Absence of damaging effects of stem cell donation in unrelated donors assessed by FISH and gene variance screening.

ASTRACT: Granulocyte-Colony-Stimulating factor (G-CSF) is currently the standard mobilising agent for peripheral blood stem cell (PBSC) donation. Concerns that it may trigger chromosome aberrations similar to those observed in leukaemia patients were refuted but long-term effects of G-CSF mobilisation on genome integrity remains unclear. In the setting of a multi-centre clinical trial we screened blood samples from 50 PBSC donors at cellular and gene level for aberrations common in haematological malignancies using fluorescence in situ hybridisation (FISH) and next generation sequencing (NGS) assays. Analysis of samples collected before, on the day of donation, 90 and 180 days after G-CSF admission confirmed the absence of short-term effects in PBSC donors on both quiescent and dividing cells. This data did not differ from the results of 50 individuals tested 3-5 years after bone marrow donation and 50 healthy persons. NGS using a panel targeting 54 genes recurrently affected in myeloid disorders (TruSight Myeloid panel, Illumina) showed that the gene profiles of samples from 48 PBSC donors remained stable throughout the study period. These data strongly indicate absence of detrimental effects on the genome integrity caused by PBSC donation.

Detection of HHV-6-specific mRNA and antigens in PBMCs of individuals with chromosomally integrated HHV-6 (ciHHV-6).

After inheritance of chromosomally integrated HHV-6 (ciHHV-6), viral DNA is found in every nucleated cell. The prevalence of ciHHV-6 is estimated to be 0.2-5% of humans. There are conflicting data on the potential for replication, possibly leading to clinical implications. We analysed peripheral blood mononuclear cells (PBMCs) from individuals with ciHHV-6 proven by fluorescence in situ hybridization (FISH) for HHV-6-specific mRNA (U94, U42, U22) and antigens by means of reverse transcription PCR and an indirect immunoperoxidase staining. U94 transcripts indicative of latent infection were detected in six (54.5%) out of 11 individuals at least once. Transcripts indicative of lytic infection (i.e. U42 and U22) were detected in four (36.4%) out of 11 individuals at least once. HHV-6 antigen was detected in seven (70%) out of 10 individuals at least once. The presence of viral mRNA and proteins supports virus gene expression from ciHHV-6, which may lead to virus replication. Considering the properties of active HHV-6 infection together with obvious replicative activity in individuals with ciHHV-6, pathophysiological effects leading to clinical consequences of chromosomally integrated viral DNA might be considered.

Genomic profile of chronic myelogenous leukemia: Imbalances associated with disease progression.

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.

Application of growth factor stimulants improves cytogenetic analysis of chronic myeloproliferative disorder patients without alteration to cell lineage or clonality.

Conventional cytogenetic methods rely on culturing bone marrow aspirates to obtain suitable and sufficient mitotic figures for G-banded analysis. Samples from patients with chronic myeloproliferative disorders (CMPD) often have increased failure rates due to reduced growth and poor morphology, all of which hamper the conventional karyotyping investigation. The application of growth factor (GF) stimulants to bone marrow aspirates has been shown to yield significant increases in both the quality and quantity of bone marrow metaphases obtained in 53 CPMD patient samples. All cultures were stimulated using the conditioned supernatant from the human bladder carcinoma cell line 5637, which contains IL-3, IL-6, and G-CSF. Results were assessed qualitatively on G-banded preparations and quantitatively by mitotic index (MI = % dividing cells). To assess whether the application of GF stimulants leads to clonal selection, culture samples from 15 patients were analyzed by fluorescence in situ hybridization, which supported the theory that clonal selection remains unaltered in GF-stimulated cultures. In addition to this immunophenotyping of cells, we demonstrated the lineage of cells propagated under these conditions. Cell markers were chosen to characterize B-lymphoid, T-lymphoid, myeloid, and primitive cell types. Results indicated that T cells were maintained in culture and B-lymphoid markers remained negative. In the myeloid subset, there was an overall reduction in the pan-myeloid markers. We believe this represents the loss of terminally differentiated cells (e.g., neutrophils) in culture. Overall, the study clearly demonstrates that the application of GF stimulants does not alter clonality or cell lineage propagated in these samples and is therefore suitable for application in diagnostic cytogenetic laboratories.

Ewing's tumour: novel recurrent chromosomal abnormalities demonstrated by molecular cytogenetic analysis of seven cell lines and one primary culture.

Conventional cytogenetics has led to the identification of the primary t(11;22)(q24;q12) translocation in the Ewing's family of tumours, and to the demonstration of certain recurring secondary aberrations that may contribute to neoplastic progression. Other important cytogenetic abnormalities may previously have been overlooked due to the limited resolution of chromosome banding. Here, we have applied the molecular cytogenetic techniques of spectral karyotyping, multiplex-fluorescence in situ hybridisation and comparative genomic hybridisation to the characterisation of seven Ewing's tumour cell lines and one primary culture. These complementary techniques have enabled us to produce a detailed description of the karyotypes of the cell lines and to demonstrate recurring numerical and structural abnormalities. In particular, we have identified a novel, unbalanced translocation involving chromosomes 16 and 17 in three of eight samples, including the primary culture. The unbalanced translocation was associated with comparative genomic hybridisation evidence of loss of 16q and 17p, copy number imbalances that were seen in five and four of the eight samples respectively. Recurrent breakpoints at 16p11.2, 16q11.1, 17p11.2 and 17q11.2 were identified. Our findings indicate that chromosomes 16 and 17 should be investigated further in the search for genes involved in the development of Ewing's family tumours.

Myeloproliferative disorders.

The myeloproliferative disorders (MPDs) are a group of pre-leukaemic disorders characterized by proliferation of one or more lineages of the myelo-erythroid series. Unlike the Philadelphia chromosome in chronic myeloid leukaemia, there is no pathognomonic chromosomal abnormality associated with the MPDs. Chromosomal abnormalities are seen in 30-40% of patients with polycythaemia vera (PV) and idiopathic myelofibrosis (IMF) and seem to indicate a poor prognosis. On the other hand, chromosomal abnormalities are rare in essential thrombocythaemia. Consistent acquired changes seen at diagnosis include deletion of the long arm of chromosome 20, del(13q), trisomy 8 and 9 and duplication of parts of 1q. Furthermore del(20q), trisomy 8 and dupl(lq) all arise in multipotent progenitor cells. Molecular mapping of 20q deletions and, to some extent, 13q deletions has identified a number of candidate target genes, although no mutations have yet been found. Finally, translocations associated with the rare 8p11 myeloproliferative syndrome and other atypical myeloproliferative disorders have permitted the identification of a number of novel fusion proteins involving fibroblast growth factor receptor-1.

Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia.

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.

Variant Philadelphia translocations in chronic myeloid leukaemia can mimic typical blast crisis chromosome abnormalities or classic t(9;22): a report of two cases.

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.

Screening for specific chromosome involvement in hematological malignancies using a set of seven chromosome painting probes. An alternative approach for chromosome analysis using standard FISH instrumentation.

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.

Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG).

Deletion of the long arm of chromosome 20 represents the most common chromosomal abnormality associated with the myeloproliferative disorders (MPDs) and is also found in other myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Previous studies have identified a common deleted region (CDR) spanning approximately 8 Mb. We have now used G-banding, FISH or microsatellite PCR to analyse 113 patients with a 20q deletion associated with a myeloid malignancy. Our results define a new MPD CDR of 2.7 Mb, an MDS/AML CDR of 2.6 Mb and a combined 'myeloid' CDR of 1.7 Mb. We have also constructed the most detailed physical map of this region to date--a bacterial clone map spanning 5 Mb of the chromosome which contains 456 bacterial clones and 202 DNA markers. Fifty-one expressed sequences were localized within this contig of which 37 lie within the MPD CDR and 20 within the MDS/AML CDR. Of the 16 expressed sequences (six genes and 10 unique ESTs) within the 'myeloid' CDR, five were expressed in both normal bone marrow and purified CD34 positive cells. These data identify a set of genes which are both positional and expression candidates for the target gene(s) on 20q.

Fluorescence in situ hybridization detection of two telomeres on the short arm of a derived chromosome 16 in an infant with thrombocytopenia.

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.

Cytogenetics of the chronic myeloid leukemia-derived cell line K562: karyotype clarification by multicolor fluorescence in situ hybridization, comparative genomic hybridization, and locus-specific fluorescence in situ hybridization.

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.

Large deletions at the t(9;22) breakpoint are common and may identify a poor-prognosis subgroup of patients with chronic myeloid leukemia.

The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene, which is usually formed as a result of the t(9;22) translocation. Patients with CML show considerable heterogeneity both in their presenting clinical features and in the time taken for evolution to blast crisis. In this study, metaphase fluorescence in situ hybridization showed that a substantial minority of patients with CML had large deletions adjacent to the translocation breakpoint on the derivative 9 chromosome, on the additional partner chromosome in variant translocations, or on both. The deletions spanned up to several megabases, had variable breakpoints, and could be detected by microsatellite polymerase chain reaction in unfractionated bone marrow and purified peripheral blood granulocytes. The deletions were likely to occur early and possibly at the time of the Philadelphia (Ph) chromosome translocation: deletions were detected at diagnosis in 11 patients, were found in all Ph-positive metaphases, and were more prevalent in patients with variant Ph chromosomes. Kaplan-Meier analysis showed a median survival time of 36 months in patients with a deletion; patients without a detectable deletion survived > 90 months. The survival-time difference was significant on log-rank analysis (P =. 006). Multivariate analysis demonstrated that the prognostic importance of deletion status was independent of age, sex, percentage of peripheral blood blasts, and platelet count. Our data therefore suggest that an apparently simple, balanced translocation may result not only in the generation of a dominantly acting fusion oncogene but also in the loss of one or more genes that influence disease progression. (Blood. 2000;95:738-743)

Novel method for the production of multiple colour chromosome paints for use in karyotyping by fluorescence in situ hybridisation.

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.

Elucidation of the mechanism of homozygous deletion of 3p12-13 in the U2020 cell line reveals the unexpected involvement of other chromosomes.

Homozygous deletions in tumor cells have been useful in the localization and validation of tumor suppressor genes. We have described a homozygous deletion in a lung cancer cell line (U2020) which is located within the most proximal of the three regions on the short arm of chromosome 3 believed to be lost in lung cancer development. Construction of a YAC contig map indicates that the deletion spans around 8 Mb, but no large deletion was apparent on conventional cytogenetic analysis of the cell line. To investigate this paradox, whole chromosome, arm-specific, and regional paints have been used. This analysis has revealed that genetic loss has occurred by complex rearrangements of chromosomes 3, rather than simple interstitial deletion. These studies emphasize the power of molecular cytogenetics to disclose unsuspected tumor-specific translocations within the extremely complex karyotypes characteristic of solid tumors.