Computer aided analysis of additional chromosome aberrations in Philadelphia chromosome positive acute lymphoblastic leukaemia using a simplified computer readable cytogenetic notation
© Bradtke et al; licensee BioMed Central Ltd. 2003
Received: 26 November 2002
Accepted: 28 January 2003
Published: 28 January 2003
The analysis of complex cytogenetic databases of distinct leukaemia entities may help to detect rare recurring chromosome aberrations, minimal common regions of gains and losses, and also hot spots of genomic rearrangements. The patterns of the karyotype alterations may provide insights into the genetic pathways of disease progression.
We developed a simplified computer readable cytogenetic notation (SCCN) by which chromosome findings are normalised at a resolution of 400 bands. Lost or gained chromosomes or chromosome segments are specified in detail, and ranges of chromosome breakpoint assignments are recorded. Software modules were written to summarise the recorded chromosome changes with regard to the respective chromosome involvement. To assess the degree of karyotype alterations the ploidy levels and numbers of numerical and structural changes were recorded separately, and summarised in a complex karyotype aberration score (CKAS). The SCCN and CKAS were used to analyse the extend and the spectrum of additional chromosome aberrations in 94 patients with Philadelphia chromosome positive (Ph-positive) acute lymphoblastic leukemia (ALL) and secondary chromosome anomalies. Dosage changes of chromosomal material represented 92.1% of all additional events. Recurring regions of chromosome losses were identified. Structural rearrangements affecting (peri)centromeric chromosome regions were recorded in 24.6% of the cases.
SCCN and CKAS provide unifying elements between karyotypes and computer processable data formats. They proved to be useful in the investigation of additional chromosome aberrations in Ph-positive ALL, and may represent a step towards full automation of the analysis of large and complex karyotype databases.
Chromosome banding analyses have become a fundamental part of the diagnostic panel in haematological neoplasias, and molecular cytogenetic methods have added to more detailed information about the karyotype changes particularly in complexly aberrant karyotypes or even in cryptic chromosome rearrangements [1, 2]. Thus, the quantity of cytogenetic data in haematological malignancies has increased not only by additional cases but also by new techniques. Large cytogenetic data pools may provide the basis to identify new rarely occurring primary chromosome aberrations in distinct disease entities. Recurring involvement of the same chromosome band in several different chromosome rearrangements may be detected and, thus, point to new translocation oncogenes which may be relevant for the leukemogenesis . Chromosome banding studies have helped to delineate minimal commonly lost chromosome regions and, thus, to identify tumour suppressor genes, e.g., the CDKN2A gene in 9p21 [2, 4–7]. Minimal regions of common chromosome gains which may harbour oncogenes important for tumour genesis have as well been narrowed down by the analyses of metaphase chromosomes [8, 9].
The degree of karyotype alterations may point to distinct pathways of genetic mechanisms of disease progression . Patients with complex karyotype alterations have been shown to have an inferior prognosis in acute myeloid leukaemia and myelodysplastic syndromes. However, the number of chromosome rearrangements which are used to define a complexly altered karyotype in different studies range from 3–5. Moreover, the number of events which are attributable to a complex chromosome rearrangement has not been defined [11–16]. E.g., unbalanced translocations (see additional file 2: glossary.doc, for explanation), triplications, or inverted duplications may count two events . Therefore, the comparison of the clinical and biological findings in this cytogenetic subgroup is hampered by the different numbers of numerical and/or structural aberrations which have been summarized to determine a complexly aberrant karyotype in the respective studies. Chromosome rearrangements are designated according to the International Standard for Human Cytogenetic Nomenclature, and in the most cases the short karyotype description is used [1, 18].
The ISCN 1995 has paid special attention to the accessibility for a computer assisted readability of the karyotypes. Therefore, numerical chromosome changes of whole chromosomes or derived ones, distinct familiar chromosome rearrangements, and chromosomal breakpoints may easily be searched for by using simple text matching in cytogenetic databases which use the short system of the ISCN . However, unbalanced chromosome translocations result in gains and also in losses of chromosomal material. Gain of a derivative chromosome resulting from an unbalanced translocation may lead to gain and also to loss of parts of the same chromosome band. For instance, in the karyotype 47,XX,der(19)t(1;19)q23;p13),+der(19)t(1;19) the gain of the derivative chromosome leads to double gain of 1q23->1qter, to loss of a part of chromosome band 19p13->19pter, and also to gain of a part of 19p13. Moreover, complex chromosome rearrangements need to be described in the detailed karyotype notation, from which gains or losses of chromosomal material may only be extracted by comparing the changes described in the single strings with the complete karyotype. Also the type of the rearrangement may be difficult to determine from the detailed karyotype description without the information of the complete karyotype. Thus, the ISCN karyotype may include metainformation about the genomic changes that is not explicitly specified in the ISCN strings and, therefore, not accessible for an automated analysis using simple text matching tools. Extensive programming would be necessary to develop a software, which is able to extract every information included in the ISCN karyotypes. In the past, Hashimoto and Kamada developed computer programs for an automated analysis of large numbers of abnormal karyotypes according to the ISCN 1978 [19, 20], but never conformed them to ISCN 1995. In tumour cytogenetics, different levels of chromosome quality are common even within the same disease entity, and the maximum banding resolutions may range from 150 to 800 or even more bands per haploid set (bphs) between different cases. To get access to the metainformation contained in the ISCN karyotypes for a computerized analysis of cytogenetic findings, to join different levels of cytogenetic resolution in a common database, and to address incomplete karyotype descriptions and questionable chromosome or breakpoint assignments we developed a simplified computer readable cytogenetic notation (SCCN). By this approach, the automatic compilation and graphical representation of the chromosome alterations became feasible. Software modules were written to calculate the frequency of gains and losses of chromosome segments, and of types and breakpoint localisations of structural rearrangements. They automatically analyse the SCCN, create proof and error tables, and present the results in separated tables and graphs. To determine the degree of the alterations of a karyotype the chromosome changes were classified according to distinct aberration categories, and a complex karyotype aberration score (CKAS) was calculated. The karyotypes of 94 Ph-positive ALL patients with additional chromosome aberrations were subjected to the computerized analyses of the chromosome findings and the degree of the karyotype alterations was assessed according to the CKAS.
Evaluation of the degree of the karyotype alterations
Evaluation of quantitative and qualitative chromosome changes
Newly developed tools for computer aided analyses were used to investigate the spectrum of additional aberrations in 94 Ph-positive ALL patients. The number of additional aberrations in a patient was determined using different aberration categories and a complex karyotype aberration score (CKAS). Single changes were recorded in only 41.5% of the cases, 13.8% had two, and 44.7% three or more events. The breakdown of the additional chromosome anomalies according to the categories of chromosome changes demonstrated that numerical aberrations contributed to 80.1% of the total events of which 68.4% resulted from chromosome gains or losses in high-hyperdiploid and near triploid karyotypes. Karyotypes >50 chromosomes have been described as additional aberrations in Ph-positive ALL [21–24]. High-hyperdiploid karyotypes in childhood ALL without Ph-translocation have been shown to result from non-disjunction of chromosomes in one single mitosis which seems to occur early in leukemogenesis [25, 26]. Thus, in Ph-positive ALL chromosome gains in high-hyperdiploid ranges may also result from one single aberration event instead of multiple numerical changes. However, numerical changes and unbalanced structural rearrangements both accounted for 92.1% of all events. Therefore, by using the proposed categories of the CKAS, changes of the dosage of genetic material were demonstrated to represent the vast majority of the additional chromosome aberrations in Ph-positive ALL. Recurrent gains were detected at 9q34 and 22q11.2 which were mainly due to the presence of additional Ph-chromosomes which has been observed in up to 26% of Ph-positive ALL-patients [21, 22, 27]. However, gains of whole chromosomes 22 also contributed to the increased dosage of 22q11.2 which may point to a role of genes of chromosome 22 in addition to the BCR-ABL gene in the development of Ph-positive ALL. Gains at chromosome 1 and 8 accumulating at the entire long arm and also loss of chromosome 7, or of 6q, 8p or 9p confirmed previous findings of recurrent secondary chromosome changes in Ph-positive ALL [22, 28–30].
However, losses at chromosome 7 were increased at the short arm which may indicate that 7p is the target of chromosome 7 deficiency in Ph-positive ALL. Moreover, loss at 6q peaked at 6q23. Although this was due to an overlap of the deleted segment in two cases, only, the efficiency of the analysis procedure to detect minimal commonly deleted segments from ISCN karyotypes was demonstrated. The analysis of the distribution of the altered chromosome regions revealed no recurring involvement of particular chromosome bands apart from 9q34 and 22q11. However, pericentromeric rearrangements became evident in 24.5% of the cases including isochromosomes in 7.4% and dicentric translocations in 5.3% of the patients. This suggests that pericentromeric regions are targets for chromosomal rearrangements in Ph-positive ALL. Agents such as mitomycin C, and ionising radiation, for instance, have been shown to induce breaks in the centromeric or heterochromatic parts of chromosomes 1, 9 and 16 [31, 32]. Moreover, a constitutional predisposition may be causative for pericentromeric rearrangements as has been demonstrated in patients with ICF (immunodeficiency, centromeric instability and facial abnormalities) syndrome and hypomethylation of satellite II DNA due to DNA methyltransferase deficiency [33, 34]. So far, it remains obscure which the causes for pericentromeric rearrangements in Ph-positive ALL may be. However, our procedure of breakpoint analysis may provide new perspectives in the unravelling of the mechanisms of the disease progression in Ph-positive ALL by pointing not only to individual chromosome bands or regions but also by providing an overview over the rearrangements of common chromosome structures of the whole karyotype.
The newly developed scoring system for the assessment of the degree of karyotype alterations (CKAS) and the simplified computer readable cytogenetic notation (SCCN) for karyotype findings, in our hands, appeared to be a helpful tool in the computer aided characterisation of the spectrum of chromosome alterations. The breakdown of the type of chromosome aberrations in the aberration categories of the CKAS may provide the basis for distinct definitions to determine the degree of karyotypic alterations. The SCCN in combination with respective software modules may be suitable for the automated analysis of chromosome findings from complex databases with respect to regions of recurrent chromosome gains, losses, or breakpoints of chromosome rearrangements. Therefore, we believe that the SCCN and CKAS may represent a step towards the development of a completely automated analysis of ISCN karyotypes of large cytogenetic data pools. The program modules for SCCN analyses including the graphical presentation facilities used in this study will be made available to interested readers upon request.
The cytogenetic data of 94 patients were chosen from the Leukaemia Cytogenetic Database (LCD) of the German competence net "Acute and Chronic Leukaemias" (see additional file 1: appendix.pdf, for detailed information)". In all patients, the diagnosis had been verified by morphological and immunological analyses. The database contained a case identifier, age, sex, the ISCN karyotypes, SCCN strings, the modal chromosome number, the counts of clones, and the aberration categories.
A commercially available database program (ACCESS 2000 Professional, Microsoft corporation, Wa) was used to program forms which guide the user through the analysing programs by menus. Modules were written using Visual Basic for Applications (VBA, Microsoft Office 2000, Microsoft corporation, Wa) to extract the respective chromosome alterations from the SCCN strings, and to compile the data for the statistical evaluation and graphical presentation of the chromosome changes.
Computerized analysis of chromosome changes
Data source containing the SCCN strings was an ACCESS 2000 database table created by a specific query, selecting data from a complexer ACCESS database. Moreover the import of all common databases, SQL- or Excel-tables was possible. The two main program units for qualitative or quantitative data analysis were editing the incoming data strings following specific algorithms belonging to the possible number of used signs and their combination. (Help-)program modules containing tables with all possible band definitions for every chromosome at a resolution of 400 bphs were stored as well as the according ideograms of all 24 chromosomes and a catalogue of all possible ISCN short terms describing the type of alterations. For the analysis of quantitative chromosome changes, gains or losses were compiled for each chromosome (sub-) band. After selection of a chromosome, the incoming data were compared with the catalogue of help-tables and every fitting string was counted in a new result table referring to the protrusive sign. Single or multiple changes of a chromosome were counted by adding the respective number of gains or losses to each of the (sub-)bands of the respective chromosome. The distribution of the qualitative changes was analysed for each chromosome by recording and counting the involved chromosome (sub)band(s) and the respective type of the rearrangement. Generally, chromosome rearrangements, which lacked a resolution at subband level and as well ranges of possible chromosome breakpoints were recorded by adding one change to each possibly involved chromosome subband. Of each chromosome, the incoming datasets containing the karyotypes and the SCCN translation as well as the processed data containing the extracted SCCN strings and the number of events recorded in each chromosome (sub)band were stored to check the results chromosome by chromosome to identify mistakes at each level of the process. Strings which did not apply to the established SCCN as well as parts of aberration strings containing question marks were excluded from the analysis and stored in error tables for revision. For each chromosome, the results of the analyses were presented as a table with the summed up changes in the order of the respective position on the chromosome. For direct visualisation of the distribution of the alterations within one chromosome, a graph was generated that showed the number of changes of each chromosome band as bars projecting onto the bands of the respective chromosome ideogram relative to the maximum value achieved in this chromosome.
Definition of a simplified computer readable cytogenetic notation
General rules for the translation of the ISCN karyotypes into a simplified computer readable cytogenetic notation (SCCN) and respective examples.
Definition of aberration categories for the assessment of the degree of karyotype alterations
Examples for the assessment of the degree of karyotype alterations according to different categories of chromosome aberrations and calculation of a complex karyotype aberration score (CKAS).
Number of aberrations
This work is part of the project "Central Cytogenetics" of the competence net "Acute and Chronic Leukaemias" and was supported by the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF), Grant 01GI9974.
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