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.

Cytogenetic and genomic characterization of cell line ARH77.

The cell line ARH77 is derived from a patient with plasma cell leukemia and has a complex and continually evolving karyotype. It is frequently used in biological studies of myeloma and plasma cell leukemia, so accurate characterization of the genome is valuable. Here we present a detailed cytogenetic investigation using G-banding and multicolor fluorescence in situ hybridization (M-FISH) in association with assessment of copy number alterations (CNAs) throughout the genome using array-based comparative genomic hybridization (aCGH). In addition to providing an accurate description of the karyotype, this complementary approach highlighted the relative merits of the individual techniques. Conventional cytogenetics and M-FISH indicated the location and types of the major chromosomal changes, whether balanced or unbalanced, and at the same time demonstrated the level of karyotypic evolution between cells. The aCGH profiles reflected the unbalanced chromosomal abnormalities detected by cytogenetics, providing refinement of their genomic breakpoint locations as well as the identification of novel genomic changes. Three aCGH platforms, comprising bacterial artificial chromosome (BAC) or oligonucleotide templates, were available for evaluation. Sixteen CNAs were consistently detected by all three platforms. Novel submicroscopic CNAs ( approximately 0.4 Mb) were detected by the highest resolution platform only, whereas the clones from the BAC arrays provided locus-specific FISH probes for confirmation of CNA.

Intrachromosomal amplification of chromosome 21 (iAMP21) may arise from a breakage-fusion-bridge cycle.

Intrachromosomal amplification of chromosome 21 (iAMP21), involving amplification of the RUNX1 gene and duplication of chromosome 21, dup(21q), defines a new cytogenetic subgroup in B-lineage acute lymphoblastic leukemia (ALL) with a poor prognosis. Characterization of this abnormality has become vital to ensure that the most accurate detection method is used. We have previously defined common regions of amplification and deletion of chromosome 21 in these patients, although the level and extent of amplification within the amplicon was highly variable. This study, using interphase fluorescence in situ hybridization (FISH) with chromosome 21 locus specific probes, substantiated these findings in a large series of patients and confirmed that the amplicon always included RUNX1. Thus, FISH with probes directed to the RUNX1 gene remains the most reliable detection method. Metaphase FISH, supported by G- and multiple color chromosomal banding (mBAND) revealed the patient specific morphology and genetic profile of the dup(21q) chromosomes, as well as the complexity of the intrachromosomal changes giving rise to them. These findings suggested that iAMP21 had arisen from a breakage-fusion-bridge cycle: a mechanism previously described in tumors, which we report for the first time in ALL.

Prognosis of children with acute lymphoblastic leukemia (ALL) and intrachromosomal amplification of chromosome 21 (iAMP21).

Patients with acute lymphoblastic leukemia (ALL) and an intrachromosomal amplification of chromosome 21 (iAMP21) comprise a novel and distinct biological subgroup. We prospectively screened 1630 (84%) patients treated on the UK MRC ALL97 protocol for iAMP21 and herein present demographic, clinical, and survival data on the 28 (2%) children found to harbor this abnormality. They had a common or pre-B ALL immunophenotype, were significantly older (median 9 years vs 5 years), and had a lower white cell count (median 3.9 vs 12.4) compared with children without this abnormality. Notably, patients with iAMP21 had a significantly inferior event-free and overall survival at 5 years compared with other patients: 29% (95% confidence interval [CI], 13%-48%) versus 78% (95% CI, 76%-80%) and 71% (95% CI, 51%-84%) versus 87% (95% CI, 85%-88%), respectively. As a result of this 3-fold increase in relapse risk, newly diagnosed patients with iAMP21 recruited to the current UK MRC ALL2003 trial are being treated on the high-risk arm and are considered for bone marrow transplantation in first remission.

Complex genomic alterations and gene expression in acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

We have previously identified a unique subtype of acute lymphoblastic leukemia (ALL) associated with a poor outcome and characterized by intrachromosomal amplification of chromosome 21 including the RUNX1 gene (iAMP21). In this study, array-based comparative genomic hybridization (aCGH) (n = 10) detected a common region of amplification (CRA) between 33.192 and 39.796 Mb and a common region of deletion (CRD) between 43.7 and 47 Mb in 100% and 70% of iAMP21 patients, respectively. High-resolution genotypic analysis (n = 3) identified allelic imbalances in the CRA. Supervised gene expression analysis showed a distinct signature for eight patients with iAMP21, with 10% of overexpressed genes located within the CRA. The mean expression of these genes was significantly higher in iAMP21 when compared to other ALL samples (n = 45). Although genomic copy number correlated with overall gene expression levels within areas of loss or gain, there was considerable individual variation. A unique subset of differentially expressed genes, outside the CRA and CRD, were identified when gene expression signatures of iAMP21 were compared to ALL samples with ETV6-RUNX1 fusion (n = 21) or high hyperdiploidy with additional chromosomes 21 (n = 23). From this analysis, LGMN was shown to be overexpressed in patients with iAMP21 (P = 0.0012). Genomic and expression data has further characterized this ALL subtype, demonstrating high levels of 21q instability in these patients leading to proposals for mechanisms underlying this clinical phenotype and plausible alternative treatments.

Interphase molecular cytogenetic screening for chromosomal abnormalities of prognostic significance in childhood acute lymphoblastic leukaemia: a UK Cancer Cytogenetics Group Study.

Summary Interphase fluorescence in situ hybridization (iFISH) was used independently to reveal chromosomal abnormalities of prognostic importance in a large, consecutive series of children (n = 2367) with acute lymphoblastic leukaemia (ALL). The fusions, TEL/AML1 and BCR/ABL, and rearrangements of the MLL gene occurred at frequencies of 22% (n = 447/2027) (25% in B-lineage ALL), 2% (n = 43/2027) and 2% (n = 47/2016) respectively. There was considerable variation in iFISH signal patterns both between and within patient samples. The TEL/AML1 probe showed the highest incidence of variation (59%, n = 524/884), which included 38 (2%) patients with clustered, multiple copies of AML1. We were thus able to define amplification of AML1 as a new recurrent abnormality in ALL, associated with a poor prognosis. Amplification involving the ABL gene, a rare recurrent abnormality confined to T ALL patients, was identified for the first time. The use of centromeric probes revealed significant hidden high hyperdiploidy of 33% and 59%, respectively, in patients with normal (n = 21/64) or failed (n = 32/54) cytogenetic results. The iFISH contributed significantly to the high success rate of 91% (n = 2114/2323) and the remarkable abnormality detection rate of 89% (n = 1879/2114). This study highlights the importance of iFISH as a complementary tool to cytogenetics in routine screening for significant chromosomal abnormalities in ALL.

ETV6/RUNX1 fusion at diagnosis and relapse: some prognostic indications.

This study was undertaken in order to compare the interphase and metaphase cytogenetics of 28 patients with ETV6/RUNX1 positive acute lymphoblastic leukemia, at diagnosis and relapse. The median time to relapse was 26 months. The significant fusion positive population heterogeneity revealed at interphase by a commercial probe for ETV6/RUNX1 fusion has not been described before. Six diagnostic samples had a single abnormal population; others had up to five each, which differed in the numbers of RUNX1 signals, and in the retention or loss of the second ETV6 signal. In contrast, the number of fusion signals was more constant. At relapse, there were fewer populations; the largest or unique clone was sometimes a re-emergence of a minor, diagnostic one, with a retained copy of ETV6 and the most RUNX1 signals. Abnormal, fusion negative clones were identified in bone marrow samples at extra-medullary relapse. Variant three or four-way translocations, which involved chromosomes 12 and 21, were prominent among the complex rearrangements revealed by metaphase FISH. The frequency of their occurrence at diagnosis and reappearance at relapse, sometimes accompanied by minor clonal evolution, was another new observation. Other recurrent cytogenetic features included a second copy of the fusion signal in six cases, partial duplication of the long arm of the X chromosome in two cases, and trisomy 10 in three cases. In comparing our data with previously reported cases, a picture is beginning to emerge of certain diagnostic features, which may provide circumstantial evidence of an increased risk of relapse.

Complex chromosomal abnormalities in utero, 5 years before leukaemia.

Prenatal acquisition of leukaemia-associated gene rearrangements is a well-established phenomenon. This is the first report of a complex cytogenetic clone, in association with an ETV6/AML1 fusion, developing in utero. Identical twin girls, aged 4 years, developed ETV6/AML1-positive acute lymphoblastic leukaemia (ALL) within 3 months of one another. Both demonstrated an identical four way, variant t(12;21). There was gain of an AML1 signal in twin 1 and loss of an ETV6 one in twin 2 at interphase. This unique case study demonstrates that ETV6/AML1 fusion and the associated complex chromosomal rearrangements occurred in utero. Clonal expansion of the abnormal cell in one twin was followed by metastasis to the other. There was a prolonged preleukaemic phase, which lasted well into childhood. The short time between the two diagnoses of ALL suggests a common precipitating event. The significance of the different secondary markers remains unclear.

Three distinct subgroups of hypodiploidy in acute lymphoblastic leukaemia.

This study of children and adults with acute lymphoblastic leukaemia (ALL) is the largest series of patients with hypodiploidy (<46 chromosomes) yet reported. The incidence of 5% was independent of age. Patients were subdivided by the number of chromosomes; near-haploidy (23-29 chromosomes), low hypodiploidy (33-39 chromosomes) and high hypodiploidy (42-45 chromosomes). The near-haploid and low hypodiploid groups were characterized by their chromosomal gains and a doubled hyperdiploid population. Structural abnormalities were more frequent in the low hypodiploid group. Near-haploidy was restricted to children of median age 7 years (range 2-15) whereas low hypodiploidy occurred in an older group of median age 15 years (range 9-54). Patients with 42-45 chromosomes were characterized by complex karyotypes involving chromosomes 7, 9 and 12. The features shared by the few patients with 42-44 chromosomes and the large number with 45 justified their inclusion in the same group. Survival analysis showed a poor outcome for the near-haploid and low hypodiploid groups compared to those with 42-45 chromosomes. Thus cytogenetics, or at least a clear definition of the modal chromosome number, is essential at diagnosis in order to stratify patients with hypodiploidy into the appropriate risk group for treatment.

t(14;19)(q32;q13): a recurrent translocation in B-cell precursor acute lymphoblastic leukemia.

The recurrent t(14;19)(q32;q13) translocation associated with chronic B-cell lymphoproliferative disorders, such as atypical chronic lymphocytic leukemia, results in the juxtaposition of the IGH@ and BCL3 genes and subsequent overexpression of BCL3. We report six patients with B-cell precursor acute lymphoblastic leukemia who have a cytogenetically identical translocation with different breakpoints at the molecular level. Fluorescence in situ hybridization with locus-specific probes confirmed the involvement of the IGH@ gene but showed that the breakpoint on 19q13 lay outside the region documented in t(14;19)(q32;q13)-positive chronic lymphocytic leukemia. This newly described translocation constitutes a distinct cytogenetic subgroup that is confined to older children and younger adults with B-cell precursor acute lymphoblastic leukemia.

Outcome heterogeneity in childhood high-hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy (HeH) (51 to 65 chromosomes) is found in one third of children with acute lymphoblastic leukemia and is associated with a good prognosis. Cytogenetic features may further refine this prognosis and identify patients with a poor outcome. We examined the effect of sex, age, individual trisomies, modal number, and structural abnormalities on survival among 700 children with HeH. Univariate analysis showed that age. sex, +4, +10, +18, and a high modal number were associated with survival. Multivariate analysis however, revealed that only age, sex, +4, and +18 were independent indicators. Hazard scores for predicting relapse and mortality were constructed. Three risk groups with 5-year event-free survival (EFS) rates of 86%, 75%, and 50% (P <.0001) were identified. The high-risk group comprised boys older than 9 years, boys aged 1 through 9 years without +18, and girls older than 9 years without +18, while girls aged 1 through 9 years with +18 had the best EFS. In terms of mortality, those younger than age 10 years with both +4 and +18 had an improved survival (96% vs 84% at 5 years, P <.0001). These findings confirm that the outcome of children with HeH is heterogeneous and that specific trisomies can identify patients with the greatest and least risk of treatment failure.

The efficacy of acarbose in type 2 diabetes mellitus in Jamaica

The efficacy and tolerability of acarbose was studied in type 2 diabetic patients eating a typical Jamaican diet. The study was an open label parallel group study without placebo control. Of the 51 subjects recruited, five (9.8 percent) did not complete the study and were excluded from further analysis. Six (13 percent) of the remaining 46 had adverse side effects and did not complete the protocol. Of the remaining 40 (Gp A), acarbose was added to their previous regime of diet alone (n=15), [Gp B], oral hypoglycaemic agents, OHAs (n=17), [Gp C], or insulin (n=8), Gp D]. In addition, during the run in period all subjects had one session each with a dietitian and a diabetes educator. Over a 3 month period, significant reductions in average glucose (mmol) were observed in Gp B 10.5 ñ 1.1 to 8.4 ñ 0.9 (p<0.027) and, from 11.0 ñ 1.0 to 8.7 ñ 0.7 (p<0.01) in Gp C. Similarly, total glycosylated haemoglobin fell from 14.8 ñ 1.1 percent to 12.2 ñ 1.0 percent (p<0.016) in Gp B, from 14.8 ñ 1.1 to 11.9 ñ 1.1 percent (p<0.002) in Gp C, and from 14.1 ñ 1.4 to 11.8 ñ 1.4 (p<0.02) in Gp D. Twenty-three per cent (23 percent) of the patients experienced flatulence; 7.5 percent changes in bowel habits and 5 percent, abdominal cramps and discomfort. Acarbose is effective as monotherapy and as combination therapy with oral hypoglycaemic agents or insulin. Side effects were common, but tolerable.(Au)

A possible aetiological model for malnutrition related diabetes mellitus (type III)

This study was undertaken to examine the diabetogenicity of linamarin, the predominant cyanoglucoside found in cassava. Cassava is a food staple, widely consumed in Jamaica, suspected of inflicting the primary insult in the onset of malnutrition related diabetes dellitus (MRDM). Male and female mongrel dogs obtained from the preclinical animal house were maintained on a diet of cornmeal cooked with pieces of chicken, fortified with Purina Laboratory Chow and water ad libitum. Undernutrition was induced by giving a very small portion of cornmeal only, for 10 days. The recovered were re-fed the normal diet with added milk and multivitamin supplements. The total dog population was 35; 15 normals, 10 undernourished and 10 recovered. On each dog, testing blood sugar along with serum insulin and insulin receptor studies were done followed by a control glucose tolerance test (GTT). After feeding linamarin, a residence time of 0.5 hour was allowed to elapse before these investigations were repeated. Linamarin dosage of 40 mg/kg body weight had no effect on the blood sugar level or receptor activity in the normal dogs. Even at dosage of 60 mg/kg the recovered dogs showed no abnormality, while in the undernourished group a dosage of 20 mg/kg induced abnormally high (p<0.05) glucose levels with a concomittant decrease in serum insulin (p<0.05) and insulin binding (p<0.05) to mononuclear leucocytes. These results point to the possible aetiology of diabetes by "Toxic" food substances in the undernourished state. (AU)

Effects of cassava cyanoglucoside, linamarin, on blood sugar levels in the dog

This study was undertaken to examine the diabetogenicity of linamarin in dogs of different nutritional status. The total dog population was 35; 15 normals, 10 undernourished, and 10 recovered. The recovered dogs were the refed undernourished dogs, On each dog a fasting plasma insulin and insulin receptor studies were done, followerd by a controlled glucose tolerance test. After feeding linamarin. 0.5 hour was allowed to elapse before these investigations were repeated. In the linamarin-treated udernourished animal, plasma insulin and insulin binding to erythrocytes and mononuclear leucocytes were significantly (P> 0.01) lower than in the control animals, and this resulted in abnormally high glucose levels. Linamarin administration had no effect on plasma insulin, insulin binding, or blood sugar levels in the normal and recovered dogs. These results point to the possible aetiology of diabetes in the undernourished state by 'toxic' foods substances such as linamarin. (AU)

The efficacy of 2-hour oral glucose tolerance test (OGTT) to identify pre-diabetes and gestational diabetes in pregnant mothers at the University Hospital of the West Indies - abstract

A pilot study was conducted at the UHWI to evaluate the OGTT as a tool for identifying gestational diabetes in our clinic population. It was performed on 21 high-risk pregnant mothers. When the current WHO criteria for the diagnosis of gestational glucose intolerance were applied, one (1) patient was considered to have gestational diabetes (4.8 per cent), seven (7) patients had impaired glucose tolerance (33.3 per cent), and thirteen (13) were considered normal (61.9 per cent). Insulin levels were determined for 13 of the patients and, within the group of patients with normal OGTT, two distinct groups were identified. Seven of the patients in this group had insulin levels determined, and of these, three had insulin levels similar to that of the impaired glucose tolerance group. Two of these three patients had foetal macrosomia. The remaining four in this group with normal OGTT had normal insulin curves and normal foetal sizes. Six of the patients with impaired glucose tolerance had insulin levels determined and two presented with foetal macrosomia (33 per cent). The results indicate that in spite of screening for impaired carbohydrate metabolism in pregnancy there are still patients who are misdiagnosed by the present WHO criteria using OGTT. Insulin assays may be a more sensitive predictor of altered carbohydrate metabolism (AU)

Hyperinsulinaemia accompanies the development of pre-elcampsia - abstract

Hyperinsulinaemia with insulin resistance has been observed in pre-eclampsia. In this longitudinal study, we examined whether this state of insulin resistance preceded the development of eclampsia. At 12, 24 and 36 weeks of gestation and at 12 weeks post-partum in twenty-five primigravidae, insulin and glucose were measured in the fasting state and in samples collected during intravenous glucose tolerance tests (IVGTT). Five women were diagnosed as pre-eclamptic (PE) at 34 - 39 weeks' gestation. In these PE, there was hyperinsulinaemia in the fasting state at 24 weeks compared to the normal pregnant (NP) women, 20.07 ñ 8.31 mU/ml vs. 13.74 ñ 5.64 mU/ml, (p<0.05). The degree of hyperinsulinaemia increased and, at 36 weeks' gestation it was 26.4 ñ 7.7 mU/l in the PE compared to 13.1 ñ 6.1 mU/l in the NP, (p<0.0001). The sensitivity of this measure for predicting pre-eclampsia at mid-pregnancy (24 weeks) was 80 per cent which increased to 100 per cent in late pregnancy (36 weeks). The specificity of this measure went from 57 per cent in mid-pregnancy to 43 per cent in late pregnancy. The glucose levels in the fasting state were not different at any time in the study. In the IVGTT, the area under the insulin curve was significantly larger in the PE as a group compared to the NP, (p<0.05). The area under the glucose curve was not different between the two groups at any time. The rate of disappearance of glucose Kg was significantly larger in the PE as a group compared to the NP, (p<0.05). Hyperinsulinaemia in the fasting state precedes the development of pre-eclampsia. It has a high sensitivity but low specificity for predicting the development of pre-eclampsia. Hyperinsulinaemia in the fasting state may have some value in screening those primigravidae at risk for developing pre-eclampsia (AU)

Zinc intake and plasma insulin-like growth factor-I (IGF-I) in Jamaican malnourished children - abstract

The effect of zinc intake on plasma immunoreactive insulin-like growth factor-I (IR-IGF-1) was studied in 24 children (aged 3 to 24 mos) recovering from severe malnutrition. The children were randomly assigned to two groups for zinc supplementation. Twelve (Zn+) were given (2mg/kg/d) added to the recovery of diet, and 12 (ZN-) were not. There was no group difference in age, sex, anthropometric measurements or plasma IR-IGF-1 at baseline. Plasma IR-IGF-1 was significantly higher in the zinc supplemented children during recovery (p = 0.031). These results suggest that zinc intake stimulates IR-IGF-1 production. (AU)

The effect of malnutrition on insulin binding to rat erythrocytes

Insulin binding to erythrocyte receptors was compared in malnourished and control rats. Percentage specific insulin binding to malnourished rat erythrocytes was significantly lower than to control erythrocytes (P<0.001). The low insulin binding in the malnourished rat erythrocytes was accompanied by low insulin receptor affinity (P=0.035).(AU)

Fasting plasma insulin and C-peptide levels in pre-eclampsia - abstract

In this study, plasma insulin, C-peptide and glucose measurements were done on the fasting blood samples obtained from primigravid pre-eclamptic (PE) women and suitably matched normal pregnant (NP) controls. This was done in order to observe any differences in the pancreatic secretion of insulin between these two groups. Fasting hyperinsulinaemia was observed as a statistically significant feature in the PE. The fasting C-peptide and glucose levels were not significantly different between the two groups. From the fasting C-peptide levels observed it would appear that the hyperinsulinaemia observed in the PE was not the result of hypersecretion of insulin by the pancreas (AU)