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This thesis proposes a novel lower-limb prosthetic device. Current prosthetics either have overly simplistic designs with inaccurate biomechanics or use delicate microprocessors that are easily damaged in harsh environments. This thesis aims to address these concerns by creating a novel device that combines a pneumatic damping system with a ball joint, resulting in a robust design with improved biomechanics. This prosthetic offers an affordable alternative that can be completely rebuilt while providing added comfort through improved biomechanics. Overall, this thesis contributes to the literature by proposing and discussing an innovative design for an affordable, comfortable, biomechanically sound alternative for lower limb prostheses.

In addition to structural chromosome aberrations resulting in fusion genes, the genome profile of haematological malignancies is characterized by copy number changes (gains and losses) relative to the ploidy level. Array comparative genome hybridization (aCGH)1 Combined with FISH and/or multiplex PCR array screening offers a reliable tool for assessment of genome complexity in haematological malignancies in diagnostic settings. The aCGH assay in our diagnostic algorithm uses 8 × 60K SurePrint G3 (Agilent, USA). The slides are scanned to measure the fluorescence ratio (FR), software evaluates imbalances. We recognize that the raw data in the form of log ratios, some 64,000 data points, contain sufficient information to identify the disease category without resorting to converting the log ratios to gains and losses and mapping them to individual chromosomes, a process open to operator interpretation. As an alternative and bearing in mind the current interest in machine learning, we attempted to auto-classify the data for the modest collection of 2,300 patient samples in our archive, according to their WHO classification. We were able to collect 175 samples for each group classified as myelodisplasia (MDS), acute myeloid leukemia (AML), myeloma (MM) and chronic lymphocytic leukemia (CLL), We used a Logistic Regression Classifier implemented in Mathematica 12. The classifier took only 2 minutes on average to complete the training. We took a total of 700 array sample files, each of 59,032 data points, of which 100 were randomly selected as a validation set. The balance of 600 files, distributed evenly and randomly over the 4 test diseases were used as a training set. We used a Confusion Matrix to evaluate and display the results as shown in the table. Confusion MatrixAMLCLLMDSMMTotalAML1609025CLL0150116MDS0116017MM1021417Predicted17162715 In spite of the small training set we correctly classified the MDS (16/17), CLL (15/16) and MM (14/17), 9 AML were reported as MDS a due to sharing genome imbalances. There was also confusion between the B cell disorders, MM and CLL known to carry overlapping genome aberrations. The auto classification offered by this method is a promising attempt to support a speedy and reliable assessment of genome complexity. There is no doubt that a better classifier would have been created with a larger training set. 1 Kallioniemi A et al., Kallioniemi OP, Sudar DA, Rutovitz D, Gray JW, Waldman F, Pinkel D (1992). “Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors”. Science. 258 (5083): 818-821). Citation Format: Colin Grace, Temenuzhka Boneva, Elisabeth P. Nacheva. Successful classification of hematological neoplasm by array profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3641.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

Philadelphia positive malignant disorders are a clinically divergent group of leukemias. These include chronic myeloid leukemia (CML) and de novo acute Philadelphia positive (Ph(+)) leukemia of both myeloid, and lymphoid origin. Recent whole genome screening of Ph(+)ALL in both children and adults identified an almost obligatory cryptic loss of Ikaros, required for the normal B cell maturation. Although similar losses were found in lymphoid blast crisis the genetic background of the transformation in CML is still poorly defined. We used Significance Analysis of Microarrays (SAM) to analyze comparative genomic hybridization (aCGH) data from 30 CML (10 each of chronic phase, myeloid and lymphoid blast stage), 10 Ph(+)ALL adult patients and 10 disease free controls and were able to: (a) discriminate between the genomes of lymphoid and myeloid blast cells and (b) identify differences in the genome profile of de novo Ph(+)ALL and lymphoid blast transformation of CML (BC/L). Furthermore we were able to distinguish a sub group of Ph(+) ALL characterized by gains in chromosome 9 and recurrent losses at several other genome sites offering genetic evidence for the clinical heterogeneity. The significance of these results is that they not only offer clues regarding the pathogenesis of Ph(+) disorders and highlight the potential clinical implications of a set of probes but also demonstrates what SAM can offer for the analysis of genome data.

The aim of this study was to investigate the distribution of CNVs in patients with coronary atherosclerosis and to assess the association between them. A total number of 31 subjects (13 Females and 18 Males) were involved in the study. They were divided into two groups according to the clinical diagnosis. The first group consisted of 21 patients with non-ST segment elevation ACS (unstable angina and non ST elevation myocardial infarction) and the second - from 10 healthy subjects.The number of CNVs observed using aCGH kit was 334. One hundred and twenty six (37.73%) are newly observed, 153 out of all 334 were from gene coding regions. The genes, which contain newly descrined CNVs, and their products were found to have role in cellular metabolism, regulation of transcription, transport and signal transduction. The present study suggests that there is a relation between CNVs described by us and the possible processes involved in the development of CAD. These observations need to be verified on a larger group of patients to clarify the role of these possible links.

The most common abnormality of chromosome 20 in haematological malignancy is deletion of the long arm [del(20q)]. These interstitial deletions are variable in size and are seen in both premalignant haematological conditions and acute myeloid neoplasia. A commonly deleted region (CDR), mapped within the 20q11.2/q13.1 segment with an estimated size of 1.7 Mbp, is considered to present a primary genetic lesion marking a gene(s), the loss of which is responsible for the pathogenesis of these haematological disorders. While a small number of recurrent translocations involving chromosome 20 have also been reported, no recurrent aberration of this chromosome has been associated with myeloid disease progression. We present nine cases of Philadelphia (Ph)-positive chronic myeloid leukaemia (CML) in which deletions of chromosome 20 were also detected by conventional karyotyping. In six cases, fluorescent in situ hybridization (FISH) mapping confirmed a del(20q) which corresponded to the myeloid CDR. In the remaining three cases however, the presumed del(20q) marker was shown to be the result of an unbalanced translocation between band 20p11 and a second copy of the Ph chromosome. This new abnormality, termed dic(20;Ph) for short, was identical to a del(20)q by G-banding, and combined duplication of the breakpoint cluster region and Abelson murine leukaemia viral oncogene homologue (BCR-ABL) fusion with loss of the 20p11-pter segment. In all three cases, the dic(20;Ph) was associated with disease progression and therefore represents a new recurrent abnormality in CML blast crisis.

The acute phase of chronic myeloid leukemia (CML) is accompanied by secondary chromosomal changes. The additional changes have a non-random pattern; however, highly abnormal (marker) chromosomes are reported in some 20% of abnormal karyotypes. These marker chromosomes have proved to be beyond the resolution of conventional G-banding analysis. We used molecular cytogenetic techniques to determine the structure of complex chromosome markers in 10 CML-derived cell lines after our investigations of CML patients in blast crisis. Multicolor fluorescence in situ hybridization identified a multitude of structural chromosome aberrations. In addition, genomic gains identified by comparative genomic hybridization (CGH) were mapped to highly complex marker chromosomes in more than one cell line. The most common genomic loss detected by CGH affected chromosome 9, whereas the most common genomic gains affected, in order of frequency, the sequences of 8q,6, and 13q. The smallest discrete amplification on 8q was identified in cell line MEG-01. This amplicon contains sequences represented by the marker D8S263/RMC08P029 but did not contain the proximal MYC gene or a more distal marker, D8S256/RMC08P025. We determined the size of the amplicon to be less than the chromosome segment 8q24.12-q24.13. The use of region- and locus-specific probes to analyze the organization of highly complex marker structures aided the identification of preferentially amplified genomic regions. The resultant amplifications could harbor gene(s) driving disease progression. (C) 2003 Wiley-Liss, Inc.

The BCR-ABL fusion in chronic myeloid leukaemia (CML) is generated by the Philadelphia (Ph) translocation t(9;22) or, in 10% of patients, variants thereof (vPh). Deletion encompassing the reciprocal product (ABL-BCR) from the derivative chromosome 9 [der(9)] occurs in 15% of all patients, but with greater frequency in vPh patients. Reports of physical separation of ABL-BCR in non-deleted patients, as well as evolution from classical to variant Ph, introduce further heterogeneity to the vPh subgroup and raise the possibility that such translocations may herald disease progression. Survival analyses, however, have thus far yielded contradictory results. We assessed the frequency of der(9) deletions, ABL-BCR abrogation, cytogenetic evolution and cryptic rearrangement in a large cohort of 54 patients with vPh CML. Deletions encompassing ABL-BCR were detected in 37% of patients, consistent with a model in which a greater number of chromosome breaks increases the risk of genomic loss. The components of ABL-BCR were physically separated in a further 52% of patients while fused in the remaining 11%. Evolution from classical to vPh was demonstrated in three patients. The difference in survival, as indicated by Kaplan-Meier analysis, was marked between classical and vPh patients (105 vs 60 months respectively; P = 0.0002). Importantly, this difference disappeared when patients with deletions were removed from the analysis. Our study showed that, despite the existence of several levels of genomic heterogeneity in variant Ph-positive CML, der(9) deletion status is the key prognostic factor.

Chronic myeloid leukemia (CML) is characterized by formation of the BCR-ABL fusion gene, usually as a consequence of the Philadelphia (Ph) translocation between chromosomes 9 and 22. Large deletions on the derivative chromosome 9 have recently been reported, but it was unclear whether deletions arose during disease progression or at the time of the Ph translocation. Fluorescence in situ hybridization (FISH) analysis was used to assess the deletion status of 253 patients with CML. The strength of deletion status as a prognostic indicator was then compared to the Sokal and Hasford scoring systems. The frequency of deletions was similar at diagnosis and after disease progression but was significantly increased in patients with variant Ph translocations. In patients with a deletion, all Ph+ metaphases carried the deletion. The median survival of patients with and without deletions was 38 months and 88 months, respectively (P = .0001). By contrast the survival difference between Sokal or Hasford high-risk and non-high-risk patients was of only borderline significance (P = .057 and P = .034). The results indicate that deletions occur at the time of the Ph translocation. An apparently simple reciprocal translocation may therefore result in considerable genetic heterogeneity ab initio, a concept that is likely to apply to other malignancies associated with translocations. Deletion status is also a powerful and independent prognostic factor for patients with CML. The prognostic significance of deletion status should now be studied prospectively and, if confirmed, should be incorporated into management decisions and the analysis of clinical trials. (C) 2001 by The American Society of Hematology.

A range of fluorescent in situ hybridization techniques have been used to reveal hidden variant Philadelphia translocations in two cases of Ph-positive chronic-phase chronic myeloid leukaemia. In one patient, a highly complex variant Ph translocation affecting four chromosomes had resulted in the formation of structures with the appearance of i(17q) and +8. Misinterpretation of these karyotypes has direct clinical relevance. Our findings illustrate that even established cytogenetic abnormalities may contain cryptic abnormalities beyond the resolution of conventional cytogenetic methods.

We report the application of multi-color fluorescence in situ hydribidization (FISH) for bone marrow metaphase cell analysis of hematological malignancies using a sub-set of the human karyotype for chromosome painting. A combination of chromosome probes labeled with three haptens enabled the construction of a “painting probe” which detects seven different chromosomes. The probe was used to screen three chronic myeloid leukemia (CML) derived cell lines and ten CML patient bone marrow samples for aberrations, additional to the Ph rearrangement, that are associated with the onset of blast crisis of CML. This approach was shown to identify karyotype changes commonly seen by conventional karyotyping, and in addition revealed chromosome changes unresolved or undetected by conventional cytogenetic analysis. The seven-color painting probe provides a useful, fast, and reliable complementary tool for chromosome analysis, especially in cases with poor chromosome morphology. This is a simple approach, since the probes can be displayed in a standard red/green/blue format accessible to standard fluorescence microscopes and image-processing software. The proposed approach using panels of locus-specific probes as well as chromosome paints will be useful in all diagnostic routine environments where analysis is directed towards screening for genetic rearrangements and/or specific patterns of chromosome involvement with diagnostic/prognostic value.

The transformation of chronic myeloid leukemia (CML) from a chronic phase to an acute phase is frequently accompanied by additional chromosome changes. Extensive chromosome G-banded studies have revealed the secondary changes are nonrandom and frequently include trisomy 8, isochromosome 17q, trisomy 19, or an extra copy of the Philadelphia chromosome. In addition to these secondary chromosome changes, complex structural rearrangements often occur to form marker structures that remain unidentified by conventional G-banded analysis. The CML-derived cell line, K562, has been widely used in research since it was originally established in 1975. The K562 karyotype however, has remained incomplete, and marker structures have never been fully described. Recent advances in fluorescence in situ hybridization (FISH) technology have introduced the possibility of chromosome classification based on 24-color chromosome painting (M-FISH). In this study, we report a clarified karyotype for K562 obtained by a combination of the following molecular cytogenetic techniques: comparative genomic hybridization (CGH), FISH mapping using locus-specific probes, and M-FISH. Multicolor FISH has identified the marker structures in this cell line. The characteristic marker chromosome in K562 has been confirmed by this study to be a der(18)t(1;18). Multicolor FISH confirmed the identity of marker structures partially identified by G-banding as der(6)t(6;6),der(17)t(9;17),der(21)t(1;21),der(5)t(5;6). In addition M-FISH has revealed a deleted 20q and a complex small metacentric marker comprised of material from chromosomes 1, 6, and 20. A cryptic rearrangement was revealed between chromosomes 12 and 21 that produced a structure that looks like a normal chromosome 12 homologue by G-banding analysis. Finally, M-FISH detected regions from chromosome 13 intercalated into two acrocentric markers.

Chronic myeloid leukemia is a clonal stem cell disorder associated with the Philadelphia (Ph) translocation [t(9; 22) (q34; q11)]. As a result of the Ph translocation, parts of the ABL and BCR genes become fused. Cytogenetic quantification of Ph+ metaphases can be used to monitor patient response to treatment but is of limited sensitivity and applies only to cycling cells. Fluorescence in situ hybridization (FISH) with probes from the BCR and ABL regions can also identify the Ph translocation in interphase cells. Established systems for the detection of fusion genes by FISH rely on colocalization of two different probes but are associated with a high rate of false-positive results. We have introduced a third probe labeled with a different fluorochrome to create a triple-probe/three-color system that permits identification of both the Ph chromosome and the derivative 9 chromosome in Ph+ cells. This system was used to determine the frequency of interphase cells carrying the BCR-ABL fusion gene in bone marrow and peripheral blood granulocytes from patients showing variable cytogenetic responses to interferon. Our data show that the triple-probe/three-color approach allows highly sensitive detection of residual disease. Moreover, this method is readily applicable to the analysis of other chromosome translocations.

Neuroblastoma tumors show a complex interaction of genetic abnormalities, among which some are of significant prognostic importance; however, analysis of chromosome changes in this tumor is often unsuccessful. Twenty primary tumors were studied by comparative genomic hybridization (CGH), and abnormalities were found in 19. While these changes included deletions of chromosome arm Ip (45%) and MYCN oncogene amplification (30%), gains of chromosome 17 material were much more frequent (75%). We also found evidence in two cases of a new amplification site at band 2p23.

Multiple myeloma (MM) is a malignant disorder characterized by neoplastic transformation of mature B cells in the bone marrow (BM), accompanied by complex genetic changes. The disease is heterogeneous at both the clinical and genomic levels. Molecular genetics and genomic investigations have demonstrated that disease evolution is associated with an accumulation of specific aberrations, mostly genome imbalances, which not only shed light on the disease pathogenesis but also allow risk assessment and treatment monitoring. We used a catalogue version of the Agilent 8x60K oligo-array with immuno-magnetically isolated CD138(+) cells from BM samples of 50 patients with myeloma to evaluate the merit of array comparative genomic hybridization (aCGH) as a diagnostic tool. We demonstrate the ability of aCGH to detect clonal imbalances to a level well below established clinically significant thresholds. aCGH, combined with target enrichment and complemented with tests for IGH rearrangements offers a cost neutral alternative to multiprobe fluorescence in situ hybridization screening. While we recognize the limitations of the standard version of the 8x60k array we demonstrate the value of aCGH as a first tier test in the diagnostic workup of MM. The array technology enables high-risk disease stratification with the added benefit of providing whole genome data to assist in establishing clinically relevant predicative markers.

Comparative genomic hybridization (CGH) represents a new technique for global analysis of a whole genome for net loss or gain of chromosome regions. It offers several advantages over alternative techniques. It permits analysis of a whole genome in a single hybridization reaction, it does not require the generation of metaphases from tumor cells, and it only requires very small numbers of tumor cells. Most previous studies have concentrated on the application of CGH to the analysis of chromosome defects associated with solid tumors. In this paper we report the use of CGH to study bone marrow samples from a patient with acute myeloid leukemia and complex karyotypic abnormalities. The results obtained using CGH were compared with G-banding analysis. Both G-banding and CGH detected a 5q deletion, a 7q deletion, additional material derived from 8q, and an HSR on 11q. However, several apparently discrepant results were also obtained. Paints for chromosomes 3, 5, 7, 8, 11, 12, 14, 17, 22, and X were therefore used to resolve these differences. Our results demonstrate that CGH detected chromosome abnormalities associated with acute myeloid leukemia and that CGH provided information that was not obtained by G-banding analysis alone. These data suggest that CGH may prove a useful adjunct to conventional cytogenetic and molecular analysis of hematologic malignancies.

Deletions of the long arm of chromosome 20 are associated with several myeloid malignancies. We have analyzed the structure of the del(20q) in 30 patients and two cell lines. Twenty-one of the patients presented with a myeloproliferative disorder and nine with a myelodysplastic syndrome. Two categories of deletions were identified. Eighteen patients had a large deletion with loss of both G(+) bands from the long arm of chromosome 20. Twelve patients had small deletions with loss of one G(+) band from the long arm of chromosome 20. A chromosome paint was generated from a del 20q marker carrying a small deletion. This probe was hybridized to normal metaphases (reverse chromosome painting) and also to metaphases from patients with a del 20q (comparative reverse chromosome painting). All six small deletions analyzed were characterized by loss of the proximal G(+) band (q12) and retention of the distal G(+) band (q13.2). These data define a minimal deleted region extending from 20q11.2-20q13.1.

Rare de novo acute leukemias of mature B cells may exhibit the chromosomal translocation t(14;18)(q32.3; q21.3). We report the preliminary characterization of two cases that exhibited not only t(14;18)(q32.3;q21.3) but also the novel translocation t(8;9)(q24.1;p13.3), involving the C-MYC locus with unknown sequences at 9p13.3. From these cases, two Epstein-Barr virus negative cell lines (Karpas 231 and 353) with features identical to those seen in fresh cells from the patient have been derived. Both cell lines have complex karyotypes: in addition to both t(14;18)(q32.3;q21.3) and t(8;9)(q24.1;p13.3), cell line Karpas 231 exhibited three-way translocation t(1;3;11) (q42.3;q27.1;q23.1), whereas Karpas 353 exhibited t(1;3;7)(p32.1;q21.1;q22.1). Both cases retained immunophenotypes characteristic of mature B cells with no evidence for commitment to other hematopoietic lineages. Both cases expressed abundant, normal-sized C-MYC transcript, but no rearrangement of C-MYC DNA sequences could be detected using probes that span 80 kb around the C-MYC coding sequences. Breakpoints within the BCL-2 gene were divergent. In Karpas 353 the BCL-2 breakpoint occurred within the 3′ untranslated major breakpoint region (mbr) of the gene, whereas, in Karpas 231, breaks in both the 3′ mbr and in the region 5′ of the gene were detected. The cytogenetic combination of t(14;18)(q32.3;q21.3) and t(8;9)(q24.1;p13.3) has been previously reported in diffuse B cell lymphomas. This combination may be a new recurrent abnormality, of central pathogenic importance in the transformation of B cells to high grade malignancies through simultaneous deregulation of BCL-2 and C-MYC genes, constitutive expression of C-MYC being driven by currently unknown DNA sequences on chromosome 9p13.3. The presence of other complex translocations including those affecting 11q23.1 may further accelerate the process of acute transformation.

The expression of the chimeric BCR/ABL1 fusion gene resulting from t(9;22)(q34;q11) in chronic myelogenous leukemia (CML) is necessary for malignant transformation, but not sufficient to maintain disease progression. The appearance of various chromosomal and molecular alterations in the accelerated and terminal phase of CML is well documented, but evidence for causal relationship is largely lacking. We carried out a genome wide screening at a resolution of 1 Mb of 54 samples at different stages of CML together with 12 CML cell lines and found that disease progression is accompanied by a spectrum of recurrent genome imbalances. Among the most frequent are losses at 1p36, 5q21, 9p21, and 9q34 and gains at 1q, 8q24, 9q34, 16p, and 22q11, all of which were located with higher precision within the genome than previously possible. These genome imbalances are unique to CML cases with clinically manifested or suspected accelerated/blast stage alike, but not seen in chronic phase samples. Previously unrecognized cryptic imbalances occurring within the Ph-chromosome were also detected, although further scrutiny is required to pin-point gene involvement and seek association with disease features. Importantly, some of these imbalances were seen in the CD34(+) cells but not in the whole BM samples of patients in accelerated phase. Taken together, these findings highlight the potential of screening CD34(+) cells for genome wide imbalances associated with disease progression. Finally, the numerous single copy number variations recorded, many unique to this cohort of patients, raise the possible association of genome polymorphism and CML. © 2007 Wiley-Liss, Inc.

The most common abnormality of chromosome 20 in haematological malignancy is deletion of the long arm [del(20q)]. These interstitial deletions are variable in size and are seen in both premalignant haematological conditions and acute myeloid neoplasia. A commonly deleted region (CDR), mapped within the 20q11.2/q13.1 segment with an estimated size of 1.7 Mbp, is considered to present a primary genetic lesion marking a gene(s), the loss of which is responsible for the pathogenesis of these haematological disorders. While a small number of recurrent translocations involving chromosome 20 have also been reported, no recurrent aberration of this chromosome has been associated with myeloid disease progression. We present nine cases of Philadelphia (Ph)-positive chronic myeloid leukaemia (CML) in which deletions of chromosome 20 were also detected by conventional karyotyping. In six cases, fluorescent in situ hybridization (FISH) mapping confirmed a del(20q) which corresponded to the myeloid CDR. In the remaining three cases however, the presumed del(20q) marker was shown to be the result of an unbalanced translocation between band 20p11 and a second copy of the Ph chromosome. This new abnormality, termed dic(20;Ph) for short, was identical to a del(20)q by G-banding, and combined duplication of the breakpoint cluster region and Abelson murine leukaemia viral oncogene homologue (BCR-ABL) fusion with loss of the 20p11-pter segment. In all three cases, the dic(20;Ph) was associated with disease progression and therefore represents a new recurrent abnormality in CML blast crisis.

The BCR-ABL fusion in chronic myeloid leukaemia (CML) is generated by the Philadelphia (Ph) translocation t(9;22) or, in 10% of patients, variants thereof (vPh). Deletion encompassing the reciprocal product (ABL-BCR) from the derivative chromosome 9 [der(9)] occurs in 15% of all patients, but with greater frequency in vPh patients. Reports of physical separation of ABL-BCR in non-deleted patients, as well as evolution from classical to variant Ph, introduce further heterogeneity to the vPh subgroup and raise the possibility that such translocations may herald disease progression. Survival analyses, however, have thus far yielded contradictory results. We assessed the frequency of der(9) deletions, ABL-BCR abrogation, cytogenetic evolution and cryptic rearrangement in a large cohort of 54 patients with vPh CML. Deletions encompassing ABL-BCR were detected in 37% of patients, consistent with a model in which a greater number of chromosome breaks increases the risk of genomic loss. The components of ABL-BCR were physically separated in a further 52% of patients while fused in the remaining 11%. Evolution from classical to vPh was demonstrated in three patients. The difference in survival, as indicated by Kaplan-Meier analysis, was marked between classical and vPh patients (105 vs 60 months respectively; P = 0.0002). Importantly, this difference disappeared when patients with deletions were removed from the analysis. Our study showed that, despite the existence of several levels of genomic heterogeneity in variant Ph-positive CML, der(9) deletion status is the key prognostic factor.

We report a case of severe thrombocytopenia with an abnormal bone marrow karyotype described by G-banding analysis as t(16;21)(p?13;q11). Using fluorescence in situ hybridization (FISH) analysis with whole chromosome paints, the chromosome rearrangement was shown to be more complex, with the additional cryptic involvement of the long arm of chromosome 3. The chromosome rearrangement involved the breakpoints 3q26, 16p13.3, and 21q11; this rearrangement has not been previously described. The size of genomic material translocated from the chromosome 16 homologue was too small to be detected by chromosome paint. A 16p-specific telomeric probe was hybridized to locate the translocated 16p material. The 16p telomeric unique sequence DNA was retained on the der(16) chromosome, indicating a more distal breakpoint. This study demonstrates that telomeric translocations can occur that would be undetected by telomeric-specific FISH probes.

The development of 24-colour fluorescence in situ hybridisation (FISH) has led to significant advances in cytogenetic research and offers the potential for automated karyotypic analysis. However, these techniques are not in routine research or clinical use because of limitations in methods of probe preparation. This article presents new probe construction protocols and strategies for multiple-colour karyotyping by chromosome painting, which makes the technique more efficient and may lead to more widespread implementation. We used paints generated by our protocols to demonstrate the presence of a cryptic translocation t(13;11;22) in the paediatric sarcoma cell line RMS 1598.

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.

Comparative genomic hybridization (CGH) is a powerful technique for detecting copy number changes throughout the genome. We describe the development of a versatile image analysis program for CGH studies. Several methods for the production of metaphases which give optimum hybridization signals have also been assessed. CGH analysis was performed on DNA samples from several different and clinically relevant specimens: amniotic fluid cells trisomic for a single chromosome, lymphoblastoid cell lines with abnormalities involving single chromosome bands, malignant cell lines and biopsy material from primary ovarian carcinomas. The results were compared with those derived from G-banding, chromosome painting, and molecular genetic techniques. Our data demonstrate that CGH was able to detect a wide range of quantitative genetic alterations including duplication or deletion of single chromosome bands. CGH analysis also indicated the presence of genetic abnormalities that were not detected by other cytogenetic or molecular approaches. Moreover, our CGH methodology allowed the ready comparison of CGH results from different tumors, a process which greatly facilitated identification of shared genetic changes.

Abstract 2561 We present the results of a study demonstrating that the genome profile of RUNX1 in MDS/AML is characterised by hitherto unreported partial deletions and absence of amplifications. This is in stark contrast to reports of chromosome 21 amplifications in ALL. We speculate that the absence of RUNX1 deletions results from them being well below a size detectable by commercial FISH probes. Extra chromosome 21 is the second most common acquired trisomy after (+) 8 in adult myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). It is rarely observed as sole abnormality but seen as part of complex karyotype in some 3–7% of the AML (Atlas of Genetics and Cytogenetics in Oncology and Haematology, http://atlasgeneticsoncology.org). Although the gene(s) in trisomy 21 associated with leukemia are unknown, the 21q22 region appears to be critical since it houses the RUNX1 gene. Multiple amplified copies of the RUNX1 carried by marker chromosomes, such as iAML21, are described in both acute lymphoblastic leukemia (ALL) and AML. A common 5.1 Mb amplification containing the RUNX1, miR-802 and genes mapping to the Down syndrome critical region identified in 91 children with iAML21, was shown to be the likely initiating event in this rare form of childhood B-cell ALL (Rand et al., Blood, 2011). In contrast, recent studies of AML in a Down syndrome and a constitutionally normal individual showed lack of RUNX1, ETS2 and ERG involvement (Canzonetta et al., BJH, 2012). Here we present 16 MDS/AML cases with imbalances of chromosome 21 identified by genomic array screening from a cohort of 83 cases. Whole genome screening (aCGH) was performed on presentation samples of MDS /AML and de novo AML cases using an oligonucleotide array platform (Agilent) at 60K, 244K, 400K and 1M density. G banding and FISH analysis were also successfully performed. Gain of an extra copy (trisomy) of chromosome 21 (+21) was found in 9 patients, all but one with complex karyotypes. In 2 AMLs high level amplifications were detected at 21q22, which involved the ETS2 and ERG but not the RUNX1 sequences. While several commercially available RUNX1 FISH probes showed gene multiple signals, custom FISH probes covering the relevant regions confirmed that the amplifications excluded the RUNX1 but affected both EST2 and ERG thus rendering the commercial probes unfit to assess CNA in this genome area. In another two cases with trisomy 12, cryptic loss of 43Kb and 98Kb resp. within the RUNX1 sequences was detected and confirmed by FISH. Furthermore, similar deletions within the 21q22.12 were also found in another 7 cases all of which had diploid set of chromosome 21 but had multiple changes at G banding level and high TGA score. These RUNX1 deletions were variable in size, ranging from 98Kb to 2.7Mb. Although our observations excluded clinical correlations it is note worthy that most of the patients with RUNX1 loss have not achieved complete cytogenetic remission. These findings suggest role for the RUNX1 loss as indicator of progressive disease and provide a novel insight into pathogenesis of MDS/AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 4562 As part of a preliminary study, we show the advantages of whole genome screening of CLL patient samples and highlight several issues. Firstly, 17p rearrangements appear to be far more complex than a simple loss of TP53; secondly, commercially available TP53 FISH probe sets may miss important genetic events for a significant number of CLL patients. Finally we show the possible role of the tumour suppressor MAP2K4 in the pathogenesis of CLL. Specific genome aberrations are recognised prognostic factors in CLL. FISH based genome risk stratifications have been used in clinical decision-making for over a decade. However molecular karyotyping is gaining acceptance as an alternative that not only overcomes the limitations of FISH but also provides a comprehensive whole genome scan. Indeed, several recent studies (Rinaldi et al., BJH 2011, Parker et al., Leukemia 2011, Quilette et al, Blood 2011) using different array platforms have revealed novel clinically relevant cryptic genome aberrations. Here we compare FISH and molecular karyotyping data of 50 diagnostic and 26 follow up samples from CLL patients. Since the loss of the TP53 gene was associated with resistance to chemotherapy, a short survival and a poor prognosis (Dohner et al, NEJM, 2000) CLL patients are routinely screened by FISH to assess its presence. Here we present 6 cases out of the 76 (5 presentation and 1 follow up), that have a deletion of the short arm of chromosome 17 (17p) by whole genome scanning with the 8×60K Agilent array platform. In 4 of the 6 cases the genome loss involved nearly the entire 17p and as expected, they were shown to carry TP53 deletion, when tested by commercial FISH probes. The remaining two cases were found to carry much smaller deletions of 17p. In one of these aCGH found a 5Mb loss within the 17p13.1 cytoband, at address chr17: 10.2–15.2. This region houses many genes, including MAP2K4, but does not include the TP53 (genome address hg19 chr17: 7.57–7.59), although FISH with commercial probes indicated TP53 loss. In the second case, the aCGH analysis identified a cryptic 120Kb loss housing the entire MAP2K4 at 17p12. As expected, commercial FISH probe analysis failed to detect any TP53 changes. However, FISH screening with BACs probes RP11–170H18 (covering the MAP2K4) and RP11–89D11 (TP53) confirmed the array results in both cases. The tumour suppressor gene MAP2K4 (mitogen-activated protein kinase kinase 4) was revealed by our aCGH study to represent the common deleted region in all cases with 17p loss. It was identified in 6 out of 76 CLL samples (8%) by the 60K genomic arrays. MAP2K4 is a putative tumor suppressor gene frequently found to be deleted in various cancer types, including solid tumors, ovarian and breast cancer (Teng DH, et al Cancer Res 1997, Davis SJ, et al BMC Cancer 2011), but to our knowledge has not been described in hematological malignancy. Further work is required to assess the frequency, type and clinical relevance of the genetic aberrations within the short arm of chromosome 17 in CLL patients. Disclosures: No relevant conflicts of interest to declare.

The Philadelphia (Ph) translocation t(9;22)(q34;q11) is found in 15% to 25% of adults and 3% to 5% of children with acute lymphoblastic leukemia (ALL)[1][1] and identifies patients with a particularly poor prognosis.[2][2] The Ph translocation is also the hallmark of chronic myeloid leukemia (CML)

Abstract 3561 Specific genome aberrations are recognised prognostic factors in CLL. FISH-based genome risk classifications used in clinical decision making for over a decade. However molecular karyotyping is gaining acceptance as an alternative, albeit cost and skill demanding, that not only overcomes the limitations of FISH but provides comprehensive whole genome scanning. Indeed, several recent studies (Rinaldi et al., BJH 2011, Parker et al., Leukemia 2011, Quilette et al, Blood 2011) using different array platforms have revealed novel clinically relevant cryptic genome aberrations. Here we compare FISH and molecular karyotyping data of 40 diagnostic and follow up samples from 22 CLL patients. The whole genome screening (WGS) was performed with 8×60K oligonucleotide arrays (Agilent) using DNA from mononuclear fraction of p.blood/bone marrow samples as well as from fixed chromosome preparations and commercial reference DNA. Following manufacturerÕs protocols and bio-informatics routines (Z score and ADM1/2 algorithms) WGS was done at a cost comparable to the expense of routine FISH screening for 13q14, p53, ATM loss and trisomy 12. Genome array screening identified clonal imbalances recognised by FISH if present above 13%. In addition, WGS provided novel information in 73% of the samples, the results of which are summarised as follows (see figure below): (i) The 13q genome loss found in 10/13 presentation samples was stratified by size and location resulting in a positive assessment for 40% of the cases positive by FISH for del 13q14 as class II/type 2 deletions. These are known to be associated with high risk for disease progression; (ii) A common deleted region of 206.2Kb at @ 50556688–50699677 (hg19) was found to encompass the DLEU2 gene and is seen in 5/13 presentation samples with 13q loss; (iii) In 3 out of 13 cases FISH-positive for 13q loss, disease progression was accompanied by a proximal extension of the deletion to include the RB1 gene region thus acquiring the status of class II/type 2 deletion (high risk) (iv) Genome loss involving IGH sequences at 14q32.32, consistent with B cell type VDJ gene rearrangements was detected in all samples, while deletions within IG kappa and IG lambda were found in 5/25 patients; (v) Cryptic deletion of the ATM gene region was detected in 2 samples undetected by FISH; (vi) Total genome imbalances (TGA) in presentation samples varied between 3 and 17 with higher levels found in samples with p53 loss and class II, type 2 13q loss; (vii) Persistent disease was found to be associated with increase of TGA, some of which appeared to be non-random, such as loss of the whole or part of chromosome 19 and deletions of 4q21 region. In conclusion, the application of 8×60K genome arrays provides established and growing genome-wide clinically relevant information as a cost neutral assay. A strategy by which array interrogation is carried out at presentation, followed by therapy response monitoring using FISH on a selected marker in CLL is a plausible alternative to routine practice. In cases where FISH has provided evidence for persistent disease, array screening will assess the genome damage, especially if carried on CD19(+)cell isolates, to assist treatment decisions. Disclosures: No relevant conflicts of interest to declare.

Abstract 4464 Philadelphia positive malignant disorders are a clinically divergent group of hemoblastoses with a unique identifying feature, the BCR/ABL1 fusion gene, usually resulting from the chromosome rearrangement t(9;22)(q34;q11) or its variants, that leads to constitutive expression of an aberrant tyrosine kinase. These include chronic myeloid leukaemia (CML) and de novo acute leukaemia of both myeloid Ph(+)AML and lymphoid origin Ph(+)ALL. The latter two disorders are clinically aggressive and therapy challenging even in the era of the powerful tyrosine kinase inhibitors. CML is a multistage progressive disease, which if untreated, inevitably ends as fatal acute leukaemia, either myeloid or lymphoid. The latter is often thought to be indistinguishable from Ph(+)ALL, the most common type of ALL in adults. We have identified DNA sequences the imbalances of which appear to be significantly associated with the disease stage and lineage origin in CML and Ph(+)ALL samples. We used array CGH at a resolution of ~2kb to explore hot spot regions obtained from 102 patient samples comprising 92 CML and controls together with 10 Ph(+)ALL and show how Significance Analysis of Microarrays (1) can be used to identify differences in the genome profile of de novo Ph(+)ALL and lymphoid blast transformation of CML. We show that lymphoid blast crisis CML differs significantly from Ph(+)ALL not only due to the presence of 9p deletions but also due to genomic gains in other chromosomes. Furthermore we identify a sub group of Ph(+)ALL with a distinctive genomic profile. Having identified genome regions of potential interest, ranked in order of significance, out of the 100's of thousands of array results, it is then a challenge to design further experiments to evaluate their contribution to the biology of the BCR/ABL positive disease. 1 Tusher V, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116-5121 (2001). Disclosures: No relevant conflicts of interest to declare.

Background Chronic myelogenous leukemia (CML) results from the neoplastic transformation of a haematopoietic stem cell. The hallmark genetic abnormality of CML is a chimeric BCR/ABL1 fusion gene resulting from the Philadelphia chromosome rearrangement t(9;22)(q34;q11). Clinical and laboratory studies indicate that the BCR/ABL1 fusion protein is essential for initiation, maintenance and progression of CML, yet the event(s) driving the transformation from chronic phase to blast phase are poorly understood. Results Here we report multiple genome aberrations in a collection of 78 CML and 14 control samples by oligonucleotide array comparative genomic hybridization. We found a unique signature of genome deletions within the immunoglobulin heavy chain (IGH) and T cell receptor regions (TCR), frequently accompanied by concomitant loss of sequences within the short arm regions of chromosomes 7 and 9, including IKZF1, HOXA7, CDKN2A/2B, MLLT3, IFNA/B, RNF38, PAX5, JMJD2C and PDCD1LG2 genes. Conclusions None of these genome losses were detected in any of the CML samples with myeloid transformation, chronic phase or controls, indicating that their presence is obligatory for the development of a malignant clone with a lymphoid phenotype. Notably, the coincidental deletions at IGH and TCR regions appear to precede the loss of IKZF1 and/or p16 genes in CML indicating a possible involvement of RAG in these deletions.

Abstract 3254 Poster Board III-1 Chronic myeloid leukaemia (CML) is characterized by the expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase that commonly results from the formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or variant rearrangement. Some 10% to 15% of CML patients present with cryptic deletions adjacent to the translocation breakpoint junction on the derivative chromosome 9. These deletions are believed to occur during the formation of the Ph translocation and are associated with a worse prognosis for CML patients treated with hydroxyurea or interferon. However there is still conflicting data regarding the significance of these deletions in patients receiving imatinib therapy, which appears to overcome the poor prognosis associated with the genome loss. Here we present data of three BCR/ABL1 positive CML patients on tyrosine kinase inhibitors (TKI) who developed therapy resistance. In all patients loss of the ABL1 sequences was identified. Surprisingly, these deletions affected not the ABL1 sequences of the ABL1/BCR fusion on the der(9) chromosome but the normal ABL1 allele on the non- translocated homologue of chromosome 9. This loss was not present at the time of diagnosis but was a new event developed during disease progression and found along with other additional chromosome rearrangements in the BCR/ABL1 positive cells. Bone marrow chromosome suspensions were studied by array comparative genomic hybridization (aCGH) analysis and/or fluorescent in situ hybridization (FISH) using and a range of commercial and bacterial artificial chromosomes (BAC) probes. While one patient carried a large deletion del(9)(q31q34) that spanned several megabases and was detectable by G-banding analysis, the other two patients had small cryptic losses (from 1.8 Mbp to 2.6 Mbp). The loss involved 3' ABL1 sequences in one patient whereas it affected the whole gene in the other two cases. Since the negative control exercised by the normal ABL1 allele over the BCR/ABL1 expression has been documented, the sensitivity to the TKI might be affected in the BCR/ABL1 positive cell clone bearing the genome loss. In summary, we report a new type of ABL1 loss in Ph positive cells - deletions at the 9q34 associated with TKI resistance in CML patients. Disclosures: No relevant conflicts of interest to declare.

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder characterized by the expression of the BCR/ABL1 fusion gene, which commonly results from formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or related variant rearrangement. BCR/ABL1 is a constitutively activated tyrosine kinase and its amplification has been described in association with resistance to imatinib in CML patients. BAC array CGH analysis on CML patients and CML cell lines (Brazma et al., 2007) revealed unexpected genomic imbalances in form of duplications and high copy number gains affecting the region immediately downstream of the ABL1 gene at the Philadelphia (Ph) chromosome in patients at the blast crisis stage. We aimed to confirm and map these amplifications by fluorescence in situ hybridization (FISH) on 19 CML patients in accelerated phase/blast crisis and 10 CML cell lines (KU812, K562, MEG-01, MC3, BV173, EM-2, LAMA-84, KCL-22, JK-1 and CML-T1) with more than 1 copy of the BCR/ABL1 fusion gene. We used a range of BAC probes and 9q and 22q sub-telomeric probes in order to do the FISH mapping. While the majority of the analysed patients and cell lines (12/19 patients and 6/10 CML cell lines) had 2 identical Ph chromosomes, 2 main groups of abnormalities were identified. Firstly, gains of the Ph chromosome taking the form of ider(22)t(9;22) chromosome were detected in 1 or more copies in a subset of bone marrow cells of 5/19 patients and, secondly, high copy number gains were seen in 2/19 patients and 2/10 cell lines (K562 and KU812). The amplified region was variable in size spanning from 400 Kb up to 1.5 Mb downstream of the ABL1 gene. In 1 patient, 7 different cell sub-clones harbouring increasing levels of amplification were found. The gains resulted in formation of different chromosome structures-from an ider(22)t(9;22) to markers with tandem amplifications, which included the BCR/ABL1 fusion with variable in length sequences downstream of the ABL1. Duplication of some 571 Kb downstream of ABL1 was also detected in 1 of the 2 apparently normal Ph chromosomes in the MC3 cell line, while a larger duplication (5.16 Mb) was found in another cell line (MEG-01). These findings confirm the presence of duplications and high level amplifications at the der(22) t(9;22) in CML patients and that the sequences involved are variable in length, indicating that the Ph chromosome is an unstable structure and vulnerable to further rearrangements during disease progression.

Deletions of the long arm of chromosome 20 (del 20q) are a recurring abnormality associated with haematological malignancy (Mitelman 1995, Sandberg 1991, Heim and Mitelman,1995, Asimakopoulos & Green, 1996). The 20q deletions are seen in approximately 10% of the cases with PV and IMF, 4% of cases with myelodyspalstic syndrome (MDS) (Fenaux et al., 1996), 1–2% of patients with acute myeloid leukaemia (AML) as well as other myeloproliferative disease (MPD) (Bench et al. 2000). Deletions of chromosome 20q are well recognised in myeloid disorders and have been seen alone or as part of a complex karyotype (Johansson et al, 1993, Schoch et al 2002). A common deleted region (CDR) of chromosome 20q was observed by fluorescence in situ hybridization (FISH) mapping within 20q11.2/q12 region (Roulston et al., 1993, Bench et al, 2000). The breakpoints within this CDR have been shown to be heterogeneous (Asimakopoulos et al., 1994) and more recently a considerable overlap of this region has been observed in myeloid malignancy (Douet-Guilbert et al, 2008). Previous studies have been performed on cases with simple deletions of chromosome 20q as assessed by G-banding and confirmed by FISH, to be interstitial and no other rearrangements involving 20q have been reported so far. Here we present 43 patients with non-random translocations of chromosome 20q that appear to have a common breakpoint within the 20q11.2 region. Karyotypically some of the cases presented with the classical del(20q) morphology while in other cases translocations were identified, such as t(1;20)(q21;q11.2). Of these cases 44% have been shown to carry an unbalanced chromosome rearrangement with loss of material at the region of the breakpoint at 20q11.2 both with and without loss material on the partner chromosome. An example of the latter is the rearrangement t(17;20)(p13;q11.2) where 20q11.2 loss was accompanied by a p53 deletion (17p13). FISH mapping located the 20q11.2 breakpoint to be within the BAC clone RP1-1J6, which covers only the sequences of one gene, Topoisomerase 1 (TOP1). This gene is reported to form a fusion with NUP98 in therapy related MDS. Indeed we confirmed by FISH the presence of this fusion in 6 of our cases, all of which carried the t(11;20)(p15;q11.2). Unexpectedly, the rest of the cases also showed breaks within the same BAC clone, where the partner chromosomes were Yp11, 1q21, 17p11 and 21q11. These findings highlight the consistent involvement of the TOP1 gene at 20q11.2 in rearrangements occurring in MDS and MPD.

In spite of the universal presence of the BCR/ABL1 fusion gene, chronic myelogenous leukemia (CML) shows remarkable clinical and genetic diversity. The consequences of der(9)t(9;22) chromosome deletions, associated with poor survival, as well as the mechanism behind their formation remain unclear, as does our understanding of the molecular events driving the disease evolution. The presence of these deletions fuelled the expectations that cryptic genome-wide aberrations may be accountable for the disease progression. Following a comprehensive BAC aCGH analysis of 48 CML samples (Brazma et al., Genes, Chromosomes & Cancer, 2007 in press) we report high-resolution oligo-nucleotide array study of a further 30 CML accelerated/ blast phase samples. We were unable to confirm the high frequency of particular single BAC imbalances (CNVs), reported both by ourselves and others, possibly due to the manufacturer’s array selection strategy. Never-the-less some of the CNVs and a wealth of new imbalances were obtained at 60kb resolution. It was possible to build a precise map of the amplicon affecting the sequences flanking the 3′ ABL1 breakpoint site, which include the LAMC3 and NUP214 genes. The presence of this amplicon was associated with therapy resistance. When assessed, at a resolution of 60 kb, the deletions of the regions flanking the ABL1/BCR breakpoint showed novel features: the genome loss affects preferentially both flanking sites as seen in 5 of the 6 ‘deleted’ samples and the 120kb deletion identified is the smallest recorded so far. Most of the major cytogenetic features of the samples were confirmed and a number of cryptic genome imbalances were detected, from 120kb to 10Mb in size, involving regions rich in genes, some already implicated in the pathogenesis of CML. Finally, recurrent micro aberrations of several adjacent oligo-nucleotides affecting non-coding sequences were detected in as many as 2/3 of the samples.

Chronic Myeloproiferative Disorders (CMPDs) form a continuum of haematological malignancies of which the molecular pathogenesis remains obscure. Chronic Myeloid Leukaemia (CML) being the only disorder with a recognisable pathogenomic abnormality, the Ph chromosome. Tyrosine kinases are clearly implicated in the disease pathogenesis, in particular the role of JAK2. It has been shown that over 80% of Polycythaemia Vera (PV) patients and 30% of Essential Thrombocythaemia (ET) patients demonstrate a clonal and recurrent mutation V617F in the JH2 pseudo-kinase domain of JAK2 located on 9p24.1. Array CGH investigations of cell lines, revealed a complex pattern of imbalances affecting the short arm of chromosome 9: on the background of 2–3-fold amplification, a loss of the 9p24.1 region, which harbours the JAK 2 along with a number of other kinases, was repeatedly observed. The aim of this study was to clarify the aCGH observations using a series of BAC DNA probes mapping from the telomere of 9p toward the centromere. Results indicate the presence of imbalances (amplifications & deletions) and rearrangements, involving the region encompassing JAK2. In all CML-BC cell lines the FISH data supports the observation of a relative loss of the 9p24.1 region on the background of amplification of chromosome 9. An on-going study of CMPD and MDS patients with both normal and abnormal karyotypes has revealed imbalances affecting the short arm of chromosome 9. In 23% (7/30) of patients there were imbalances; the majority of these (56%) demonstrated a relative loss of information in this region. The pathegenomic significance of imbalances involving 9p remains unclear. It may however represent an acquired homozygousity as a consequence of mitotic recombination, with a ‘second hit’ removing a wild type allele, leading on to further, as yet un-clarified changes.

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase. The fusion gene is a result of t(9;22)(q34;q11) or related variant rearrangements leading to formation of the Philadelphia chromosome (Ph). In less than 2% of the CML patients the bone marrow cells appear to have a normal karyogram although the fusion gene can be identified by molecular means and located by FISH on 22q11 and less frequently on 9q34 or on a third chromosome. The biology and clinical significance of the genetic rearrangements in Ph negative BCR/ABL1 positive disease are controversial and widely discussed. Two alternative mechanisms have been postulated but not confirmed: simple insertion or multiple step model. Although sequencing analysis as early as 1990 (Morris and Heisterkamp) showed that a large part of the 9q34 region is present in the masked Ph, little has been done to clarify the underlying rearrangements. Aided by genome array CGH and FISH, we constructed a comprehensive map of the regions flanking ABL1 and BCR genes in 9 patients and one cell line with this form of CML. The main results show: the formation of the BCR/ABL1 fusion is associated with relocation of sequences well in excess of the ABL1 and BCR gene in all 10 samples, the involvement of downstream 3′ ABL1 is diversely associated with distal breakpoint clusters in the regions of the RAP-GEF1 and RXRA genes, the disease progression can be accompanied by sequential breakages/reunions within the regions flanking the BCR/ABL1 fusion and the presence of multiple cryptic rearrangements of both 9q34 and 22q11 segments identifies a sub-group of patients with shorter survival thus demonstrating the clinical significance of genome instability in Ph negative, BCR/ABL1 positive CML.

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR-ABL fusion gene, a constitutively activated tyrosine kinase. The fusion gene commonly results from formation of the Philadelphia chromosome after a t(9;22)(q34;q11) or related variant rearrangement. The disease follows a biphasic course with an initial benign chronic phase (CP) of 3–4 years treatable with agents such as Imatinib, followed by a rapidly fatal transformation resembling an acute leukaemia (blast crisis, BC). The CML blast crisis has been linked to a number of secondary chromosome changes leading to genome imbalances, such as additional Ph, +8, +9, iso17q. Prognostic scores based on clinical criteria (Sokal and Hasford scores) have been replaced with the discovery of other cytogenetic abnormalities occurring simultaneously at the time of translocation, such as deletions flanking the ABL/BCR breakpoint at the der(9)t(9;22) chromosome that lead to a significantly worse prognosis. We undertook array CGH analysis using an oligo based platform (Agilent) on 46 CML patients (25 CP, 19 BC & 8 in both phases) and 12 CML cell lines revealed a range of recurrent genomic imbalances. Two sets of recurrent cryptic imbalances were identified. These represented both gains and high-level amplifications affecting the 9q34.12 region immediately downstream of ABL gene, as well as losses or gains in the distal part of the 9q34-qter segment. These imbalances were found only in patients at the BC stage. Furthermore, in cases with follow-up samples, the 9q34 imbalances were present in the advanced stage of disease thus confirming their secondary nature. Among the genes found to be amplified in CML/BC is NUP-214, a nucleoporin gene, recently implicated in T-cell acute lymphoblastic leukaemia, thus suggesting an important role for cell clone survival. Confirmation of these amplifications was sought using fluorescent in situ hybridisation (FISH). A dual fusion probe targeting the ABL-NUP214 region was applied to a selection of twenty three patients with confirmed CML and features such as deletion of der(9) chromosome or resistance to treatment with Imatinib, shown to be more prone to variation within the CML phenotypeAmplification of the NUP214 region was found in five patients. These findings represent the first reports of NUP214 amplifications in CML, paving the way for future work.

Samples from 20 CML patients and 12 CML derived cell lines were studied by matrix comparative genomic hybridization (CGH) using 1Mbp BAC micro-arrays with increased density at the telomere regions (Spectral Genomics, Houston). The micro-arrays reproducibly delineated previously characterised gains and losses (Gribble et al., 1999 & 2003). Novel non random imbalances, gains and losses were revealed to affect either single loci or short regions summarised as follows: i) Cryptic deletions The cryptic deletions affected either a single BAC clone or a group of several consecutive clones. These losses were seen in chromosome areas with either normal banding or within regions of apparently balanced translocations. These include the BAC clone RP11-259N12 in the short arm of chromosome 1, found as a single BAC loss or as part of a small segment in 1p35 sub-band. Other examples include deletions of up to five consecutive BAC clones within the 9p21 region known to contain tumour suppressor genes, deletions within 14q11.2, 16p13.1 and 20q11.2/12 regions. ii) Cryptic gain Gain of extra copy of the BAC clone RP11-130P22 was in patients samples, which did not appear to have abnormalities of the short of chromosome 2; similarly additional copy of the RP11-96F19 clone were detected in 4 patients and 3 cells lines without evidence for aberrations of 12q24 region. Other examples of cryptic gains include regions of 16p11.2, 19p13.1 and 19q13. iii) High level amplification Array analysis successfully refined common amplicons within regions of known high level amplifications from the regions of 9q34.1, 22q11.2, and 8q24.13. In the 8q region three separate common amplicons were identified, all of which do not include the cMyc oncogene. iv) Single locus nullisomy Heterozygous deletions were found to affect a single locus located within a large chromosome segment present in only one copy. There are two such examples - (i) lack of the RP11-87O1 clone in the cell line K562, where the whole of the short arm of chromosome 9 is present in one copy only and (ii) deletion of both copies of the RP11-79J17 clone seen in the cell like KYO-1, where the short arm of chromosome 6 is monosomic. v) Deletions of regions as large as 5Mbp adjacent to the chromosome telomeres These were revealed by the loss of more than one BAC probe and estimated to range in size from 1 to 5 Mbp. The para-telomeric deletions frequently affected the short arms of chromosomes 4 and 12 as well as the long arm of chromosome 3. vi) Low level imbalances Low levels of gain or loss, indicated by ratio values more than 0.5 or less than 1.5, for large regions, spanning several chromosome bands, were seen throughout the whole chromosome complement. Most frequently involved areas include the 14q22-qter, 15q22-qter, 20p12-p12 ter as well as 20q12-qter, 19p13.1-c-q13.3 and 10p12-pter. These reflect genetic aberrations in cell clones presented at low levels (20–30%). vii) Deletions flanking translocation breakpoints Deletion of single BAC clone covering sequences upstream of the ABL gene was found in the cell line MC3 and 3 patients. The loss of this region on der(9)t(9;22) is well characterised and we have known by FISH that it extends 3 Mbp from ABL exon 1a in MC3 cells (Reid et al., 2003). The micro-arrays faithfully reproduced the mapping data and allowed us to size the deletions in the der(9) chromosome in the patients samples. This demonstrates the ability of the array CGH to detect cryptic deletions accompanying balanced translocations.

Background Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder, almost always characterized by the presence of the Philadelphia chromosome (Ph), usually due to t(9;22)(q34;q11) or its variants. The Ph results in the formation of the BCR/ABL1 fusion gene, which is a constitutively activated tyrosine kinase. Around 1% of CML patients appear to have a Ph negative karyotype but carry a cryptic BCR/ABL1 fusion that can be located by fluorescence in situ hybridisation (FISH) at chromosome 22q11, 9q34 or a third chromosome. Here we present FISH mapping data of BCR and ABL1 flanking regions and associated chromosomal rearrangements in 9 Ph negative BCR/ABL1 positive CML patients plus the cell line CML-T1. Results BCR/ABL1 was located at 9q34 in 3 patients, 22q11 in 5 patients and CML-T1 and 22p11 in 1 patient. In 3 of 6 cases with the fusion at 22q11 a distal breakpoint cluster was found within a 280 Kb region containing the RAPGEF1 gene, while in another patient and the CML-T1 the distal breakpoint fell within a single BAC clone containing the 3' RXRA gene. Two cases had a duplication of the masked Ph while genomic deletions of the flanking regions were identified in 3 cases. Even more complex rearrangements were found in 3 further cases. Conclusion BCR/ABL1 formation resulted from a direct insertion (one step mechanism) in 6 patients and CML-T1, while in 3 patients the fusion gene originated from a sequence of rearrangements (multiple steps). The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in Ph negative BCR/ABL1 positive CML.

ETV6-ABL1 is a rare gene fusion with oncogenic properties, reported so far in 28 patients presenting a variety of haematological malignancies associated with clinical outcome, including chronic myeloid leukaemia (CML), acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and chronic myeloproliferative neoplasm (cMPN). Here we report on a 46-year-old female who presented with Philadelphia negative CML, positive for the ETV6-ABL1 fusion. Whole genome screening carried out with oligonucleotide arrays showed a subtle loss at 12p13 and cryptic imbalances within the 9q34.3 region in a highly unstable genome. FISH mapping with custom BAC probes identified two breakpoints 5 Mb apart within the 9q34 region, together with a break at 12p13. While FISH with commercial BCR-ABL1 probes failed to detect any ABL1 changes, the ETV6 break-apart probe conclusively identified the ETV6-ABL1 fusion thus determining the probe’s role as the primary diagnostic FISH test for this chimeric oncogene. In addition, we confirm the association of the ETV6-ABL1 fusion with imatinib resistance reported so far in three other patients, while recording excellent response to the 2nd generation tyrosine kinase inhibitor (TKI) nilotinib. In summary, we highlight the value of ETV6 FISH as a diagnostic test and the therapy resistance of ETV6-ABL1 positive disorders to imatinib.