Katrina Rack

Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16

We have completed a long-range restriction map of the terminal region of the short arm of human chromosome 16 (16p13.3) by physically linking a distal genetic locus (alpha-globin) with two recently isolated probes to telomere-associated repeats (TelBam3.4 and TelBam-11). Comparison of 47 chromosomes has revealed major polymorphic length variation in this region: we have identified three alleles in which the alpha-globin genes lie 170 kb, 350 kb, or 430 kb from the telemere. The two most common alleles contain different terminal segments, starting 145 kb distal to the alpha-globin genes. Beyond this boundary these alleles are nonhomologous, yet each contains sequences related to other (different) chromosome termini. This chromosome size polymorphism has probably arisen by occasional exchanges between the subtelomeric regions of nonhomologous chromosomes; analogous length variation is likely to be present at other human telomeres.

Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders

The European Myeloma Network has organized two workshops on fluorescence in situ hybridization in multiple myeloma. The first aimed to identify specific indications and consensus technical approaches of current practice. A second workshop followed a quality control exercise in which 21 laboratories analyzed diagnostic cases of purified plasma cells for recurrent abnormalities. The summary report was discussed at the EHA Myeloma Scientific Working Group Meeting 2010. During the quality control exercise, there was acceptable agreement on more than 1,000 tests. The conclusions from the exercise were that the primary clinical applications for FISH analysis were for newly diagnosed cases of MM or frank relapse cases. A range of technical recommendations included: 1) material should be part of the first draw of the aspirate; 2) samples should be sent at suitable times to allow for the lengthy processing procedure; 3) most importantly, PCs must be purified or specifically identified; 4) positive cut-off levels should be relatively conservative: 10% for fusion or break-apart probes, 20% for numerical abnormalities; 5) informative probes should be combined to best effect; 6) in specialist laboratories, a single experienced analyst is considered adequate; 7) at least 100 PC should be scored; 8) essential abnormalities to test for are t(4;14), t(14;16) and 17p13 deletions; 9) suitable commercial probes should be available for clinically relevant abnormalities; 10) the clinical report should be expressed clearly and must state the percentage of PC involved and the method used for identification; 11) a retrospective European based FISH data bank linked to clinical data should be generated; and 12) prospective analysis should be centralized for upcoming trials based on the recommendations made. The European Myeloma Network aims to build on these recommendations to establish standards for a common European data base to define subgroups with prognostic significance.

Telomere length in myelodysplastic syndromes.

We have studied telomere length in the bone marrow cells or the granulocyte and lymphocyte cell fractions of 54 patients with myelodysplastic syndromes (MDS) by Southern blot hybridization using the (TTAGGG)4 probe. The average telomere length expressed as the peak telomere repeat array (TRA) in the peripheral blood, or bone marrow samples obtained from a group of 21 healthy age‐matched controls (26–89 years old, mean age 55), ranged between 7.5 and 9.5 kb (mean peak TRA 8.6 kb). Twenty‐four patients with refractory anemia (RA) were studied; 10/24 (42%) had telomere reduction (<7.5 kb) relative to age‐matched controls and the mean peak TRA was 7.5 kb (range 4.0–9.0 kb). Eleven patients with RA with excess blasts (RAEB) were studied; 5/11 (45%) had reduced telomeres relative to age‐matched controls and the mean peak TRA was 7.1 kb (range 5.0–9.0 kb). Eighteen patients with MDS in transformation to AML, comprising 15 with RAEB in transformation (RAEBt) and 3 with CMML in transformation (CMMLt), were also studied. Thirteen of eighteen patients (72%) had telomere reduction relative to age‐matched controls and the mean peak TRA was 6.1 kb (range 3.5–9.0 kb). Thirty‐six patients included in the study had either a normal karyotype or a simple karyotype (1 karyotypic change) and 20/36 (55%) of these had telomere reduction and the mean peak TRA was 7.1 kb (range 4.3–9.0 kb); 8 patients had a complex karyotype (3 or more karyotypic changes) and 5/8 (62%) of these had telomere reduction and the mean peak TRA was 6.1 kb (range 3.5–9.0 kb). We conclude, firstly that there is heterogeneity of telomere length in MDS and that this is observed throughout the spectrum of FAB‐subtypes. Secondly, these data show that a marked reduction in telomere length in MDS if often associated with leukemic transformation and with the presence of complex karyotypic abnormalities. Am. J. Hematol. 56:266–271, 1997.

Clonality of cell populations in refractory anaemia using combined approach of gene loss and X‐linked restriction fragment length polymorphism‐methylation analyses

We have used X‐linked restriction fragment length polymorphism (RFLP)‐methylation and gene deletion analyses to investigate the nature of the progenitor cell of origin in the myelodysplastic syndromes (MDS). Gene deletion studies were performed on the granulocyte and T‐lymphocyte fractions of six women with refractory anaemia (RA) and either a partial deletion of the long arm of chromosome 5 (5q‐) or monosomy 7. All six showed gene loss in the granulocyte but not the T‐lymphocyte fractions, indicating monoclonality of the granulocytes but not the T‐lymphocytes. In order to further investigate this finding, we subsequently performed X‐RFLP‐methylation studies using the probe M27β, and also a probe for the phosphoglycerate kinase (PGK) gene. These studies have confirmed the monoclonality of the granulocytes and the polyclonality of the T‐lymphocytes in these cases. Our findings suggest that in this group of patients with MDS the T‐lymphocytes were not involved in the disorder, and furthermore, in the one case where B‐lymphocytes were also available, that the progenitor cell of origin was restricted to the myeloid lineage.

17q21.31 microduplication patients are characterised by behavioural problems and poor social interaction

Background: Microdeletions at 17q21.31 have recently been shown to cause a novel syndrome. Here we identify the reciprocal 17q21.31 duplication syndrome in 4 patients. Method: Patients with the 17q21.31 duplication were identified by screening a large cohort of patients (n = 13 070) with mental retardation and congenital malformation by comparative genomic hybridisation microarray. Parental origin was investigated in 3 patients by quantitative polymerase chain reaction and microsatellite genotyping. Results: In three cases it was possible to show that duplication arose de novo. Intellectual skills range from normal to mild mental retardation. Patients are characterised by poor social interaction, with relationship difficulties, reminiscent of autistic spectrum disorders. Other features are rather variable with no striking common phenotypic features. Parental origin was investigated for 3 patients. In all cases duplication was of maternal origin either through interchromosomal (2 cases) or interchromatid (1 case) rearrangement. The 3 mothers are all carriers of the inverted H2 haplotype, emphasising the role of local genomic architecture alteration as a predisposing factor for this duplication. Conclusion: Autistic features observed in our patients suggest that genes in the duplicated interval should be considered as candidates for disorders in the autistic spectrum. Other phenotypic observations are rather variable or aspecific. This adds 17q21.31 duplications to a growing group of recently identified genomic disorders with variable penetrance and expressivity.

Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin-2 stimulation: A Belgian multicentric study.

We performed a multicentric study to assess the impact of two different culture procedures on the detection of chromosomal abnormalities in 217 consecutive unselected cases with chronic lymphocytic leukemia (CLL) referred for routine analysis either at the time of diagnosis (n = 172) or during disease evolution (n = 45). Parallel cultures of peripheral blood or bone marrow were set up with the addition of either the conventional B‐cell mitogen 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) or a combination of CpG oligonucleotide (CpG) and interleukin‐2 (IL‐2). Cytogenetic analyses were performed on both cultures. Clonal abnormalities were identified in 116 cases (53%). In 78 cases (36%), the aberrant clone was detected in both cultures. Among these, the percentages of aberrant metaphases were similar in both conditions in 17 cases, higher in the CpG/IL‐2 culture in 43 cases, and higher in the TPA culture in 18 cases. Clonal aberrations were detected in only one culture, either in CpG/IL‐2 or TPA in 33 (15%) and 5 (2%) cases, respectively. Taken together, abnormal karyotypes were observed in 51% with CpG/IL‐2 and 38% with TPA (P < 0.0001). Application of FISH (n = 201) allowed the detection of abnormalities not visible by conventional cytogenetic analysis in 80 cases: del(13q) (n = 71), del(11q) (n = 5), +12 (n = 2), del(14q) (n = 1), and del(17p) (n = 1). In conclusion, our results confirm that CpG/IL‐2 stimulation increases the detection rate of chromosomal abnormalities in CLL compared with TPA and that further improvement can be obtained by FISH. However, neither conventional cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques.

Fluorescence In Situ Hybridization Analysis of Masked (8;21)(q22;q22) Translocations

The translocation (8;21)(q22;q22) is associated with acute myeloblastic leukemia (AML M2). The accurate detection of this chromosomal rearrangement is vital due to its association with a favorable prognosis. Variant translocations exist; these may be hidden within an unusual or complex karyotype. In such cases, it is often difficult to confirm the presence of t(8;21)(q22;q22) by conventional cytogenetic analysis alone. The molecular detection of the AML1/ETO fusion gene is possible by reverse transcriptase polymerase chain reaction (RT-PCR) or dual-color fluorescence in situ hybridization (FISH) using probes specific for AML1 and ETO. Four cases of AML M2, with unusual or complex structural chromosomal abnormalities, without cytogenetic evidence of the classical t(8;21)(q22;q22), were studied by FISH. Two were AML1/ETO positive by RT-PCR, one showed a rearrangement by AML1 by Southern analysis, and the fourth had morphological features characteristic of t(8;21). The FISH results showed a co-localization of one AML1 and one ETO signal in interphase and metaphase nuclei in all four cases, demonstrating the presence of variant t(8;21)(q22;q22) rearrangements. Therefore, FISH analysis with the AML1 and ETO probes is extremely valuable, in cases of AML M2, because of its ability to reveal masked t(8;21)(q22;q22) translocations and thus quickly confirm the diagnosis, allowing patients to be assigned to the correct risk group in terms of treatment.

PRDM16 (1p36) translocations define a distinct entity of myeloid malignancies with poor prognosis but may also occur in lymphoid malignancies

The PRDM16 (1p36) gene is rearranged in acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) with t(1;3)(p36;q21), sharing characteristics with AML and MDS with MECOM (3q26.2) translocations. We used fluorescence in situ hybridization to study 39 haematological malignancies with translocations involving PRDM16 to assess the precise breakpoint on 1p36 and the identity of the partner locus. Reverse‐transcription polymerase chain reaction (PCR) was performed in selected cases in order to confirm the partner locus. PRDM16 expression studies were performed on bone marrow samples of patients, normal controls and CD34+ cells using TaqMan real‐time quantitative PCR. PRDM16 was rearranged with the RPN1 (3q21) locus in 30 cases and with other loci in nine cases. The diagnosis was AML or MDS in most cases, except for two cases of lymphoid proliferation. We identified novel translocation partners of PRDM16, including the transcription factors ETV6 and IKZF1. Translocations involving PRDM16 lead to its overexpression irrespective of the consequence of the rearrangement (fusion gene or promoter swap). Survival data suggest that patients with AML/MDS and PRDM16 translocations have a poor prognosis despite a simple karyotype and a median age of 65 years. There seems to be an over‐representation of late‐onset therapy‐related myeloid malignancies.

Translocation t(14;18) is not associated with inferior outcome in chronic lymphocytic leukemia

Translocation t(14;18) is not associated with inferior outcome in chronic lymphocytic leukemia

Translocation t(1;6)(p35.3;p25.2): a new recurrent aberration in ‘unmutated’ B-CLL

Although reciprocal chromosomal translocations are not typical for B-cell chronic lymphocytic leukemia (B-CLL), we identified the novel t(1;6)(p35.3;p25.2) in eight patients with this disorder. Interestingly, all cases showed lack of somatically mutated IgVH. Clinical, morphological, immunologic, and genetic features of these patients are described. Briefly, the age ranged from 33 to 81 years (median: 62.5 years) and the sex ratio was 6M:2F. Most of the patients (6/8) presented with advanced clinical stage. Therapy was required in seven cases. After a median follow-up of 28 months, five patients are alive and three died from disease evolution. Three cases developed transformation into diffuse large B-cell lymphoma. Translocation t(1;6) was found as the primary karyotypic abnormality in three patients. Additional chromosomal aberrations included changes frequently found in unmutated B-CLL, that is, del(11)(q), trisomy 12 and 17p aberrations. Fluorescence in situ hybridization analysis performed in seven cases allowed us to map the t(1;6) breakpoints to the 1p35.3 and 6p25.2 chromosomal bands, respectively. The latter breakpoint was located in the genomic region coding for MUM1/IRF4, one of the key regulators of lymphocyte development and proliferation, suggesting involvement of this gene in the t(1;6). Molecular characterization of the t(1;6)(p35.3;p25.2), exclusively found in unmutated subtype of B-CLL, is in progress.