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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.

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.

Changes in gene dosage are a major driver of cancer, known to be caused by a finite, but increasingly well annotated, repertoire of mutational mechanisms1. 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)2, 3. 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.

This paper discusses changing patterns of resource utilisation over time in the locality of Chibuene, Vilankulos, situated on the coastal plain of southern Mozambique. The macroscopic charcoal, bone and shell assemblages from archaeological excavations are presented and discussed against the off-site palaeoecological records from pollen, fungal spores and microscopic charcoal. The Chibuene landscape has experienced four phases of land use and resource utilisation that have interacted with changes in the environment. Phase 1 (a.d. 400–900), forest savanna mosaic, low intensity cattle herding and cultivation, trade of resources for domestic use. Phase 2 (a.d. 900–1400), forest savanna mosaic, high intensity/extensive cultivation and cattle herding. Phase 3 (a.d. 1400–1800), savanna woodland and progressive decrease in forests owing to droughts. Decline of agricultural activities and higher reliance on marine resources. Possible trade of resources with the interior. Phase 4 (a.d. 1800–1900), open savanna with few forest patches. Warfare and social unrest. Collapse of trade with the interior. Decline in marine resources and wildlife. Loss of cattle herds. Expansion of agriculture locally and introduction of New World crops and clearing of Brachystegia trees. The study shows the importance of combining different environmental resources for elucidating how land use and natural variability have changed over time.

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.

The management of Helicobacter pylori negative patients with dyspepsia in primary care has not been studied in placebo-controlled studies.H. pylori negative patients with dyspepsia symptoms of at least moderate severity (or =4 on a seven-point Likert scale) were recruited from 35 centers. Patients were randomized to a 4-wk treatment of omeprazole 20 mg od, ranitidine 150 mg bid, cisapride 20 mg bid, or placebo, followed by on-demand therapy for an additional 5 months. Treatment success was defined as no or minimal symptoms (scoreor = 2 out of 7), and was assessed after 4 wk and at 6 months.Five hundred and twelve patients were randomized and included in the intention-to-treat (ITT) analysis. At 4 wk, success rates (95% CI) were: omeprazole 51% (69/135; 43-60%), ranitidine 36% (50/139, 28-44%), cisapride 31% (32/105, 22-39%), and placebo 23% (31/133, 16-31%). Omeprazole was significantly better than all other treatments (p0.05). The proportion of patients who were responders at 4 wk and at 6 months was significantly greater for those receiving omeprazole 31% (42/135, 23-39%) compared with cisapride 13% (14/105, 7-20%), and placebo 14% (18/133, 8-20%) (p= 0.001), but not ranitidine 21% (29/139, 14-27%) (p= 0.053). The mean number of on-demand study tablets consumed and rescue antacid used was comparable across groups. Economic analysis showed a trade-off between superior efficacy and increased cost between omeprazole and ranitidine.Treatment with omeprazole provides superior symptom relief compared to ranitidine, cisapride, and placebo in the treatment of H. pylori negative primary care dyspepsia patients.

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.

Background & Aims. The acid inhibitory effect of proton pump inhibitors is reported to be greater in the presence than in the absence of an H. pylori infection. This study was undertaken to test the hypothesis that the acid inhibitory effect of omeprazole given twice a day is greater in H. pylori infected healthy volunteers than in the same individuals following eradication because of differences in the pharmacodynamics of omeprazole, greater duodenogastric reflux, the effects of ammonia produced by the H. pylori, or lower gastric juice concentrations of selected cytokines, which may inhibit gastric acid secretion. Materials and Methods. We undertook 24-hour pH-metry in 12 H. pylori-positive healthy volunteers: (1) when on no omeprazole; (2) when on omeprazole 20 mg bid for 8 days; (3) 2 months after eradication of H. pylori and when on no omeprazole; and (4) after eradication of H. pylori and when on omeprazole 20 mg twice a day. Results. In subjects given omeprazole, eradication of H. pylori reduced pH and percentage pH ≥ 3, as well as increasing the area under the H+ concentration-time curve. These differences were not due to alterations in (1) gastric juice concentrations of IL-1α, IL-8, IL-13, epidermal growth factor, or bile acids; (2) serum gastrin concentrations; or (3) the pharmacokinetics of omeprazole. There was no change in the difference in the H+ concentration-time curve ‘without omeprazole’ minus ‘with omeprazole’, when comparing ‘after’ versus ‘before’ eradication of H. pylori. Conclusions. Eradication of H. pylori was not associated with an alteration in the acid inhibitory potency when comparing the difference in gastric acidity ‘with’ versus ‘without’ omeprazole. When the results were expressed by simply taking into account the acid measurements while on omeprazole before versus after eradication of H. pylori, the acid inhibition with omeprazole was greater in the presence than in the absence of a H. pylori infection. The clinical significance of the small difference is not clear.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of 2% childhood acute lymphoblastic leukaemia (ALL). iAMP21-ALL patients have precursor B-cell ALL, are older (median age 9 years), generally present with low white cell counts and have an inferior outcome when treated with standard therapy. Stratification to high risk treatment arms has significantly reduced their relapse risk, thus accurate diagnosis is essential. We have identified iAMP21 as a complex structure of one copy of chromosome 21, comprising multiple regions of gain, amplification, inversion and deletion, initiated through breakage-fusion-bridge cycles and chromothripsis. Currently, fluorescence in situ hybridisation (FISH), using probes directed to RUNX1, is the most reliable and convenient detection method, in which four or more copies of RUNX1 on a single abnormal chromosome 21 defines iAMP21. From our examination of several hundred cases of iAMP21-ALL, we have noted that, in the absence of metaphase FISH and/or in cases with an unusual cytogenetic presentation, reliance on FISH alone for accurate detection may be problematic. Among a collection of 210 patients with iAMP21-ALL, we performed SNP6.0 and Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA kit P327 iAMP21-ERG (MRC Holland) on 57 and 45 patients, respectively. Although chromosome 21 structure is highly variable between patients, a characteristic copy number profile emerged from the SNP6.0 data (Figure 1). In common, all cases were amplified across a 5.1Mb region, between 32,813,553 and 37,941,425bp that includes RUNX1 and 46 other known protein-coding genes. Asub-telomeric deletion occurred in 88% patients. We propose that this distinctive chromosome 21 copy number profile is used in addition to FISH for the definitive diagnosis of iAMP21-ALL in problematic cases. Support for this approach is provided by results from 5 patients from the Children's Oncology Group (North America, Australia and New Zealand) and ALL2003 (UK) treatment trials who met the iAMP21 FISH criteria but had atypical karyotypes or an unusual distribution of the additional RUNX1 signals that made confident diagnosis challenging. Clinical and cytogenetic data were collected and SNP6.0 and MLPA were performed to clarify the genomic alterations present in these cases. In patient #1, although five RUNX1 signals were observed per cell, two normal copies of chromosome 21 (each with one RUNX1 signal) were present with only three signals located to the abnormal chromosome 21. In patient #2, the additional signals were distributed between 2 different abnormal copies of chromosome 21. Metaphase FISH of patient #3 indicated that the RUNX1 signals were distributed between one normal copy of chromosome 21 and four small chromosomes, which were identified to originate from chromosome 21 by chromosome painting with a chromosome 21 specific probe (wcp21). In a further 2 cases (patients #4 and #5) the signals were too tightly clustered for the number to be discerned. In these 5 cases the characteristic SNP6.0 profile definitively confirmed the suspected diagnosis of iAMP21-ALL (Figure 2). Our previous SNP6.0 data have shown that the copy number profile of the iAMP21 chromosome remains stable between diagnosis and relapse, as well as in serial xenografts successfully transplanted with iAMP21-ALL cells over several generations of mice. However, while FISH analysis of patient #6 at diagnosis and relapse showed the same signal pattern at both time points, there was a change in the karyotype, involving translocation of part of the iAMP21 chromosome onto chromosome 11 at relapse. These observations indicate that the iAMP21 chromosome may become fragmented and distributed throughout the genome without changing its genomic profile. Collectively these observations indicate that the amplified segments resulting from the formation of iAMP21 chromosomes, with typical copy number profiles, can be distributed throughout the genome, either by translocation or fragmentation. Thus FISH together with chromosome 21 copy number profiling provide more accurate diagnosis of iAMP21-ALL than cytogenetics, which may be misleading. For laboratories with no access to SNP6.0 or other copy number arrays, we have shown that MLPA, with a kit specifically designed to detect chromosome 21 copy number changes, provides a reliable alternative (Figure 3). Disclosures No relevant conflicts of interest to declare.

The 8p11 myeloproliferative syndrome (EMS) is associated with three translocations, t(8;13)(p11;q12), t(8;9)(p11;q33), and t(6;8)(q27;p11), that fuse unrelated genes (ZNF198, CEP110, and FOP, respectively) to the entire tyrosine kinase domain of FGFR1. In all cases thus far examined (n = 10), the t(8;13) results in an identical mRNA fusion between ZNF198 exon 17 and FGFR1 exon 9. To determine if consistent fusions are also seen in the variant translocations, we performed RT-PCR on four cases and sequenced the products. For two patients with a t(8;9), we found that CEP110 exon 15 was fused to FGFR1 exon 9. For two patients with a t(6;8), we found that FOP exon 5 (n = 1) or exon 7 (n = 1) was fused to FGFR1 exon 9. To determine if FGFR1 might be involved in other myeloid disorders with translocations of 8p, we developed a two-color FISH assay using two differentially labeled PAC clones that flank FGFR1. Disruption of this gene was indicated in a patient with a t(8;17)(p11;q25) and Ph-negative chronic myeloid leukemia in association with systemic malignant mast cell disease, a patient with acute myeloid leukemia with a t(8;11)(p11;p15), and two cases with T-cell lymphoma, myeloproliferative disorder, and marrow eosinophilia with a t(8;12)(p11;q15) and ins(12;8)(p11;p11p21), respectively. For the patient with the t(8;11), the chromosome 11 breakpoint was determined to be in the vicinity of NUP98. We conclude that 1) all mRNA fusions in EMS result in splicing to FGFR1 exon 9 but breakpoints in FOP are variable, 2) two-color FISH can identify patients with EMS, and 3) the t(8;17)(p11;q25), t(8;11)(p11;p15), t(8;12)(p11;q15), and ins(12;8)(p11;p11p21) are novel karyotypic changes that most likely involve FGFR1.

In-depth meetings of the XIth International Workshop on Gastroduodenal Pathology andHelicobacter pyloriled to the presentation and discussion of extensive new data onH pyloriand its diseases. The mode of transmission ofH pyloriremains unclear, and it remains unknown why only a small proportion of infected individuals develop duodenal or gastric ulcer disease and even fewer develop gastric cancer. The role ofH pylorieradication in persons with uninvestigated dyspepsia remains controversial. New clinical trials ofH pyloritreatment show symptom relief and improvement in the quality of life of persons with functional dyspepsia, especially in those with ulcer-like or reflux-like dyspepsia. Clearly the move is toward symptom-based management of persons with dyspepsia, with fewer endoscopies being needed in the otherwise healthy young dyspeptic patients. It remains controversial whether eradicatingH pyloriin duodenal ulcer or functional dyspepsia increases the risk of subsequent development of gastroesophageal reflux disease. The one-week proton pump inhibitor-based triple regimens remain the gold standard ofH pyloritherapy, but some of the ranitidine bismuth citrate plus two antibiotic regimens also achieve an 80%H pylorieradication rate on an intention-to-treat basis. While the urea breath test remains the noninvasive test of choice, interesting new data are available on the use of stool antigen testing to diagnoseH pyloriinfection. The number ofH pylori-associated gastroduodenal diseases grows to include possible liver, vascular, immune and skin conditions.

The Xth International Workshop on Gastroduodenal Pathology andHelicobacter pyloriwas held in Lisbon, Portugal, from September 12 to 14, 1997. State-of-the-art reviews and research findings were presented to over 2000 participants. This review focuses on important new developments and serves as a rapid communication of clinically relevant material.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is accompanied by genomic mutations and rearrangements that commonly affect cytokines, transcription factors or signalling molecules that drive B-cell development or contribute to the pre-B cell receptor (pre-BCR) checkpoint. Deletions of the long arm of chromosome 6 [del(6q)] occur in ~10% of BCP-ALL and are also frequent in mature B and T-cell malignancies. Loss of function of the 6q genes EPHA7 and PRDM1, have been implicated in the genesis of lymphoma and BACH2, as a mediator of pre-BCR negative selection, is functionally a candidate tumour suppressor gene. However loss of these or other 6q genes have not been demonstrated, for example through biallelic inactivation, to contribute to BCP-ALL. Analysis of our own and published SNP6.0 data from ALL patients defined 5 focal recurrent regions of deletion on 6q, 4 mapping to 6q15-6q21, coincident with previously published common regions of deletion in ALL. These 4 regions contain 22 candidate genes, including EPHA7 but not BACH2 or PRDM1, which nevertheless mapped close to focal deletions and were also classed as candidate tumour suppressors. To develop the clone tracking assay, we adapted the SIN-SIEW lentiviral construct that expresses EGFP under the control of a spleen focus forming virus (SFFV) promoter. Candidate gene consensus coding sequence (CCDS) or a control luciferase cDNA were cloned between the promoter and an internal ribosomal entry site immediately upstream of EGFP. Transduction of the control (pSLIEW) or candidate gene SIN-SIEW-CCDS constructs consistently expressed EGFP in 697, a BCP-ALL cell line with del(6)(q14.1-22.3). For clone tracking, SIN-SIEW-CCDS constructs were assigned to 4 pools that also included pSLIEW. Pools were transduced into 697 cells that were both cultured in vitro and transplanted by intra-femoral injection into NOD/LtSz-scid IL2Rƴ null (NSG) mice. DNA was isolated from transduced cells immediately before transplant and then at 3 to 5 day intervals from cultured cells or from cells recovered from mouse bone marrow, spleen or liver at end stage disease. The pSLIEW construct facilitated monitoring of disease progression by in vivo imaging and also served as a control to measure CCDS construct copy number changes against. To quantify changes in integrated SIN-SIEW-CCDS, we developed a multiplex targeted Illumina sequencing approach. In vitro, highly significant (p Disclosures No relevant conflicts of interest to declare.

Folding of gene loci organized in tandem arrays reveals variation of chromatin structure as a function of cell differentiation., Interphase chromatin compaction well above the 30-nm fiber is well documented, but the structural motifs underlying this level of chromatin folding remain unknown. Taking a reductionist approach, we analyzed in mouse embryonic stem (ES) cells and ES-derived fibroblasts and erythroblasts the folding of 10–160-megabase pair engineered chromosome regions consisting of tandem repeats of bacterial artificial chromosomes (BACs) containing ∼200 kilobases of mammalian genomic DNA tagged with lac operator (LacO) arrays. Unexpectedly, linear mitotic and interphase chromatid regions formed from noncontiguously folded DNA topologies. Particularly, in ES cells, these model chromosome regions self-organized with distant sequences segregating into functionally distinct, compact domains. Transcriptionally active and histone H3K27me3-modified regions positioned toward the engineered chromosome subterritory exterior, with LacO repeats and the BAC vector backbone localizing within an H3K9me3, HP1-enriched core. Differential compaction of Dhfr and α- and β-globin transgenes was superimposed on dramatic, lineage-specific reorganization of large-scale chromatin folding, demonstrating a surprising plasticity of large-scale chromatin organization.

Much attention has recently been focused onHelicobacter pylori(formerlyCampylobacter pylori). It is strongly implicated as the causative agent in chronic gastritis, and may be involved in gastric and duodenal ulcers, although the latter has not been confirmed. Several virulence factors have been documented, although their involvement in the pathogenes is ofH pyloriis not proven.H pyloriwould appear to have one natural reservoir, ie, the gastric mucosa of humans. To avoid this harsh environment, it is postulated thatH pyloripossesses several characteristics which enhance survival. Strong urease enzymes produced by these organism reduce urea to ammonia and appear to create a locally elevated environment with respect to pH. The spiral shape of the cells and their flagellar motility allow them to wind themselves into the mucous layer of the stomach. Some evidence exists for the production of strong proteolytic activity, hence degrading the mucous barrier and increasing permeability for the organism. Cyroroxin excreted by the bacteria may have some effect on the surrounding cells, with the possible lysis and release of bacterial growth factors. There is evidence that a chemotactic response is present due to these growth factors and their higher concentration in the intracellular spaces. The presence of specific and nonspecific adhesion has also been demonstrated, thus allowing the bacterium, once at the epithelial cell surface, to attach and avoid being washed off by movement within the stomach. Although treatment with antimicrobials eradicates the organism and improves symptoms of peptic ulcer patients, there is no indication that the same occurs in nonulcer dyspepsia patients. Further work is essential to describe the virulence mechanisms ofH pyloriand the possible pathogenic role of the organism.

The mammalian inactive X chromosome (Xi) is a model for facultative heterochromatin. Increased DNA compaction for the Xi, and for facultative heterochromatin in general, has long been assumed based on recognition of a distinct Barr body using nucleic-acid staining. This conclusion has been challenged by a report revealing equal volumes occupied by the inactive and active X chromosomes. Here, we use light and electron microscopy to demonstrate in mouse and human fibroblasts a unique Xi ultrastructure, distinct from euchromatin and constitutive heterochromatin, containing tightly packed, heterochromatic fibers/domains with diameters in some cases approaching that of prophase chromatids. Significant space between these packed structures is observed even within condensed regions of the Xi. Serial-section analysis also reveals extensive contacts of the Xi with the nuclear envelope and/or nucleolus, with nuclear envelope association being observed in all cells. Implications of our results for models of Xi gene silencing and chromosome territory organization are discussed.

Acute lymphoblastic leukemia (ALL) is characterized by the expansion of immature hematopoietic cells in the bone marrow and blood. It is the most common childhood malignancy and is one of the major causes of death in children. Treatment improvements have increased the cure rate to more than 80% for children and about 40% for adults. However, current chemotherapy causes acute and long-term toxicity. Hence, there is a compelling need to understand the development of ALL and identify key players that may be used in targeted therapy. The B-cell receptor (BCR) and its precursor, pre-BCR, control B cell development, which is arrested in ALL. This blockade occurs at the first quality control checkpoint of B-cell development; the pre BCR checkpoint. To enhance our understanding of B-cell precursor (BCP)-ALL specific membrane associated signaling, we have investigated the functions of the recently discovered Chemokine factor like Marvel like Trans Membrane proteins (CMTM) that interact with BCR in pre-BCR checkpoint. We have identified heterozygous focal deletions of CMTM family genes, specifically CMTM6, 7 and 8 in BCP-ALL, including the subtype with intrachromosomal amplification of chromosome 21 (iAMP21). Similar focal deletions were found in iAMP21 xenograft models after serial passage, indicating their possible link to survival advantage. Although CMTM family proteins were first described in 2003, little is known about their physiological functions. The loss of a small gene cluster at chromosome 3p22, including CMTM6 and 7 has been reported in several cancers, including esophageal squamous cell, nasopharyngeal and lung carcinomas, indicating their potential roles as tumor suppressor genes. Among the CMTM family members, CMTM3 and 7 were initially identified as interacting partners of B-cell receptors. To characterize the CMTM mediated macromolecular assemblage in BCP-ALL, immunoprecipitation (IP) studies were performed using CMTM7 antibody. Initially, the expression of CMTM7 and the sensitivity of the CMTM7 antibodies were tested using various BCP-ALL cell lines. Due to their positive expression levels, the pre-B697 and NALM6 cell lines were selected for the IP studies. When the CMTM7-mediated membrane protein complex was isolated using CMTM7 antibody, we determined that the well-established tumor suppressor, B-cell linker (BLNK), interacted with CMTM7 in pre-B697 and NALM6. CMTM7-interaction partners are being verified by mass spectrometry. Next, to identify possible physiological functions, we performed a phylogenetic analysis and discovered that the CMTM family genes were homologous to myelin and lymphocyte (MAL) proteins, tricellulins, plasmolipins and occludin families, which comprise the tetra-spanin trans-membrane domain known as MARVEL (MAL and related protein for vesicle trafficking and membrane linking). These proteins are associated with cell communication and intracellular transport. To investigate the molecular mechanism of action of CMTM6, 7 and 8 in BCP-ALL and B cell development, we cloned, expressed and purified all three members and preliminary functional studies in-vitro indicated that they formed oligomers. Taken together, these data identify a critical membrane regulator, CMTM7, which may function as a tumor suppressor, communicating signals from membrane to cytosolic components through BLNK signaling in BCP-ALL. Disclosures No relevant conflicts of interest to declare.

Deletions of the short arm of chromosome 12 (12p) are found in around 6% of acute myeloid leukaemia (AML). Particularly in paediatric AML they often occur as the sole cytogenetic change and impart a poor prognosis (Harrison et al J Clin Oncol 2010). Despite multiple deletion mapping studies, a single gene has not been identified from this region that is responsible for driving leukaemic progression, thus it is clear that a functional approach is required. This study aimed to functionally implicate a significant gene through the use of a competitive selection assay. Publicly available array data from 8 studies were used to determine copy number from AML patient samples to define a minimal region of 12p deletion (MRD). Data were obtained from 866 samples, which had been analysed on either the Affymetrix SNP Array 6.0 or the Affymetrix GeneChip 500K Array platforms. Data files from matched remission samples were also analysed in 576 cases. In total 58 samples (6.7%) were found to have deletions of 12p and were used to determine the MRD. Multiplex ligation dependent probe amplification, a polymerase chain reaction based technique to determine copy number was utilised to further define the MRD. A set of probes targeted to genes from within the MRD was designed and 75 karyotypically normal AML patients were screened. 5 samples (6.6%) were found to have deletions of the MRD region, and these were confirmed by fluorescence in situ hybridisation. AML cell lines with deletions of 12p (NKM-1 and GDM-1) were selected to investigate the functional relevance of the genes within the MRD. Both were used in vitro and in immunocompromised mouse models, where leukaemia was established by intrafemoral injection and monitored by luminescent imaging of luciferase expression constructs. Taking a pooled approach, genes within the MRD were expressed from integrated lentiviral vectors in these cell lines. To evaluate effects of expression on leukaemia growth or survival, changes in construct copy number after cell line expansion in vitro and in vivo were determined by targeted high throughput sequencing on the Illumina MiSeq platform. The effects on growth of 11 candidate genes were assessed. Demonstrating a strong anti leukaemic effect for expression of this gene, a construct for cyclin dependent kinase inhibitor 1B (CDKN1B) was rapidly selected against in the assay. Expression levels of CDKN1B were measured in a range of AML cell lines and patient samples with and without 12p deletion, and the downstream effects of high and low CDKN1B protein levels were investigated. Overexpression of CDKN1B in AML cell lines carrying a 12p deletion was shown to inhibit growth through G1 phase cell cycle arrest. The sensitivity of AML cell lines to treatment with the cyclin dependent kinase inhibitor Flavopiridol was assessed and low expression levels of CDKN1B were found to moderately correlate with increased sensitivity to Flavopiridol (n = 8, Spearman’s rho = 0.4048). Data obtained from this project provide valuable insight into the mechanisms giving rise to this complex disease and represent the initial step towards developing novel targeted therapies specifically tailored to individual patients with high risk AML. These assays may also be used to study other regions of interest as well as other cancers. Disclosures No relevant conflicts of interest to declare.

Expression of BCR/ABL, a constitutively active tyrosine kinase, is a primary event in the pathogenesis of chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph+ALL). Inhibition of the BCR/ABL kinase activity in the BV173 CML cell line with STI571 resulted in a significant overexpression of a 10-kb novel mRNA, found to be the human ortholog of the murine Bach2, a B-cell-specific transcription factor. The human BACH2 cDNA is >9,120 bp long and includes an open reading frame of 2,526 bp encoding a protein with a basic leucine zipper (bZip) and a BTB/POZ domain, mediating DNA-binding and heterodimerization. BACH2 was consistently upregulated (2-10-fold) in all 10 Ph+ lymphoid lines tested following BCR/ABL inhibition. In CML myeloid cell lines (n = 8) and BCR/ABL-negative lines (n = 6), BACH2 was either undetectable by Northern blotting or did not change in response to STI571, suggesting that BACH2 repression by BCR/ABL may be specifically relevant to lymphoid transformation. Quantitative RT/PCR revealed a significantly lower level of BACH2 expression in leukocytes from patients with CML (n = 24) as compared to normal individuals (n = 23) (P < 0.0005). Moreover, CD34+ cells treated in vitro with STI571 exhibited a consistent upregulation of BACH2 in 8 of 10 CMLs but in none of the 9 normal individuals tested. Transcription regulation of BACH2 in BCR/ABL-positive cells was exerted via the MEK pathways, as shown by their responses to the U0126-specific inhibitor. Radiation hybrid mapping and FISH revealed that BACH2 is located on chromosome 6, band q15, a region frequently associated with deletions in ALL and non-Hodgkin's lymphoma, suggesting its possible role as a tumor suppressor gene. However, no rearrangement or loss of signal was observed by Southern blotting in 34 lymphomas, 10 B-cell ALLs, or seven reactive lymph nodes. The pattern of BACH2 expression in BCR/ABL-positive cells suggests that transcriptional repression by this regulator is impaired in CML and may contribute to the emergence of lymphoid blast crisis.

Insight into the role of recurrent genomic changes arising in leukemia has depended largely on the functional analysis of cell lines and transgenic animals. However not all acquired abnormalities are represented in available cell lines and a few are too complex to be faithfully engineered in animal models. One such abnormality is intrachromosomal amplification of chromosome 21 (iAMP21), a heterogeneous cytogenetic rearrangement, with a distinct clinical profile, occurring in 2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The most highly amplified segment of iAMP21 varies in size and copy number but has always included a 5.1 Mb common region of amplification (CRA). Regions flanking the most highly amplified segment have profiles that may be step-like, or more complex combinations of lower level amplification, normal copy number and deletion. Abnormalities at other chromosomal locations such as deletions of IKZF1, CDKN2A/B or RB1 and rearrangements activating CRLF2 co-occur with iAMP21 but have never been shown to precede its formation. To investigate clonal evolution and to provide a resource for future functional studies, we established xenografts from three cases of iAMP21 BCP-ALL in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Signs of disease were seen between 6 and 8 months after intrafemoral injection and autopsy revealed marked splenomegaly in all cases. Cells isolated from the bone marrow and spleen, were positive for human B cell markers CD19 and CD10. Disease onset was more rapid (3-6 months) when primografts were transplanted and cells from these secondary mice were used to establish leukemia in a third generation. Whole body imaging of mice transplanted with xenograft cells, transduced with a lentivirus expressing luciferase, revealed the rapid spread of disease from the injection site to the contralateral femur, sternum, pelvis, ribs, spine and skull, with the spleen becoming the major site of engraftment at late stage disease. Comparison of SNP 6.0 array profiles of presentation and xenografts samples revealed several examples of post-transplant genomic progression which, with the exception of a biallelic deletion of the CDKN2A/B locus, had not previously been reported in iAMP21 patients. Additional copies of chromosomes 9 and 12 in patient samples were lost in xenografts resulting in copy number neutral loss of heterozygosity (CNN-LOH) for chromosome 12 but not 9. Analysis of further primary iAMP21 BCP-ALL cases identified two with CNN-LOH for part of the long arm of chromosome 12, suggesting involvement of an imprinted locus in this region. iAMP21 chromosomes were retained in all xenografts, supporting evidence that it is a primary abnormality. However, as these rearrangements have been considered to be stable once formed, we were surprised to observe acquisition of a non–contiguous deletion of chromosome 21 in one secondary mouse and all of its tertiary recipients. The deletion was proximal to the CRA and resulted in extension of pre-existing haploinsufficient regions by 6.6 Mb. Evolution of this iAMP21 chromosome was confirmed by fluorescence in-situ hybridisation (FISH) using probes targeting the deletion, with loss of signal observed in all cells from mice carrying the deletion but in no cells from the presentation sample or unaffected mice. Although we cannot rule out that the deletion arose in response to adaptive pressure specific to the xeno-environment, opportunities to observe similar progression in patients have been limited because FISH analysis has generally targeted only the CRA and few paired presentation/relapse samples have been analysed by genomic array. While it remains likely that overrepresentation of oncogenes within the CRA acts as a primary driver of iAMP21 BCP-ALL, the additional deletion may highlight the position of tumour suppressor genes involved in leukemia progression. As these patients are treated as a single clinical entity, an important implication of these data is that the position and extent of deletions in iAMP21 rearrangements may influence response to therapy. In conclusion, xenografts of iAMP21 BCP-ALL closely resemble human primary disease and will make valuable pre-clinical models for basic and translational research. As models for the study of clonal evolution they support previous evidence that iAMP21 is a primary abnormality and have revealed novel forms of clonal progression. Disclosures: No relevant conflicts of interest to declare.

The molecular basis for blast transformation of chronic myeloid leukemia (CML) remains poorly understood. Cytogenetic alterations associated with CML blast crisis have previously been extensively studied by conventional G-banding analysis. However the complexity of some chromosome abnormalities or poor chromosome morphology or both has exceeded the resolution of G-banding analysis in a significant proportion of CML cases, and complex chromosome rearrangements have remained unidentified. In this study, comparative genomic hybridization (CGH) was used to elucidate genome imbalances in chronic phase or blast crisis samples or both from 12 CML patients. CGH and G-banding results were compared, and discrepancies were further clarified by using multipaint chromosome analysis and locus-specific DNA probes. No imbalances were detected in the 4 early disease phase samples studied. Eleven blast crisis samples were analyzed by G-banding and CGH, and the commonest genomic abnormality detected was overrepresentation of the long arm of chromosome 8, which was detected in 5 patients. This overrepresentation was attributable to trisomy 8 in 4 patients, whereas amplification of the entire long arm of chromosome 8 was detected in 1 patient. The formation of isochromosomes of the long arm of chromosome 8 was observed as a mechanism for gene amplification in this patient. Additional material originating from chromosome 8 was also observed intercalated into three marker chromosomes in peripheral blood metaphase spreads from this patient. These markers may further define areas on chromosome 8 that harbor oncogenes implicated in transformation of chronic myeloid leukemia.

OBJECTIVE: To study whether an intravenous infusion dose of omeprazole (80 mg + 8 mg/h) during 24 h can be subsequently reduced with maintained effect. Second, to study the effect of oral omeprazole 20 mg given once or twice daily up to day 10, after cessation of a 3-day intravenous infusion (80 mg + 8 mg/h). DESIGN: Prospective, randomized, partly blinded study. METHODS: Twelve Helicobacter pylori(+) patients and 12 H. pylori(-) subjects were included. In part I the patients received omeprazole, 80 mg + 8 mg/h, during 24 h followed by 8, 4 or 2 mg/h. In part II the subjects received 80 mg + 8 mg/h during 3 days followed by 20 mg omeprazole orally, once or twice daily until day 10. Intragastric pH was measured. RESULTS: All H. pylori(+) patients showed a rapid increase of intragastric pH with a mean intragastric pH of 6.7 during the second half of the first day. After the subsequent dose reduction, the mean pH decreased to 6.1-6.2. Patients continuing on 8 mg/h showed the best results. Likewise, all H. pylori(-) subjects showed a rapid and sustained reduction of intragastric acidity during the infusion. Subsequent dose reduction to 20 mg once daily led to a stable fraction of time with pH > 3 of 72%. CONCLUSIONS: Omeprazole given as a continuous infusion of 80 mg + 8 mg/h for 72 h followed by omeprazole 20 mg once daily raised the intragastric pH to and above levels alleged to allow haemostasis in patients with peptic ulcer bleeding and subsequent healing of the ulcer.

With the exponential increase in research in the field of Helicobacter pylori a paradigm shift has occurred. It is now recognized that H pylori is a chronic infection of the stomach causing inflammation. Some patients remain asymptomatic, while others may develop dyspepsia, duodenal or gastric ulcer, gastric cancer or a mucosa-associated lymphoid tissue lymphoma. However, the role of H pylori in contributing to nonulcer dyspepsia or nonsteroidal anti-inflammatory drug gastropathy remains controversial. An effective vaccine against H pylori is years away. Major interest has focused on the questions "who should be investigated and therefore treated" and "what is the latest gold standard for eradication of H pylori"? In Europe, guidelines have been developed to help the practitioner answer these important questions. Canadian guidelines will soon be available. For persons with known peptic ulcer disease there should be unequivocal acceptance that the good clinical practice of eradicating H pylori will result in substantial savings in health care expenses. The original 'classical triple therapy' (bismuth, metronidazole and tetracycline [BMT]) has now been surpassed by the combination of a proton pump inhibitor (PPI) plus two antibiotics (metronidazole plus clarithromycin; amoxicillin plus clarithromycin; or amoxicillin plus metronidazole), each given twice a day for one week. In Canada, the regimen of omeprazole plus one antibiotic (amoxicillin or clarithromycin) was approved recently but gives an eradication rate that is lower than the current target of 90%. According to the European (Mäastricht) recommendations, if a single treatment attempt with PPI plus two antibiotics fails, PPI plus BMT is recommended.

Abstract 581 Patients with iAMP21 (intrachromosomal amplification of chromosome 21) represent a distinct cytogenetic subgroup of childhood B cell precursor ALL (BCP-ALL; incidence ∼2%) with a poor prognosis on standard therapy (29% EFS at 5 years). From cytogenetic studies the amplification of chromosome 21 appears to be the primary genetic change, however, whether this or associated abnormalities provide the initiating event of leukaemogenesis remains to be elucidated. Currently fluorescence in situ hybridisation (FISH) with probes directed to the RUNX1 gene provides the only reliable detection method. Knowledge of the underlying genetic mechanism will lead to improved diagnosis and determine those patients at high risk of relapse who require more intensive therapy. Our previous study using genomic arrays (1Mb aCGH, n=10; NimbleGen chromosome 21 custom oligonucleotide array, n=5) identified a common region of amplification (CRA) on 21q of 6.6 Mb and a common region of deletion (CRD) of 3.3 Mb within the telomeric region in 100% and 70% of patients, respectively. However, expression profiling (Affymetrix U133A, n=8) indicated that no important genes within the CRA or CRD were differentially expressed. We now have clinical, cytogenetic and FISH data on 94 iAMP21 patients (44 F, 50 M; median age 9.5y, range 2-30); the largest iAMP21 cohort investigated to date. Higher resolution analysis of 17/94 diagnostic samples by aCGH (Agilent 185K platform) have refined these regions to 5.1 Mb and 2.5 Mb in 100% and 88% of patients, respectively. Recent genome mapping has highlighted the presence of the microRNA miR-802 within the CRA and shown this region overlaps with the Down Syndrome Critical Region at 21q22.3 encompassing the genes DSCR1, DSCR3 and DYRK1. Detailed analysis of chromosome 21 identified recurrent breakpoints centromeric of the CRD within three genes: PDE9A (n=2), COL6A2 (n=2) and DSCAM (n=2). The role of these genes in the initiation of the chromosomal instability associated with iAMP21 is currently under investigation. Global analysis of the iAMP21 genome identified an average of 13 copy number alterations (CNA) per case with recurrent deletions of the leukaemia associated genes PAX5 (n=2), IKZF1 (n=4) and CDKN2A (n=4). To determine the incidences of PAX5, IKZF1 and CDKN2A deletions among the cohort of 94 iAMP21 patients, FISH using probes targeting these genes was carried out on availabel samples. IKZF1 (exons 3-6) was deleted at the highest incidence of 21% (13/62), while CDKN2A and PAX5 were deleted at a lower incidence of 17% (12/71) and 8% (4/48), respectively. The high incidence of IKZF1 deletions found in this patient group correlates with their association with high risk ALL. We have recently reported a deletion within the PAR1 region of the sex chromosomes, centromeric of the CRLF2 gene involving CSF2RA and IL3RA, which leads to deregulated expression of CRLF2. This deletion occurred at an incidence of 23% (17/73), as determined by FISH. Among pairs of matched diagnostic and relapse samples; one had this deletion at both diagnosis and relapse while the second had a deletion in the diagnostic sample only. Gain of an additional × chromosome was identified in 28% (18/65). Of note is that the 3 aberrations: abnormalities of chromosome 21, gain of the × chromosome and high incidence of deletions centromeric of CRLF2 in this patient group mirror the findings in Down Syndrome (DS) ALL, although we have not identified any mutations of the JAK2 kinase domain among this cohort of iAMP21 patients to date. This genome-wide study provides further support for the proposal that amplification of genes within the CRA on chromosome 21 is the primary event driving leukaemogenesis in iAMP21 patients. It has also highlighted that the 3 abnormalities in common with DS ALL and/or deletions of IKZF1, which all occur at a high incidence, contribute towards the progression of this high risk subtype of childhood BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

A 42-year-old male patient with a history of occupational exposure to benzene presented with pancytopenia. His bone marrow showed evidence of trilineage dysplasia and cytogenetic analysis revealed a unique t(9;13)(q34;q12) translocation. Five months after diagnosis he developed secondary AML. He was treated with four courses of chemotherapy and an autologous bone marrow transplantation (BMT). Four years post-transplantation he remains in haematological and morphological remission though the cytogenetic abnormality is still present in all metaphases examined.

Using FISH and LOH in ALL cases with microscopically visible deletions of the long arm of chromosome 6q, we previously defined a region of minimal deletion (RMD) in a spectrum of lymphoid malignancies and with other investigators have suggested that the region may harbour several tumour suppressor genes. (Sinclair et al 2004; Cancer Research, 64: 4089). 15 recognised genes were positioned in the RMD and three were found expressed in bone marrow: GRIK2, C6orf111 and Cyclin-C, the investigation of which is reported in this study. GRIK2 analysis. Combining published RMD in ALL with our own data, we extensively analysed GRIK2 in ALL patients carrying heterozygous 6q deletion. Although a paternally inherited non-silent mutation of GRIK2 was identified in one of 14 patients, we concluded that loss of one or more other genes in the 6q15-q21 region was likely to contribute to ALL pathogenesis. Cyclin-C (CCNC) analysis. Lymphoid cell lines without and with del (6q)(Peer, Molt-4 and NKL) were assessed for expression levels of C6orf111 and Cyclin-C and other candidate tumour suppressor genes within the region (FLASH, Q9NR58, Cx43, BACH2, BLIMP1, APG5L, TAK1 and AIM1) using real-time quantitative RT-PCR (RQ-PCR). While easily detectable in all other cell lines, no expression of CCNC was demonstrated in NKL (by RT-PCR and Western blotting), this in spite of the presence of at least one copy of the gene (as demonstrated by FISH using PAC P13743 which contains CCNC) suggesting that expression of the second allele was ablated through either micro-deletion, point mutation or epigenetic silencing. Expression levels of other candidate genes were similar, albeit with minor variations, in all cell lines. We further analysed expression of CCNC in whole PB and BM, a variety of haemopoietic cells isolated from PB and in 66 de novo ALL patients (9 T ALL; 48 B cell ALL (predominantly common ALL) and 9 other ALL). Expression was relatively high in all cell lines, intermediate in normal T-lymphocytes and low in other normal cells. Leukemic cells collected from diagnostic material from adult acute lymphoid malignancies showed a wide variation in levels of CCNC expression. To exclude the possibility that low expression in some cases may be due to the presence of a high proportion of normal cells CCNC levels were normalised against Ki67 expression (as an indicator of mitotic index) by RQ-PCR. At least 27 patients showed a CCNC level below the median value. Twenty-five cases showed value above the median value. Levels of expression of CCNC and Ki67 were correlated with survival, immunophenotype or other clinical parameters, but no statistically significant correlation was demonstrated.

While deletions of the long arm of chromosome 6 (6q) are common in patients with ALL, translocations are rare, most are considered sporadic and none have been cloned. Among myeloid malignancies, the recurrent translocations: t(6;11)(q27;q23), t(6;11)(q21;q23) and t(6;8)(q27;p11) give rise to known fusion oncogenes. Other uncharacterised balanced or unbalanced 6q abnormalities have occasionally been reported. Using fluorescence in-situ hybridisation (FISH) with HGMP mapped PAC and BAC clones, we investigated chromosomal rearrangements of 6q in acute leukemia. Previously we reported 24 cases with deletions of 6q (Sinclair et al 2004, Can Res 64; 4089), here we describe the analysis of 11 patients with novel balanced rearrangements. Six of seven breakpoints in ALL and two in a single case of AML were localised to a 10.5 Mb ‘hotspot’ within 6q22–q23. Of these, five were analysed down to the level of a single clone. In two cases of childhood T ALL, both carrying a t(6;7)(q23;q31~36), split FISH signals were produced by adjacent PACs, RP1–32B1 and RP3–388E23, mapping the breakpoints to within a ~150Kb region. A search of published karyotypes identified five similar rearrangements, four also in pediatric T ALL, all present as a sole abnormality or represented in the major clone. Of genes in the region, two (c-MYB and AHI1) are tightly regulated during normal hemopoietic development and over-expression of both has been reported to occur in hemopoietic malignancies. Moreover, clusters of retroviral insertion sites occur within both genes and immediately proximal to c-MYB in murine and feline leukemias and lymphomas. Thus we have defined a new primary recurrent translocation in T ALL that is likely to target either c-MYB and/or AHI1.Three other non recurrent rearrangements of 6q22–q23, mapped in detail, interrupted regions without known genes.. Therefore, these abnormalities were unlikely to have promoted leukemia through classical oncogene activation, but may have influenced expression of one or more common target genes through a long-range position effect. Alternatively these clustered rearrangements may mark the position of a new fragile site on 6q. Additional 6q breakpoints in three cases of AML and one of ALL mapped to 6q15, 6q21, 6q25 and 6q26–q27, and were coincident with the positions of a known oncogene partner AF6q21, the haemopoietic transcription factor BACH2 and two cloned fragile sites FRA6E and FRA6F. Putative partner genes of four translocations were investigated by FISH and the involvement of MLL was demonstrated in one case with t(6;11)(q15;q23).

Objective: To determine whether a “test for Helicobacter pylori and treat” strategy improves symptoms in patients with uninvestigated dyspepsia in primary care. Design: Randomised placebo controlled trial. Setting: 36 family practices in Canada. Participants: 294 patients positive for Hpylori (13C- urea breath test) with symptoms of dyspepsia of at least moderate severity in the preceding month. Intervention: Participants were randomised to twice daily treatment for 7 days with omeprazole 20 mg, metronidazole 500 mg, and clarithromycin 250 mg or omeprazole 20 mg, placebo metronidazole, and placebo clarithromycin. Patients were then managed by their family physicians according to their usual care. Main outcome measures: Treatment success defined as no symptoms or minimal symptoms of dyspepsia at the end of one year. Societal healthcare costs collected prospectively for a secondary evaluation of actual mean costs. Results: In the intention to treat population (n=294), eradication treatment was significantly more effective than placebo in achieving treatment success (50% v 36%; P=0.02; absolute risk reduction=14%; number needed to treat=7, 95% confidence interval 4 to 63). Eradication treatment cured H pylori infection in 80% of evaluable patients. Treatment success at one year was greater in patients negative for H pylori than in those positive for H pylori (54% v 39%; P=0.02). Eradication treatment reduced mean annual cost by $C53 (86 to 180) per patient. Conclusions: A “test for H pylori with 13C-urea breath test and eradicate” strategy shows significant symptomatic benefit at 12 months in the management of primary care patients with uninvestigated dyspepsia. What is already known on this topic Dyspepsia is a common problem in primary health care, although controversy exists about its definition Studies of H pylori eradication in patients with uninvestigated dyspepsia have shown reduced need for endoscopy and thus significant cost savings compared with a strategy of prompt endoscopy The “test for H pylori and treat” strategy has been recommended for uninvestigated dyspepsia, but there have been no randomised controlled trials showing improvement in symptoms What this study adds When given eradication treatment in primary care, H pylori positive patients with uninvestigated dyspepsia show improvement in overall dyspepsia symptoms at 12 months This supports the “test for H pylori and treat” strategy

How chromatin folds into mitotic and interphase chromosomes has remained a difficult question for many years. We have used three generations of engineered chromosome regions as a means of visualizing specific chromosome regions in live cells and cells fixed under conditions that preserve large-scale chromatin structure. Our results confirm the existence of large-scale chromatin domains and fibers formed by the folding of 10-nm and 30-nm chromatin fibers into larger, spatially distinct domains. Transcription at levels within severalfold of the levels measured for endogenous loci occur within these large-scale chromatin structures on a condensed template linearly compacted several hundred fold to 1000-fold relative to B-form DNA. However, transcriptional induction is accompanied by a severalfold decondensation of this large-scale chromatin structure that propagates hundreds of kilobases beyond the induced gene. Examination of engineered chromosome regions in mouse embryonic stem cells (ESCs) and differentiated cells suggests a surprising degree of plasticity in this large-scale chromatin structure, allowing long-range DNA interactions within the context of large-scale chromatin fibers. Recapitulation of gene-specific differences in large-scale chromatin conformation and nuclear positioning using these engineered chromosome regions will facilitate identification of cis and trans determinants of interphase chromosome architecture.