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.