The genomic landscape of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

Disruption to the FOXO-PRDM1 axis resulting from deletions of chromosome 6 in acute lymphoblastic leukaemia.

A common problem in the study of human malignancy is the elucidation of cancer driver mechanisms associated with recurrent deletion of regions containing multiple genes. Taking B-cell acute lymphoblastic leukaemia (B-ALL) and large deletions of 6q [del(6q)] as a model, we integrated analysis of functional cDNA clone tracking assays with patient genomic and transcriptomic data, to identify the transcription factors FOXO3 and PRDM1 as candidate tumour suppressor genes (TSG). Analysis of cell cycle and transcriptomic changes following overexpression of FOXO3 or PRDM1 indicated that they co-operate to promote cell cycle exit at the pre-B cell stage. FOXO1 abnormalities are absent in B-ALL, but like FOXO3, FOXO1 expression suppressed growth of TCF3::PBX1 and ETV6::RUNX1 B-ALL in-vitro. While both FOXOs induced PRDM1 and other genes contributing to late pre-B cell development, FOXO1 alone induced the key transcription factor, IRF4, and chemokine, CXCR4. CRISPR-Cas9 screening identified FOXO3 as a TSG, while FOXO1 emerged as essential for B-ALL growth. We relate this FOXO3-specific leukaemia-protective role to suppression of glycolysis based on integrated analysis of CRISPR-data and gene sets induced or suppressed by FOXO1 and FOXO3. Pan-FOXO agonist Selinexor induced the glycolysis inhibitor TXNIP and suppressed B-ALL growth at low dose (ID50 < 50 nM).

hiPSC-derived bone marrow milieu identifies a clinically actionable driver of niche-mediated treatment resistance in leukemia.

Leukemia cells re-program their microenvironment to augment blast proliferation and enhance treatment resistance. Means of clinically targeting such niche-driven treatment resistance remain ambiguous. We develop human induced pluripotent stem cell (hiPSC)-engineered niches to reveal druggable cancer-niche dependencies. We reveal that mesenchymal (iMSC) and vascular niche-like (iANG) hiPSC-derived cells support ex vivo proliferation of patient-derived leukemia cells, affect dormancy, and mediate treatment resistance. iMSCs protect dormant and cycling blasts against dexamethasone, while iANGs protect only dormant blasts. Leukemia proliferation and protection from dexamethasone-induced apoptosis is dependent on cancer-niche interactions mediated by CDH2. Consequently, we test CDH2 antagonist ADH-1 (previously in Phase I/II trials for solid tumors) in a very aggressive patient-derived xenograft leukemia mouse model. ADH-1 shows high in vivo efficacy; ADH-1/dexamethasone combination is superior to dexamethasone alone, with no ADH-1-conferred additional toxicity. These findings provide a proof-of-concept starting point to develop improved, potentially safer therapeutics targeting niche-mediated cancer dependencies in blood cancers.

SH2B3 inactivation through CN-LOH 12q is uniquely associated with B-cell precursor ALL with iAMP21 or other chromosome 21 gain.

In more than 30% of B-cell precursor acute lymphoblastic leukaemia (B-ALL), chromosome 21 sequence is overrepresented through aneuploidy or structural rearrangements, exemplified by intrachromosomal amplification of chromosome 21 (iAMP21). Although frequent, the mechanisms by which these abnormalities promote B-ALL remain obscure. Intriguingly, we found copy number neutral loss of heterozygosity (CN-LOH) of 12q was recurrent in iAMP21-ALL, but never observed in B-ALL without some form of chromosome 21 gain. As a consequence of CN-LOH 12q, mutations or deletions of the adaptor protein, SH2B3, were converted to homozygosity. In patients without CN-LOH 12q, bi-allelic abnormalities of SH2B3 occurred, but only in iAMP21-ALL, giving an overall incidence of 18% in this sub-type. Review of published data confirmed a tight association between overrepresentation of chromosome 21 and both CN-LOH 12q and SH2B3 abnormalities in B-ALL. Despite relatively small patient numbers, preliminary analysis linked 12q abnormalities to poor outcome in iAMP21-ALL (p = 0.03). Homology modelling of a leukaemia-associated SH2 domain mutation and in vitro analysis of patient-derived xenograft cells implicated the JAK/STAT pathway as one likely target for SH2B3 tumour suppressor activity in iAMP21-ALL.

Dynamic clonal progression in xenografts of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

Intrachromosomal amplification of chromosome 21 is a heterogeneous chromosomal rearrangement occurring in 2% of cases of childhood precursor B-cell acute lymphoblastic leukemia. These abnormalities are too complex to engineer faithfully in animal models and are unrepresented in leukemia cell lines. As a resource for future functional and preclinical studies, we have created xenografts from the leukemic blasts of patients with intrachromosomal amplification of chromosome 21 and characterized them by in-vivo and ex-vivo luminescent imaging, flow immunophenotyping, and histological and ultrastructural analyses of bone marrow and the central nervous system. Investigation of up to three generations of xenografts revealed phenotypic evolution, branching genomic architecture and, compared with other B-cell acute lymphoblastic leukemia genetic subtypes, greater clonal diversity of leukemia-initiating cells. In support of intrachromosomal amplification of chromosome 21 as a primary genetic abnormality, it was always retained through generations of xenografts, although we also observed the first example of structural evolution of this rearrangement. Clonal segregation in xenografts revealed convergent evolution of different secondary genomic abnormalities implicating several known tumor suppressor genes and a region, containing the B-cell adaptor, PIK3AP1, and nuclear receptor co-repressor, LCOR, in the progression of B-cell acute lymphoblastic leukemia. Tracking of mutations in patients and derived xenografts provided evidence for co-operation between abnormalities activating the RAS pathway in B-cell acute lymphoblastic leukemia and for their aggressive clonal expansion in the xeno-environment. Bi-allelic loss of the CDKN2A/B locus was recurrently maintained or emergent in xenografts and also strongly selected as RNA sequencing demonstrated a complete absence of reads for genes associated with the deletions.

CREBBP knockdown enhances RAS/RAF/MEK/ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response.

Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP, are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors.

The ability to cross the blood-cerebrospinal fluid barrier is a generic property of acute lymphoblastic leukemia blasts.

Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

Constitutional and somatic rearrangement of chromosome 21 in acute lymphoblastic leukaemia.

Changes in gene dosage are a major driver of cancer, known to be caused by a finite, but increasingly well annotated, repertoire of mutational mechanisms. This can potentially generate correlated copy-number alterations across hundreds of linked genes, as exemplified by the 2% of childhood acute lymphoblastic leukaemia (ALL) with recurrent amplification of megabase regions of chromosome 21 (iAMP21). We used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. Here we show that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have approximately 2,700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by a chromothripsis event involving both sister chromatids of the Robertsonian chromosome, a novel mechanism for cancer predisposition. In sporadic iAMP21, breakage-fusion-bridge cycles are typically the initiating event, often followed by chromothripsis. In both sporadic and rob(15;21)c-associated iAMP21, the final stages frequently involve duplications of the entire abnormal chromosome. The end-product is a derivative of chromosome 21 or the rob(15;21)c chromosome with gene dosage optimized for leukaemic potential, showing constrained copy-number levels over multiple linked genes. Thus, dicentric chromosomes may be an important precipitant of chromothripsis, as we show rob(15;21)c to be constitutionally dicentric and breakage-fusion-bridge cycles generate dicentric chromosomes somatically. Furthermore, our data illustrate that several cancer-specific mutational processes, applied sequentially, can coordinate to fashion copy-number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.

The 2012 SAGE wait times program: Survey of Access to GastroEnterology in Canada.

BACKGROUND: Periodically surveying wait times for specialist health services in Canada captures current data and enables comparisons with previous surveys to identify changes over time. METHODS: During one week in April 2012, Canadian gastroenterologists were asked to complete a questionnaire (online or by fax) recording demographics, reason for referral, and dates of referral and specialist visits for at least 10 consecutive new patients (five consultations and five procedures) who had not been seen previously for the same indication. Wait times were determined for 18 indications and compared with those from similar surveys conducted in 2008 and 2005. RESULTS: Data regarding adult patients were provided by 173 gastroenterologists for 1374 consultations, 540 procedures and 293 same-day consultations and procedures. Nationally, the median wait times were 92 days (95% CI 85 days to 100 days) from referral to consultation, 55 days (95% CI 50 days to 61 days) from consultation to procedure and 155 days (95% CI 142 days to 175 days) (total) from referral to procedure. Overall, wait times were longer in 2012 than in 2005 (P<0.05); the wait time to same-day consultation and procedure was shorter in 2012 than in 2008 (78 days versus 101 days; P<0.05), but continued to be longer than in 2005 (P<0.05). The total wait time remained longest for screening colonoscopy, increasing from 201 days in 2008 to 279 days in 2012 (P<0.05). DISCUSSION: Wait times for gastroenterology services continue to exceed recommended targets, remain unchanged since 2008 and exceed wait times reported in 2005.

A survey of the practice of after-hours and emergency endoscopy in Canada.

OBJECTIVE: To determine staffing and practice patterns for after-hours endoscopy service in Canada. METHODS: A link to a web-based survey was sent by e-mail to all clinical members of the Canadian Association of Gastroenterology in February 2011. A priori, it was planned to compare variations in practice among gastroenterologists (GIs) performing endoscopy in different regions of Canada, between pediatric and adult GIs, and between university and community hospitals. RESULTS: Of 422 potential respondents, 168 (40%) responded. Of the 139 adult GIs, 61% performed after-hours endoscopy in the endoscopy suite where daytime procedures were performed, 62% had a trained endoscopy nurse available for all procedures, 38% had access to propofol sedation, 12% reprocessed the endoscopes themselves or with the help of a resident, 4% had out-of-hospital patients come directly to their endoscopy suite and 53% were highly satisfied. The adult endoscopists practising at community hospitals were more likely to have an anesthetist attend the procedure. Regional differences were noted, with more involvement of anesthetists (13%) and availability of propofol (50%) in Ontario, more frequent reprocessing of endoscopes in the central reprocessing units in British Columbia (78%) and almost universal availability of a trained endoscopy nurse (96%) with concomitant higher endoscopist satisfaction (84% highly satisfied) in Alberta. CONCLUSIONS: More than one-third of surveyed endoscopists across the country do not have a trained endoscopy nurse to assist in after-hours endoscopy - the time period when urgent patients often present and typically require therapeutic endoscopic interventions. There are significant regional differences in the practice of after-hours endoscopy in Canada.

Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that has a dismal outcome when treated with standard therapy. For improved diagnosis and risk stratification, the initiating genetic events need to be elucidated. To investigate the genetic basis of BCP-ALL, genomes of 94 iAMP21 patients were interrogated by arrays, FISH, and multiplex ligation-dependent probe amplification. Most copy number alterations targeted chromosome 21, reinforcing the complexity of this chromosome. The common region of amplification on chromosome 21 was refined to a 5.1-mb region that included RUNX1, miR-802, and genes mapping to the Down syndrome critical region. Recurrent abnormalities affecting genes in key pathways were identified: IKZF1 (22%), CDKN2A/B (17%), PAX5 (8%), ETV6 (19%), and RB1 (37%). Investigation of clonal architecture provided evidence that these abnormalities, and P2RY8-CRLF2, were secondary to chromosome 21 rearrangements. Patient outcome was uniformly poor with standard therapy irrespective of the presence or absence of these changes. This study has provided evidence that chromosome 21 instability is the only anomaly among those so far investigated that is common to all iAMP21 patients, and therefore the initiating event is likely to be found among the complex structural rearrangements of this abnormal chromosome.

Analysis of a breakpoint cluster reveals insight into the mechanism of intrachromosomal amplification in a lymphoid malignancy.

A distinct sub-group of B-cell precursor acute lymphoblastic leukemia, defined by intrachromosomal amplification of chromosome 21 (iAMP21), is restricted to older children and has been associated with a poor outcome. Accurate diagnosis is important for appropriate risk stratification for treatment. It could be improved by understanding the initiating mechanism. iAMP21 is characterized by amplification of a 5.1-24 Mb region of chromosome 21, which includes the RUNX1 gene. It is thought to arise through a breakage-fusion-bridge (BFB) mechanism. Breakpoints initiating BFB cycles were determined from recent array data from 18 patients. Three occurred within the PDE9A gene. Other patients with breakpoints in PDE9A were identified by fluorescence in situ hybridization and molecular copy number counting. Sequencing defined a 1.7 Kb breakpoint cluster region, positioned 400 bp distal to an extensive region enriched for CA repeats with the potential to form Z-DNA. None of the rearranged sequences showed the inverted repeat structure characteristic of BFB; instead PDE9A was fused to intergenic regions of chromosome 21 or to genes on other chromosomes. These observations indicated that previously unrecognized complex events, involving microhomology-mediated end joining, preceded or accompanied initiation of the BFB cycle. A chi-like heptomer, CCTCAGC, contained four of the breakpoints, two within PDE9A and two within partner Alu-repeat sequences. This heptomer was closely homologous to a breakpoint hotspot within the TCF3 gene, suggesting involvement of a common novel recombinogenic mechanism that might also contribute to the recombinogenic potential of Alu repeats. These findings provide insight into potential mechanisms involved in the formation of iAMP21.

Point-of-care, peer-comparator colonoscopy practice audit: The Canadian Association of Gastroenterology Quality Program--Endoscopy.

BACKGROUND: Point-of-care practice audits allow documentation of procedural outcomes to support quality improvement in endoscopic practice. OBJECTIVE: To evaluate a colonoscopists' practice audit tool that provides point-of-care data collection and peer-comparator feedback. METHODS: A prospective, observational colonoscopy practice audit was conducted in academic and community endoscopy units for unselected patients undergoing colonoscopy. Anonymized colonoscopist, patient and practice data were collected using touchscreen smartphones with automated data upload for data analysis and review by participants. The main outcome measures were the following colonoscopy quality indicators: colonoscope insertion and withdrawal times, bowel preparation quality, sedation, immediate complications and polypectomy, and biopsy rates. RESULTS: Over a span of 16 months, 62 endoscopists reported on 1279 colonoscopy procedures. The mean cecal intubation rate was 94.9% (10th centile 84.2%). The mean withdrawal time was 8.8 min and, for nonpolypectomy colonoscopies, 41.9% of colonoscopists reported a mean withdrawal time of less than 6 min. Polypectomy was performed in 37% of colonoscopies. Independent predictors of polypectomy included the following: endoscopy unit type, patient age, interval since previous colonoscopy, bowel preparation quality, stable inflammatory bowel disease, previous colon polyps and withdrawal time. Withdrawal times of less than 6 min were associated with lower polyp removal rates (mean difference -11.3% [95% CI -2.8% to -19.9%]; P=0.01). DISCUSSION: Cecal intubation rates exceeded 90% and polypectomy rates exceeded 30%, but withdrawal times were frequently shorter than recommended. There are marked practice variations consistent with previous observations. CONCLUSION: Real-time, point-of-care practice audits with prompt, confidential access to outcome data provide a basis for targeted educational programs to improve quality in colonoscopy practice.

A one-year economic evaluation of six alternative strategies in the management of uninvestigated upper gastrointestinal symptoms in Canadian primary care.

BACKGROUND: The cost-effectiveness of initial strategies in managing Canadian patients with uninvestigated upper gastrointestinalsymptoms remains controversial. OBJECTIVE: To assess the cost-effectiveness of six management approaches to uninvestigated upper gastrointestinal symptoms in the Canadian setting. METHODS: The present study analyzed data from four randomized trials assessing homogeneous and complementary populations of Canadian patients with uninvestigated upper gastrointestinal symptoms with comparable outcomes. Symptom-free months, qualityadjusted life-years (QALYs) and direct costs in Canadian dollars of two management approaches based on the Canadian Dyspepsia Working Group (CanDys) Clinical Management Tool, and four additional strategies (two empirical antisecretory agents, and two prompt endoscopy) were examined and compared. Prevalence data, probabilities, utilities and costs were included in a Markov model, while sensitivity analysis used Monte Carlo simulations. Incremental cost-effectiveness ratios and cost-effectiveness acceptability curves were determined. RESULTS: Empirical omeprazole cost $226 per QALY ($49 per symptom-free month) per patient. CanDys omeprazole and endoscopy approaches were more effective than empirical omeprazole, but more costly. Alternatives using H2-receptor antagonists were less effective than those using a proton pump inhibitor. No significant differences were found for most incremental cost-effectiveness ratios. As willingness to pay (WTP) thresholds rose from $226 to $24,000 per QALY, empirical antisecretory approaches were less likely to be the most costeffective choice, with CanDys omeprazole progressively becoming a more likely option. For WTP values ranging from $24,000 to $70,000 per QALY, the most clinically relevant range, CanDys omeprazole was the most cost-effective strategy (32% to 46% of the time), with prompt endoscopy-proton pump inhibitor favoured at higher WTP values. CONCLUSIONS: Although no strategy was the indisputable cost effective option, CanDys omeprazole may be the strategy of choiceover a clinically relevant range of WTP assumptions in the initial management of Canadian patients with uninvestigated dyspepsia.

Survey of access to gastroenterology in Canada: the SAGE wait times program.

BACKGROUND: Assessment of current wait times for specialist health services in Canada is a key method that can assist government and health care providers to plan wisely for future health needs. These data are not readily available. A method to capture wait time data at the time of consultation or procedure has been developed, which should be applicable to other specialist groups and also allows for assessment of wait time trends over intervals of years. METHODS: In November 2008, gastroenterologists across Canada were asked to complete a questionnaire (online or by fax) that included personal demographics and data from one week on at least five consecutive new consultations and five consecutive procedure patients who had not previously undergone a procedure for the same indication. Wait times were collected for 18 primary indications and results were then compared with similar survey data collected in 2005. RESULTS: The longest wait times observed were for screening colonoscopy (201 days) and surveillance of previous colon cancer or polyps (272 days). The shortest wait times were for cancer-likely based on imaging or physical examination (82 days), severe or rapidly progressing dysphagia or odynophagia (83 days), documented iron deficiency anemia (90 days) and dyspepsia with alarm symptoms (99 days). Compared with 2005 data, total wait times in 2008 were lengthened overall (127 days versus 155 days; P<0.05) and for most of the seven individual indications that permitted data comparison. CONCLUSION: Median wait times for gastroenterology services continue to exceed consensus conference recommended targets and have significantly worsened since 2005.

International consensus recommendations on the management of patients with nonvariceal upper gastrointestinal bleeding.

DESCRIPTION: A multidisciplinary group of 34 experts from 15 countries developed this update and expansion of the recommendations on the management of acute nonvariceal upper gastrointestinal bleeding (UGIB) from 2003. METHODS: The Appraisal of Guidelines for Research and Evaluation (AGREE) process and independent ethics protocols were used. Sources of data included original and published systematic reviews; randomized, controlled trials; and abstracts up to October 2008. Quality of evidence and strength of recommendations have been rated by using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. RECOMMENDATIONS: Recommendations emphasize early risk stratification, by using validated prognostic scales, and early endoscopy (within 24 hours). Endoscopic hemostasis remains indicated for high-risk lesions, whereas data support attempts to dislodge clots with hemostatic, pharmacologic, or combination treatment of the underlying stigmata. Clips or thermocoagulation, alone or with epinephrine injection, are effective methods; epinephrine injection alone is not recommended. Second-look endoscopy may be useful in selected high-risk patients but is not routinely recommended. Preendoscopy proton-pump inhibitor (PPI) therapy may downstage the lesion; intravenous high-dose PPI therapy after successful endoscopic hemostasis decreases both rebleeding and mortality in patients with high-risk stigmata. Although selected patients can be discharged promptly after endoscopy, high-risk patients should be hospitalized for at least 72 hours after endoscopic hemostasis. For patients with UGIB who require a nonsteroidal anti-inflammatory drug, a PPI with a cyclooxygenase-2 inhibitor is preferred to reduce rebleeding. Patients with UGIB who require secondary cardiovascular prophylaxis should start receiving acetylsalicylic acid (ASA) again as soon as cardiovascular risks outweigh gastrointestinal risks (usually within 7 days); ASA plus PPI therapy is preferred over clopidogrel alone to reduce rebleeding.

Analysis of balanced rearrangements of chromosome 6 in acute leukemia: clustered breakpoints in q22-q23 and possible involvement of c-MYB in a new recurrent translocation, t(6;7)(q23;q32 through 36).

BACKGROUND AND OBJECTIVES: Many clinically important oncogenes and tumor suppressor genes have been identified through analysis of recurrent chromosomal rearrangements in acute leukemia. The contribution of sporadic rearrangements to malignancy is less clear and few have been mapped in detail. In this study we investigated the significance of novel translocations and inversions of 6q in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). DESIGN AND METHODS: Breakpoints of balanced 6q rearrangements were mapped in sequential fluorescent in situ hybridization (FISH) experiments with BAC and PAC clones in 11 patients. RESULTS: Six of seven breakpoints in ALL and two in a single case of AML were localized to within a 10.5 Mb hotspot at 6q22-q23 with five analyzed to the level of a single probe. In two cases of childhood T-ALL, both carrying a t(6;7)(q23;q32 through 36), split FISH signals were produced by adjacent PAC, mapping the breakpoints to within an approximately 150 Kb region containing the genes c-MYB and AHI1. Five similar rearrangements, four also in pediatric T ALL were identified in the literature. Other 6q22-q23 translocations mapped in detail interrupted regions containing no recognized genes. 6q breakpoints outside the q22-q23 region were widely dispersed and in two were mapped to positions overlapping the cloned fragile sites FRA6E and FRA6F. The involvement of MLL was demonstrated in one case with t(6;11)(q15;q23). INTERPRETATION AND CONCLUSIONS: We identified a new primary recurrent translocation t(6;7) (q22;q23 through q26) in pediatric T-ALL. Other translocations interrupting the 6q22-q23 breakpoint cluster region did not appear to be recurrent and may contribute to leukemogenesis through a novel mechanism. Key words; chromosome 6, translocation, c-Myb, AHI1.

A fluorescence in situ hybridization map of 6q deletions in acute lymphocytic leukemia: identification and analysis of a candidate tumor suppressor gene.

With the objective of identifying candidate tumor suppressor genes, we used fluorescence in situ hybridization to map leukemia-related deletions of the long arm of chromosome 6 (6q). Twenty of 24 deletions overlapped to define a 4.8-Mb region of minimal deletion between markers D6S1510 and D6S1692 within chromosome 6 band q16. Using reverse transcription-PCR, we found evidence of expression in hematopoietic cells for 3 of 15 genes in the region (GRIK2, C6orf111, and CCNC). Comparison between our own and published deletion data singled out GRIK2 as the gene most frequently affected by deletions of 6q in acute lymphocytic leukemia (ALL). Sequence analysis of GRIK2 in 14 ALL cases carrying heterozygous 6q deletions revealed a constitutional and paternally inherited C to G substitution in exon 6 encoding for an amino acid change in one patient. The substitution was absent among 232 normal alleles tested, leaving open the possibility that heterozygous carriers of such mutations may be susceptible to ALL. Although low in all normal hematopoietic tissues, quantitative reverse transcription-PCR showed higher baseline GRIK2 expression in thymus and T cells than other lineages. Among T-cell ALL patients, 6q deletion was associated with a statistically significant reduction in GRIK2 expression (P = 0.0001). By contrast, elevated GRIK2 expression was measured in the myelomonocytic line THP-1 and in one patient with common ALL. Finally, we detected significant levels of GRIK2 expression in prostate, kidney, trachea, and lung, raising the possibility that this gene may be protective against multiple tumor types.

Idiopathic arterial calcification in childhood.

Idiopathic arterial calcification in infancy is usually fatal with death in early life and diagnosis at post mortem. This report describes a unique, late presentation with hypertension and cardiac failure in a child aged 33 months, found to have widespread arterial calcification at radiological imaging. The calcium-phosphate axis was normal and there was no other demonstrable cause for calcification. Additionally, the histological features of arterial calcification at renal biopsy paralleled the findings in infants with this disorder. The late presentation in this case is unusual and has not been previously reported. Ultrasound and CT are sensitive for calcification, and the disease should be suspected in children presenting with cardiac or respiratory manifestations and features of arterial calcification, where no metabolic cause is established.