than in patients, increased with tilt in both groups, and remained lower in the control subjects throughout the experiment. Baseline systolic and diastolic blood pressure was similar in both groups. Diastolic blood pressure initially increased with tilt in all subjects and decreased significantly thereafter in patients. Pulse pressure was lower in patients throughout the experiment. The heart rate power spectra displayed a higher baseline level of low frequency fluctuations in the control group. The high frequency fluctuations component was similar in all subjects. The results of the test, regarding haemodynamic parameters and autonomic control of the heart rate, as expressed by low and high frequency fluctuations, are consistent with a reduced sympathetic reserve in the individuals with previous episodes of syncope.
Abstract: Congenital defect of the muscular layer of the small intestine is a rare cause of spontaneous bowel perforation in premature infants. During the last 12 years we have observed four similar cases. We describe the most recent one, a premature infant who developed two abdominal events. On her 2nd day of life, spontaneous perforation of the distal ileum due to focal absence of the muscular layer occurred. Several weeks later she developed the typical clinical and histological picture of necrotizing enterocolitis. The clinical and histological characteristics of the two different conditions are compared, and the 24 cases reported in the literature are discussed. We conclude that focal absence of intestinal musculature may be not such a rare entity as is commonly believed.
Abstract: 1. The value of low dosage of pyridostigmine (30 mg three times daily) in preventing peripheral anti-muscarinic side effects of a transdermal controlled-release formulation of hyoscine, was tested in a double-blind placebo-controlled study, involving 47 healthy subjects. 2. Salivary excretion was repeatedly measured during 48 h of combined therapy of two transdermal hyoscine patches with pyridostigmine and 14 h after its cessation. Blood acetylcholinesterase activity was also measured, serving as an index of pyridostigmine bioavailability. 3. The adjunctive therapy with pyridostigmine was highly effective in preventing the substantial impairment in salivary flow caused by the transdermal formulation. An associated 23% inhibition of blood acetylcholinesterase activity was observed. 4. Small doses of pyridostigmine may therefore have a role in increasing the tolerability of transdermal hyoscine therapy. In some patients this drug combination might also allow some increment of the hyoscine dose.
Abstract: The power spectrum of instantaneous heart rate fluctuations was used to determine the optimal doses of atropine that induce a maximal vagolytic or vagomimetic effect. In a crossover placebo controlled study, eight volunteers received increasing bolus doses of intravenous atropine (0.1 to 2.3 mg per subject) or placebo, and frequency bands of the power spectrum were integrated. During atropine administration a significant bimodal dose dependence was observed for the respiratory peak (0.2 to 0.4 Hz, p = 0.0006), the midfrequency band (0.09 to 0.15 Hz, p = 0.0035), and mean heart rate (p < 0.0001). Low doses (< 0.4 mg per subject) increased the respiratory and midfrequency band power, with maximal response at 0.2 mg per subject. Larger doses of atropine, 0.5 to 2.3 mg per subject, markedly reduced the power in all frequency bands in a dose-dependent way. The corresponding changes in mean heart rate were simultaneous, but in the opposite direction. We suggest that the respiratory peak of the power spectrum can be used to optimize drug effects on cardiac parasympathetic control.
Abstract: The polymerase chain reaction (PCR) has become a standard method for highly sensitive detection of the bcr/abl rearrangement in patients with chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). The exquisite sensitivity of the PCR facilitates the detection of residual leukemic cells after chemotherapy or after bone marrow transplantation. However, the detection of minimal residual disease does not yield any information on the malignant potential of the bcr/abl-rearranged cells. Qualitative PCR is therefore of limited prognostic value in the monitoring of residual leukemia. We have adapted the PCR for quantitative evaluation of cells carrying the bcr/abl rearrangement and by means of two exemplary cases of CML patients after bone marrow transplantation and under treatment with alpha-interferon, respectively, we show that this new technique is suitable for the long term follow-up of the activity of the residual bcr/abl rearranged clone. Longitudinal monitoring of residual disease by the technique presented provides a novel tool for detection of incipient relapse at a very early stage.
Abstract: The t(1;19)(q23;p13) is the most common recurring chromosomal translocation in childhood acute lymphoblastic leukemia (ALL) and has been associated with adverse prognosis. It involves the rearrangement of two genes, PBX1 and E2A, resulting in the production of transforming chimeric DNA-binding proteins. In all previous reports in which the presence of a chimeric transcript was described, the fusion point between the coding sequences of E2A and PBX1 was found to be constant at the RNA level. We have used RNA-based polymerase chain reaction (PCR) for the detection of E2A/PBX1 messenger RNAs (mRNAs) in children with ALL at the time of diagnosis. Of 21 patients exhibiting this rearrangement, 3 (14%) expressed a variant E2A/PBX1 transcript in addition to the expected one. The relative amounts of the two chimeric mRNAs varied between the patients, but remained constant in the same patient during different stages of the disease. Sequence analysis showed an identical insertion of 27 bp at the E2A/PBX1 junction of the variant RNA species, the translation of which would result in the replacement of Val478 by 10 amino acids. The inserted sequence has not been detected in any other human transcript besides the variant E2A/PBX1 RNA species and probably represents a splicing variant of the chimeric RNA. We conclude that a subset of pediatric patients with ALL that carry the E2A/PBX1 rearrangement express two types of the chimeric mRNA. The biologic significance of this additional E2A/PBX1 transcript is discussed.
Abstract: The syndrome of Albright hereditary osteodystrophy (AHO), pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) is clinically and genetically heterogeneous. Classically, patients with PHP have the skeletal features of AHO, resistance to multiple hormones that work via cAMP such as parathyroid hormone and thyroid stimulating hormone, and deficient activity of Gs protein, the guanine nucleotide-binding protein that stimulates adenylate cyclase. However, patients without hormone resistance but with AHO and Gs deficiency were described (PPHP), as well as patients with multiple hormone resistance but without AHO or Gs deficiency. In a few patients with deficient Gs activity, hypothyroidism rather than hypocalcemia was the initial presentation of the disorder. We describe here a new variant of the syndrome, affecting 5 individuals in a 3 generation family with AHO, normal Gs activity and hypothyroidism. In the first 2 generations, mild features of AHO were present. The 2 sibs in the third generation had severe manifestations of AHO, including mild mental retardation as well as hypothyroidism. Diagnosis of congenital osteoma cutis at birth of the proband led to the diagnosis of the family. Elucidation of the molecular defect will shed light on the relationship between hormone resistance and AHO, as well as on the physiological mechanism of hormonal signal transduction.
Abstract: The interaction between a low-dose cholinesterase inhibitor, pyridostigmine (PYR), and atropine was investigated by spectral analysis of heart rate fluctuations in eight healthy humans. Each subject was given increasing boluses of IV atropine during treatment with PYR (30 mg.3/day) or placebo. PYR attenuated the bimodal dose-dependent changes in the respiratory peak (which respresents the parasympathetic control) in response to atropine. We suggest that spectral analysis can be used for quantifying the complex dose-dependent cholinergic agonist-antagonist interactions, and may help to disclose an asymptomatic low-dose intoxication with acetylcholinesterase inhibitors.
Abstract: The autonomic cardiac control was studied as a sensitive parameter of anticholinergic treatment in humans, using heart-rate (HR) power spectrum. A cross-over placebo controlled study was performed in 8 young volunteers who received increasing bolusdoses of IV atropine (from 1.3 micrograms/kg to 29.9 micrograms/kg) or placebo. Computing the HR power spectrum and integrating over specific frequency bands, we focused in particular on the respiratory frequency band (usually between 0.2-0.4 Hz) which is purely of vagal mediation. At small atropine doses (less than 5.2 micrograms/kg), the respiratory peak increased, relative to baseline, with maximal response at 2.6 micrograms/kg (from 1.0 to 1.9 +/- 0.9). Larger doses of atropine (greater than or equal to 6.5 micrograms/kg) reduced the power of the respiratory peak, by a few orders of magnitude, in a dose-dependent way. Corresponding changes were observed in mean HR but in the opposite direction i.e., a maximal bradycardia at 2.6 micrograms/kg and a nearly two fold increase in mean HR at 29.9 micrograms/kg. We conclude that atropine has a bimodal dose-dependent effect on parasympathetic cardiac control. Since the use of HR spectral analysis has been demonstrated in various animal species, we suggest that it can be used as a sensitive noninvasive probe for animal to man transformation studies.
Abstract: The genital mycoplasmas: Ureaplasma urealyticum and Mycoplasma hominis have recently assumed an increasing importance as neonatal pathogens. The aim of the present survey was to determine the prevalence of infections with these organisms in preterm infants in two neonatal intensive care units in Israel. Among 99 preterm infants, 24 (24%) harboured mycoplasmas in their throats shortly after birth. U. urealyticum was the most common organism. M. hominis was isolated only from 3 infants. Six out of 27 (22%) mechanically ventilated infants secreted U. urealyticum in their lower airways. The rate of colonization was inversely correlated with gestational age; 80% of infants younger than 28 weeks gestation were found to be colonized as opposed to 17.9% at 28-36 weeks of gestation. No mycoplasmas were isolated in blood cultures drawn from 146 infants and CSF cultures obtained from 47 preterm infants. Neonatal mortality, respiratory complications and intraventricular haemorrhage grade 3-4 were significantly increased in colonized infants. However, above gestational age of 27 weeks, colonization with mycoplasmas was not associated with a worse prognosis. We conclude that colonization with U. urealyticum is common in Israeli preterm infants, correlates inversely with gestational age and has no detrimental effect on neonatal morbidity and mortality of infants older than 27 wks of gestation.
Abstract: The effect of repeated doses of 30 mg pyridostigmine bromide every 8 h on flight skills in an A-4 simulator was tested in this crossover double-blind placebo-controlled study on 10 pilots experienced in actual and simulated A-4 flights. The pilots flew two test simulator flights 2 h after the fourth dose of pyridostigmine or placebo. The flight profile included navigation, rapid ascent, 360 degrees turns, and instrument landing. Each flight lasted approximately 20 min. Flight parameters measured included indicated air speed, true heading, barometric altitude, vertical velocity, and bank. The mean whole blood cholinesterase inhibition level was 29%. There was no decrement in performance under treatment with pyridostigmine in the percent of deviation time from the prescribed limits or in the average duration or magnitude of the deviation in each of the flight parameters. We conclude that pyridostigmine bromide in repeated doses of 30 mg every 8 h does not appear to influence pilot performance during short A-4 missions.
Abstract: We studied urinary acidification daily during the hospital course of 16 infants with acute gastroenteritis and metabolic acidosis. Urine pH value on admission was higher than 5.5 in 14 (87%) patients. We hypothesized that inappropriate urinary acidification was due to sodium deficiency and inadequate sodium delivery to the distal nephron. Forty-one urinary samples were collected during metabolic acidosis. The mean pH of 24 urine samples with sodium concentration less than 10 mmol/L was significantly higher than the pH of 17 samples with sodium concentration greater than 10 mmol/L (6.04 +/- 0.06 vs 5.19 +/- 0.1; p less than 0.001). The urine ratios of titratable acid to creatinine and of total acidity to creatinine were significantly higher in urine samples containing more sodium (p less than 0.02), whereas the ammonium/creatinine ratio was not. After administration of furosemide or correction of the sodium deficit, appropriate acidification was observed. We conclude that impaired urinary acidification is frequently found during metabolic acidosis in infants with acute gastroenteritis and results from a sodium deficit rather than from transient distal renal tubular acidosis.
Abstract: A 9-year-old boy developed severe positional vertigo, unsteadiness, vomiting and nystagmus during active mumps parotitis. Audiometric examination showed complete right sensorineural hearing loss. Brain stem evoked response to audiometric stimulation of the left ear was normal, but was absent on stimulation of the right ear. CT scan of the brain was normal. While vestibular symptoms gradually improved during 1 week, the deafness on the right was permanent. Acute vestibulitis during mumps is very rare, but permanent unilateral deafness might be a much more common, severe, persistent complication than is generally believed. Such cases call for an urgent change in Israel's vaccination policy so that vaccination against mumps is included.
Abstract: Time of useful consciousness (TUC) was determined in 17 subjects exposed twice to 25,000 ft (7,620 m) in an altitude chamber. The criterion for TUC determination was inability to add two-digit numbers correctly. Median values of TUC were 267.5 seconds (s) in the first exposure and 240 s in the second. The intraindividual variability between the two exposures was 40.6 s. The probability of remaining in "useful" consciousness as a function of time at 25,000 ft (7,620 m) was similar in both exposures. The need for a more scientific approach towards the determination of time of useful consciousness in simulated high altitudes is raised.
Abstract: Data related to risk factors for catheter-acquired bacteriuria were collected prospectively on 112 patients consecutively catheterized for greater than 24 hours at the Hadassah University Hospital. Logistic regression analysis indicated that factors independently associated (p less than or equal to 0.05) with a higher risk of catheter-acquired bacteriuria were as follows: hospitalization in orthopedics or urology, ethnic origin (Arabs greater than Jews), insertion of a catheter after the sixth day of hospitalization, catheterization outside the operating theatres, lack of administration of systemic antibiotics, unsatisfactory catheter care, and prolonged duration (greater than or equal to 7 days) of catheterization before infection occurred. The risk associated with catheterization outside the operating theater could be explained by its correlate, that is, catheterization for incontinence/obstruction as opposed to output measurement. Life-table analyses demonstrated that the daily risk for acquiring bacteriuria during the first six days of catheterization was higher among patients ultimately catheterized for greater than or equal to 7 days than among those ultimately catheterized for less than 7 days (P less than 0.05).
