Diana Brazma

Transmission of Integrated Human Herpesvirus 6 through Stem Cell Transplantation: Implications for Laboratory Diagnosis

We identified a stem cell donor with chromosomally integrated human herpesvirus (HHV)-6 and monitored the recipient for HHV-6 after transplantation. The appearance and subsequent increase in HHV-6 load paralleled engraftment and an increase in white blood cell count. Fluorescent in situ hybridization analysis showed integrated HHV-6 on chromosome band 17p13.3 in the donor and in the recipient after transplantation but not in the recipient before transplantation. The increase in viral load due to the genetic transmission of integrated HHV-6 could have been misinterpreted as substantial active infection and, thus, led to the administration of toxic antiviral therapy. We suggest that the confounding influence of integration be considered in laboratory investigations associating HHV-6 with disease.

Human herpesvirus 6 integrates within telomeric regions as evidenced by five different chromosomal Sites

Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal integration sites of human herpesvirus 6 (HHV‐6) in phytohemagglutinin‐stimulated leukocytes and B lymphocytes from Epstein–Barr virus transformed lymphoblastoid cell lines (LCLs). Five different chromosomal integration sites were found in nine individuals. Only one site was identified in each individual, each site was in the vicinity of the telomeric region and was on either the p or q arm of only one of the two chromosome homologues. The sites were 9q34.3, 10q26.3, 11p15.5, 17p13.3, and 19q 13.4, of which three have not been previously identified. For 9q34.3 the site of integration was further mapped using a locus‐specific probe for 9q34.3 together with a pan‐telomeric probe and both co‐localized with the HHV‐6 signal. Similarly an arm‐specific telomeric probe for 19q co‐localized with the HHV‐6 signal. It was therefore concluded that the site of integration is actually within the telomere. The number of viral DNA copies/cell was calculated in blood, LCL cells and hair follicles and was one or more in every case for each of the nine individuals. This result was confirmed by FISH where 100% of cells gave an HHV‐6 signal. These findings add to previous reports suggesting that integrated HHV‐6 DNA is found in every cell in the body and transmitted vertically. Finally, including our data, worldwide seven different chromosomal sites of HHV‐6 integration have now been identified. Large epidemiological studies of chromosomal integration are required to identify further telomeric sites, geographical or racial variation and possible clinical consequences. J. Med. Virol. 80:1952–1958, 2008.

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.

Does BCR/ABL1 positive acute myeloid leukaemia exist?

The BCR/ABL1 fusion gene, usually carried by the Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) or variants, is pathognomonic for chronic myeloid leukaemia (CML). It is also occasionally found in acute lymphoblastic leukaemia (ALL) mostly in adults and rarely in de novo acute myeloid leukaemia (AML). Array Comparative Genomic Hybridization (aCGH) was used to study six Ph(+)AML, three bi‐lineage and four Ph(+)ALL searching for specific genomic profiles. Surprisingly, loss of the IKZF1 and/or CDKN2A genes, the hallmark of Ph(+)ALL, were recurrent findings in Ph(+)AML and accompanied cryptic deletions within the immunoglobulin and T cell receptor genes. The latter two losses have been shown to be part of ‘hot spot’ genome imbalances associated with BCR/ABL1 positive pre‐B lymphoid phenotype in CML and Ph(+)ALL. We applied Significance Analysis of Microarrays (SAM) to data from the ‘hot spot’ regions to the Ph(+)AML and a further 40 BCR/ABL1(+) samples looking for differentiating features. After exclusion of the most dominant markers, SAM identified aberrations unique to de novo Ph(+)AML that involved relevant genes. While the biological and clinical significance of this specific genome signature remains to be uncovered, the unique loss within the immunoglobulin genes provides a simple test to enable the differentiation of clinically similar de novo Ph(+) AML and myeloid blast crisis of CML.

Deletions of Immunoglobulin heavy chain and T cell receptor gene regions are uniquely associated with lymphoid blast transformation of chronic myeloid leukemia

BackgroundChronic 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.ResultsHere 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.ConclusionsNone 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.

Can genome array screening replace FISH as a front-line test in multiple myeloma?

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.

Genome gains at chromosome 21q21/22 segment leads to co-amplification of Down Syndrome Critical Regions and known oncogenes in a case of donor cell-derived acute myeloid leukaemia following allogeneic sex mismatched umbilical cord blood transplantation for chronic myeloid leukaemia

Donor cell-derived leukaemia (DCL) following umbilical cord blood transplantation (UCBT) is a rare phenomenon with only a handful of cases appearing in the literature. Potential mechanisms for DCL development post-haematopoietic cell transplantation include occult pre-leukaemic clones in donor cells, immune surveillance defect(s), therapy-related stromal abnormalities, excess cytokine stimulation, and DNA replication/repair errors associated with post-transplant expansion (Ruiz-Argüelles et al, 2007). We agree with the view that a common mechanism is not likely. Although case numbers are small, no consistent chromosome abnormality has been identified thus far, with normal karyotype in approximately half the cases, and a mixture of numerical and/or complex structural aberrations in the remainder. We performed detailed analysis on patient and cord blood samples from a 32-year-old female with chronic myeloid leukaemia and a history of acute megakaryoblastic transformation (AMKL), who developed DCL following UCBT. Cytogenetic analysis at presentation identified t(9;22) (q34q11) and a marker inv(7)(q?22q?35) in all dividing cells (Fig. 1A). Following transformation to AMKL and the attainment of morphological remission with induction chemotherapy, the patient underwent reduced intensity conditioned single antigen mismatched UCBT from a male donor. Despite achieving complete molecular response, and full donor chimaerism [200 nuclei showing male signal pattern by XY fluorescent in situ hybridization (FISH)] at 1 month post-UCBT, bone marrow (BM) examination at 17 months revealed tri-lineage dysplasia, erythroid hyperplasia (53% of cells) and increased blasts (16% of total cells; 33% of nonerythroid cells). Immunophenotyping confirmed a clonal population of myeloblasts (CD34 30%, CD13 73%, CD33 73%, cMPO 46%, TdT 0%, CD7 45%, CD13:34 30%, CD7:33 30%). All cells were BCR/ABL1 negative by nested polymerase chain reaction (PCR) and of male origin by XY FISH. The patient died from overwhelming sepsis following the diagnosis of an evolving donor cell-derived acute myeloid leukaemia (AML). Analysis of BM aspirate unveiled an abnormal karyotype 44,XY,-7, der(17)t(17;21)(p13?p/q?),-21 confirmed by FISH (Fig. 1B). Genome-wide oligonucleotide array comparative genomic hybridization (aCGH) analysis identified loss of chromosome 7, and partial losses and gains of chromosome 21, whilst chromosome 17 showed no changes (Fig. 2). Genomic imbalances at the 21q21/q22 region involved gene rich sequences including:

Copy Number Variants: Distribution in Patients with Coronary Atherosclerosis

ABSTRACT 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 described 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.