A Scalable Computational Approach for Simulating Complexes of Multiple Chromosomes.

Significant efforts have been recently made to obtain the three-dimensional (3D) structure of the genome with the goal of understanding how structures may affect gene regulation and expression. Chromosome conformational capture techniques such as Hi-C, have been key in uncovering the quantitative information needed to determine chromatin organization. Complementing these experimental tools, co-polymers theoretical methods are necessary to determine the ensemble of three-dimensional structures associated to the experimental data provided by Hi-C maps. Going beyond just structural information, these theoretical advances also start to provide an understanding of the underlying mechanisms governing genome assembly and function. Recent theoretical work, however, has been focused on single chromosome structures, missing the fact that, in the full nucleus, interactions between chromosomes play a central role in their organization. To overcome this limitation, MiChroM (Minimal Chromatin Model) has been modified to become capable of performing these multi-chromosome simulations. It has been upgraded into a fast and scalable software version, which is able to perform chromosome simulations using GPUs via OpenMM Python API, called Open-MiChroM. To validate the efficiency of this new version, analyses for GM12878 individual autosomes were performed and compared to earlier studies. This validation was followed by multi-chain simulations including the four largest human chromosomes (C1-C4). These simulations demonstrated the full power of this new approach. Comparison to Hi-C data shows that these multiple chromosome interactions are essential for a more accurate agreement with experimental results. Without any changes to the original MiChroM potential, it is now possible to predict experimentally observed inter-chromosome contacts. This scalability of Open-MiChroM allow for more audacious investigations, looking at interactions of multiple chains as well as moving towards higher resolution chromosomes models.

The functional epigenetic landscape of aberrant gene expression in molecular subgroups of newly diagnosed multiple myeloma.

BACKGROUND: Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease. METHODS: Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate. RESULTS: We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes. CONCLUSIONS: Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies.

The impact of cytogenetics on duration of response and overall survival in patients with relapsed multiple myeloma (long-term follow-up results from BSBMT/UKMF Myeloma X Relapse [Intensive]): a randomised, open-label, phase 3 trial.

The Myeloma X trial (ISCRTN60123120) registered patients with relapsed multiple myeloma. Participants were randomised between salvage autologous stem cell transplantation (ASCT) or weekly cyclophosphamide following re-induction therapy. Cytogenetic analysis performed at trial registration defined t(4;14), t(14;16) and del(17p) as high-risk. The effect of cytogenetics on time to progression (TTP) and overall survival was investigated. At 76 months median follow-up, ASCT improved TTP compared to cyclophosphamide (19 months (95% confidence interval [95% CI] 16-26) vs. 11 months (9-12), hazard ratio [HR]: 0·40, 95% CI: 0·29-0·56, P < 0·001), on which the presence of any single high-risk lesion had a detrimental impact [likelihood ratio test (LRT): P = 0·011]. ASCT also improved OS [67 months (95% CI 59-not reached) vs. 55 months (44-67), HR: 0·64, 95% CI: 0·42-0·99, P = 0·0435], with evidence of a detrimental impact with MYC rearrangement (LRT: P = 0·021). Twenty-one (24·7%) cyclophosphamide patients received an ASCT post-trial, median OS was not reached (95% CI: 39-not reached) for these participants compared to 31 months (22-39), in those who did not receive a post-trial ASCT. The analysis further supports the benefit of salvage ASCT, which may still be beneficial after second relapse in surviving patients. There is evidence that this benefit reduces in cytogenetic high-risk patients, highlighting the need for targeted study in this patient group.

Modified cIg-FISH protocol for multiple myeloma in routine cytogenetic laboratory practice.

The International Myeloma Working Group recommends that fluorescence in situ hybridization (FISH) be performed on specifically identified plasma cells (PC). This is because chromosomal abnormalities are not frequently detected by traditional karyotyping due to the low proliferative rate of PC in multiple myeloma (MM). Conventional FISH enhances the sensitivity but lacks the specificity, as it does not distinguish PC from other hematopoetic cells. To fulfill this recommendation, PC need to be selected either by flow cytometry or immunomagnetic bead-based PC sorting or by concomitant labeling of the cytoplasmic immunoglobulin light chain, which allows for unambiguous identification. These techniques require expertise, time, and funding and are not easily incorporated into the routine workflow of the cytogenetic laboratory. We have modified and refined the technique using fixed cell pellets to achieve nicely separated and easily identifiable PC. With immunostaining and subsequent FISH (i.e., cytoplasmic immunoglobulin FISH, cIg-FISH), this technique can be easily incorporated into every cytogenetic laboratory. Twenty samples from patients with MM were subjected to routine FISH, cIg-FISH, and chromosomal karyotyping and the results were compared. Three FISH probes, which enabled detection of the t(4;14), t(14;16) and deletion of TP53, were used to validate this modified technique successfully.

Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms.

Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of myelodysplasia (MDS) and myeloproliferative neoplasms (MPN), underlying pathogenic molecular lesions are unknown. We identified recurrent areas of somatic copy number-neutral loss of heterozygosity (LOH) and deletions of chromosome 4q24 in a large cohort of patients with myeloid malignancies including MDS and related mixed MDS/MPN syndromes using single nucleotide polymorphism arrays. We then investigated genes in the commonly affected area for mutations. When we sequenced TET2, we found homozygous and hemizygous mutations. Heterozygous and compound heterozygous mutations were found in patients with similar clinical phenotypes without LOH4q24. Clinical analysis showed most TET2 mutations were present in patients with MDS/MPN (58%), including CMML (6/17) or sAML (32%) evolved from MDS/MPN and typical MDS (10%), suggesting they may play a ubiquitous role in malignant evolution. TET2 mutations affected conserved domains and the N terminus. TET2 is widely expressed in hematopoietic cells but its function is unknown, and it lacks homology to other known genes. The frequency of mutations in this candidate myeloid regulatory gene suggests an important role in the pathogenesis of poor prognosis MDS/MPN and sAML and may act as a disease gene marker for these often cytogenetically normal disorders.

Two consecutive immunophenotypic switches in a child with MLL-rearranged acute lymphoblastic leukemia.

An 18-month-old girl was diagnosed with pre-pre-B ALL/t(4;11) leukemia, which during the treatment and after matched bone marrow transplantation (BMT), underwent two consecutive switches from lymphoid to myeloid lineage and vice versa. The high expression of HOXA9 and FLT3 genes remaining genotypically stable in a leukemia throughout phenotypic switches, suggests that this leukemia may have originated as a common B/myeloid progenitors.

Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited.

BACKGROUND: Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. METHODS: Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. RESULTS: In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. CONCLUSION: The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.

The hypereosinophilic syndrome: fluorescence in situ hybridization detects the del(4)(q12)-FIP1L1/PDGFRA but not genomic rearrangements of other tyrosine kinases.

BACKGROUND AND OBJECTIVES: According to WHO criteria, the idiopathic hypereosinophilic syndrome (HES) is defined as persistent eosinophilia (>1.5x10(9)/L) without underlying causes, which is associated with signs or symptoms of organ involvement. Increased bone marrow blasts (>5%) or cytogenetic/genetic markers indicate chronic eosinophilic leukemia (CEL). A cryptic deletion of 4q12, i.e. del(4)(q12), producing the FIP1L1/PDGFRA fusion gene, identifies a distinct CEL subgroup (4q-/CEL). Our aims were: a) to use interphase-fluorescent in situ hybridization (FISH) to detect the cryptic 4q12 deletion; b) to compare the clinico-hematologic features of 4q-/CEL with other HES; c) to investigate whether PDGFRB, FGFR1, ABL1, and ETV6-activated tyrosine kinases are rearranged in CEL/HES. DESIGN AND METHODS: This multicenter study included 20 patients fulfilling the WHO criteria for HES and 6 patients without signs/symptoms of end-organ involvement. Double-color FISH was applied in all cases to investigate del(4)(q12). Further interphase-FISH assessed whether PDGFRB/5q33, FGFR1/8p11, ABL1/9q34, and ETV6/12p13, undergo rearrangements in HES. RESULTS: Ten of the 26 patients (9 males and 1 female) had a cryptic del(4)(q12)-FIP1L1/PDGFRA which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis in four. Hepatomegaly and splenomegaly were significantly more frequent in these 10 than in the other 16 patients. Seven of these 10 patients received imatinib mesylate therapy and all achieved hematologic remission. In 3 of the patients interphase-FISH and RT-PCR demonstrated cytogenetic and molecular remission. Improvements were observed in signs and symptoms of cardiac and central nervous system involvement in 2 and 1 patient, respectively. Rearrangements of PDGFRB, FGFR1, ABL1, or ETV6 were not detected in this study. INTERPRETATION AND CONCLUSIONS: FISH is a reliable diagnostic test for differentiating 4q-/CEL from other forms of HES, allowing an early diagnosis of good responders to imatinib mesylate therapy. For the first time we show that PDGFRB, FGFR1, ABL1 and ETV6 are not rearranged in HES and 4q-/CEL cases we studied.

CD221 (IGF-1R) is aberrantly expressed in multiple myeloma, in relation to disease severity.

We investigated the expression of insulin-like growth factor-1 receptor (CD221) in normal, reactive and malignant plasma cells. We show that CD221 is aberrantly expressed on human myeloma cells, that higher levels of CD221 are observed in patients and human myeloma cell lines with the most aggressive 14q32 translocations, and that CD221 expression has a negative prognostic impact in patients with multiple myeloma.

Chromosome aberration and lipid peroxidation in chromium-exposed workers.

Chromosome aberration frequency and lipid peroxidation levels were analyzed to investigate their efficacy as biological markers for monitoring the genotoxicity and oxidative damage in Korean chromium (Cr)-exposed workers. Fifty-one Cr-exposed workers and 31 age-matched controls in ten chrome-plating plants were sampled. The Cr level was measured in the workers' blood and urine, and in the ambient air at the workplaces. The conventional Giemsa staining method and fluorescence in situ hybridization (FISH) technique were used for chromosome aberration analysis. Spectrum green whole chromosome paint specific for chromosome 4 was used in the FISH procedure. As for lipid peroxidation, malondialdehyde (MDA) was measured in the blood plasma as thiobarbituric acid-reactive substances (TBARS). The blood Cr concentration was statistically correlated with both the frequency of chromatid exchange and the total frequency of chromosome/chromatid breaks and exchanges, as detected by the Giemsa staining. Meanwhile, the frequency of translocation, as detected by the FISH technique, was significantly higher in the Cr-exposed workers than in the controls and it correlated with the blood Cr concentration. Although the concentration of MDA, the metabolite of lipid peroxidation, in the exposed workers was higher than that of the controls, no statistically significant correlation between the MDA level and the blood or urine Cr levels was observed. Accordingly, the genotoxicity and oxidative damage (plasma lipid peroxidation) in the Korean Cr-exposed workers were consequential at quite low exposure levels, plus chromosome rearrangement, especially translocation, was clearly evident as a biological response marker for Cr exposure based on a significant positive correlation between the translocations detected by FISH and the Cr in the blood.

MLL-AF4 gene rearrangement in a child with Epstein-Barr virus-related posttransplant B-cell lymphoma.

Recipients of solid organ allografts are known to be at increased risk of developing Epstein-Barr virus-related posttransplant lymphoproliferative diseases. A 28-month-old boy who had received a heterotopic liver transplant presented with lymphadenopathy in the abdomen, multiple nodules in the liver, and bilateral renal infiltration 19 months after transplantation. He was diagnosed with a Burkitt-like lymphoma based on bone marrow examination and the finding that the blastic cells in bone marrow were EBER-1 positive. Cytogenetic analysis of the bone marrow cells showed an MLL-AF4 rearrangement. He was treated with a combined chemotherapy regimen. He has been in continuous complete remission for 15 months now.

PMX2B, a new candidate gene for Hirschsprung's disease.

Hirschsprung's (HSCR) disease is a congenital intestinal malformation of the enteric nervous system. It is a multigenic malformation and until now, eight genes have been involved in the etiology of this disease: genes encoding proteins of the RET signaling pathway (RET, GDNF and NTN), genes participating in the endothelin (EDN) type B receptor pathway (EDNRB, EDN3 and ECE-1), the SOX10 gene and the SIP1 gene that is mutated in syndromic forms of HSCR. Mutations of these genes are found in not more than 50-60% of affected individuals. Here, we report on the results of a molecular cytogenetic study performed in a girl who presented with a syndromic short segment HSCR associated with a de novo t(4;8)(p13;p22) translocation. A comparative genomic hybridization (CGH) study found a 4p12p13 deletion. A molecular characterization of this rearrangement showed that the 4p13 deletion was 5 Mb in length and included the paired mesoderm homeobox gene (PMX2B) (MIM 603851), a gene expressed in the human embryonic gut and essential for the development of autonomic neural crest derivatives. The present observation suggests that PMX2B haploinsuffciency might predispose to HSCR.

Clinical heterogeneity in childhood acute lymphoblastic leukemia with 11q23 rearrangements.

To assess the clinical heterogeneity among patients with acute lymphoblastic leukemia (ALL) and various 11q23 abnormalities, we analyzed data on 497 infants, children and young adults treated between 1983 and 1995 by 11 cooperative groups and single institutions. The substantial sample size allowed separate analyses according to age younger or older than 12 months for the various cytogenetic subsets. Infants with t(4;11) ALL had an especially dismal prognosis when their disease was characterized by a poor early response to prednisone (P=0.0005 for overall comparison; 5-year event-free survival (EFS), 0 vs 23+/-+/-12% s.e. for those with good response), or age less than 3 months (P=0.0003, 5-year EFS, 5+/-+/-5% vs 23.4+/-+/-4% for those over 3 months). A poor prednisone response also appeared to confer a worse outcome for older children with t(4;11) ALL. Hematopoietic stem cell transplantation failed to improve outcome in either age group. Among patients with t(11;19) ALL, those with a T-lineage immunophenotype, who were all over 1 year of age, had a better outcome than patients over 1 year of age with B-lineage ALL (overall comparison, P=0.065; 5-year EFS, 88+/-+/-13 vs 46+/-14%). In the heterogeneous subgroup with del(11)(q23), National Cancer Institute-Rome risk criteria based on age and leukocyte count had prognostic significance (P=0.04 for overall comparison; 5-year EFS, 64+/-+/-8% (high risk) vs 83+/-+/-6% (standard risk)). This study illustrates the marked clinical heterogeneity among and within subgroups of infants or older children with ALL and specific 11q23 abnormalities, and identifies patients at particularly high risk of failure who may benefit from innovative therapy.

Genomic characterization of the chromosomal breakpoints of t(4;14) of multiple myeloma suggests more than one possible aetiological mechanism.

Using FISH-based techniques, rearrangements of the immunoglobulin heavy-chain (IgH) locus at 14q32 have been found in the majority of cases of multiple myeloma (MM). Some of these IgH translocations are recurrent and we have characterized the genomic breakpoints of seven t(4;14) translocations from MM patients, using a combination of vectorette and conventional polymerase chain reaction methods, the aim being to understand the molecular mechanism leading to MM. Conventionally, the chromosome 14q32 breakpoints in these reciprocal translocations are believed to be located in the IgH mu switch (S) region and a further downstream S region with deletion of intervening DNA occurring as a result of aberrant class switch recombination (CSR); this was seen in five of the cases analysed. However, in two patients it was possible to demonstrate that the rearranged hybrid switch region sequence was joined to DNA from chromosome 4p16, suggesting that IgH translocations can occur in B cells that have already undergone legitimate CSR. The complex nature of these rearrangements leads us to speculate that primary IgH translocations may occur at different time points in the development in MM plasma cells, either at the time of physiological CSR or at a later stage, possibly involving a different mechanism.

Premature chromosome condensation associated with fluorescence in situ hybridisation detects cytogenetic abnormalities after a CT scan: evaluaton of the low-dose effect.

The purpose of this study was to assess the cytogenetic effects of the X ray irradiation used during a CT scan in order to estimate the mean absorbed dose in circulating lymphocytes. Chromosomal aberrations were scored in blood lymphocytes of ten patients undergoing CT scans, by applying fluorescence in situ hybridisation (FISH) to metaphase cells and premature chromosome condensation (PCC) with chromosomes 1, 3 and 4 painting probes immediately after exposure. This generated a dosimetric index that reflects the dose to the circulating lymphocytes. By using PCC a significant increase in the frequency of chromosomal fragment was observed immediately after a CT scan. However, no significant increase in chromosomal aberration was detected in metaphase cells. The mean dosimetric index immediately after exposure was 0.057 Gy (95% CI: 0.052-0.082 Gy). This dosimetric index depends essentially on the size of the examined and exposed blood volumes. This dose is in close agreement with the dose length product (DLP) (Gy cm) (R = 0.80). It should be kept in mind when justifying requests for diagnostic CT scan especially in young patients. The presence of chromosomal fragments after a CT scan indicated the cytogenetic effect of a low dose. PCC associated with chromosome painting is a method for detecting the cytogenetic effect of a low dose immediately after exposure.

Inv(4)(p14q27) in a case with de novo acute nonlymphocytic leukemia.

An inversion, inv(4)(p14q27), was found as the sole karyotypic anomaly at diagnosis in the bone marrow cells from a 65-year-old male patient with an M4 acute nonlymphocytic leukemia (ANLL). To our knowledge, the breakpoints observed in this case appear to be different from other inversions of chromosome 4 previously described in ANLL. The patient we described had a poor response to chemotherapy and had a short survival.

A new translocation that rearranges the AML1 gene in a patient with T-cell acute lymphoblastic leukemia.

The AML1 gene (also known as RUNX1 or CBFA2), located in chromosome band 21q22, encodes a transcription factor which heterodimerizes with the CBFbeta protein forming a complex called human core binding factor (CBF). The CBF complex appears to regulate a number of genes important for hematopoiesis. AML1 is one of the most common targets of chromosomal rearrangements in human leukemias and has been involved in 14 chromosomal translocations to date. Here we report a new chromosomal translocation, t(4;21)(q31;q22) that disrupts the AML1 gene in a 12-year-old boy with newly diagnosed T-cell acute lymphoblastic leukemia (ALL). This is the first reported chromosomal translocation where AML1 is rearranged in childhood T-cell ALL. By metaphase fluorescence in situ hybridization analysis, the AML1 breakpoint was mapped using recombinant phage clones, and shown to be either immediately upstream or downstream of exon 5.