Julie Howard-Reeves

p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53s transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.

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: