Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome.

The distinction between acquired aplastic anemia (AA) and hypocellular myelodysplastic syndrome (hMDS) is often difficult, especially nonsevere AA. We postulated that somatic mutations are present in a subset of AA, and predict malignant transformation. From our database, we identified 150 AA patients with no morphological evidence of MDS, who had stored bone marrow (BM) and constitutional DNA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. The initial cohort of 57 patients was screened for 835 known genes associated with BMF and myeloid cancer; a second cohort of 93 patients was screened for mutations in ASXL1, DNMT3A, BCOR, TET2, and MPL. Somatic mutations were detected in 19% of AA, and included ASXL1 (n = 12), DNMT3A (n = 8) and BCOR (n = 6). Patients with somatic mutations had a longer disease duration (37 vs 8 months, P < .04), and shorter telomere lengths (median length, 0.9 vs 1.1, P < .001), compared with patients without mutations. Somatic mutations in AA patients with a disease duration of >6 months were associated with a 40% risk of transformation to MDS (P < .0002). Nearly one-fifth of AA patients harbor mutations in genes typically seen in myeloid malignancies that predicted for later transformation to MDS.

MicroRNA-196a promotes an oncogenic effect in head and neck cancer cells by suppressing annexin A1 and enhancing radioresistance.

Radiotherapy is a major treatment modality for head and neck squamous cell carcinoma (HNSCC). Up to 50% of patients with locally advanced disease relapse after radical treatment and there is therefore a need to develop predictive bomarkers for clinical use that allow the selection of patients who are likely to respond. MicroRNA (miRNA) expression profiling of a panel of HNSCC tumours with and without recurrent disease after surgery and radiotherapy detected miR-196a as one of the highest upregulated miRNAs in the poor prognostic group. To further study the role of miR-196a, its expression was determined in eight head and neck cancer cell lines. Overexpression of miR-196a in HNSCC cells, with low endogenous miR-196a expression, significantly increased cell proliferation, migration and invasion, and induced epithelial to mesenchymal transition. Conversely, miR-196a knockdown in cells with high endogenous expression levels significantly reduced oncogenic behaviour. Importantly, overexpression of miR-196a increased radioresistance of cells as measured by gamma H2AX staining and MTT survival assay. Annexin A1 (ANXA1), a known target of miR-196a, was found to be directly modulated by miR-196a as measured by luciferase assay and confirmed by Western blot analysis. ANXA1 knockdown in HNSCC exhibited similar phenotypic effects to miR-196a overexpression, suggesting the oncogenic effect of miR-196a may at least be partly regulated through suppression of ANXA1. In conclusion, this study identifies miR-196a as a potential important biomarker of prognosis and response of HNSCC to radiotherapy. Furthermore, our data suggest that miR-196a and/or its target gene ANXA1 could represent important therapeutic targets in HNSCC.

Apoptin interacts with and regulates the activity of protein kinase C beta in cancer cells.

Apoptin, the VP3 protein from chicken anaemia virus (CAV), induces tumour cell-specific cell death and represents a potential future anti-cancer therapeutic. In tumour but not in normal cells, Apoptin is phosphorylated and translocates to the nucleus, enabling its cytotoxic activity. Recently, the ß isozyme of protein kinase C (PKCß) was shown to phosphorylate Apoptin in multiple myeloma cell lines. However, the exact mechanism and nature of interaction between PKCß and Apoptin remain unclear. Here we investigated the physical and functional link between PKCß and CAV-Apoptin as well as with the recently identified Apoptin homologue derived from human Gyrovirus (HGyV). In contrast to HCT116 colorectal cancer cells the normal colon mucosa cell lines expressed low levels of PKCßI and showed reduced Apoptin activation, as evident by cytoplasmic localisation, decreased phosphorylation and lack of cytotoxic activity. Co-immunoprecipitation and proximity ligation assay studies identified binding of both CAV- and HGyV-Apoptin to PKCßI in HCT116 cells. Using Apoptin deletion constructs the N-terminal domain of Apoptin was found to be required for interacting with PKCßI. FRET-based PKC activity reporter assays by fluorescence lifetime imaging microscopy showed that expression of Apoptin in cancer cells but not in normal cells triggers a significant increase in PKC activity. Collectively, the results demonstrate a novel cancer specific interplay between Apoptin and PKCßI. Direct interaction between the two proteins leads to Apoptin-induced activation of PKC and consequently activated PKCßI mediates phosphorylation of Apoptin to promote its tumour-specific nuclear translocation and cytotoxic function.

Utility of peripheral blood for cytogenetic and mutation analysis in myelodysplastic syndrome.

Recent studies have shown that more than 80% of bone marrow (BM) samples from patients with myelodysplastic syndrome (MDS) harbor somatic mutations and/or genomic aberrations, which are of diagnostic and prognostic importance. We investigated the potential use of peripheral blood (PB) and serum to identify and monitor BM-derived genetic markers using high-resolution single nucleotide polymorphism array (SNP-A) karyotyping and parallel sequencing of 22 genes frequently mutated in MDS. This pilot study showed a 100% SNP-A karyotype concordance and a 97% mutation concordance between the BM and PB. In contrast, mutation analysis using Sanger sequencing of PB and serum-derived DNA showed only 65% and 42% concordance to BM, respectively. Our results show the potential utility of PB as a surrogate for BM for MDS patients, thus avoiding the need for repeated BM aspirates particularly in elderly patients and those with fibrotic or hypocellular marrows.

A functional assay for microRNA target identification and validation.

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis.

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9.4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0.001) and it retained significance in multivariable model (Hazard Ratio 3.8, 95%CI 2.3-6.3,P < 0.001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in non-responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q- and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein.

Next-generation sequencing of the TET2 gene in 355 MDS and CMML patients reveals low-abundance mutant clones with early origins, but indicates no definite prognostic value.

Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using "next-generation" sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chronic myelomonocytic leukemia patients (P < .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. "Deeper" sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34(+) and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Delivery of therapeutic proteins as secretable TAT fusion products.

The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TATkappa-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TATkappa-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins.

MicroRNA-9 inhibits growth and invasion of head and neck cancer cells and is a predictive biomarker of response to plerixafor, an inhibitor of its target CXCR4.

Head and neck squamous cell carcinomas (HNSCC) are associated with poor morbidity and mortality. Current treatment strategies are highly toxic and do not benefit over 50% of patients. There is therefore a crucial need for predictive and/or prognostic biomarkers to allow treatment stratification for individual patients. One class of biomarkers that has recently gained importance are microRNA (miRNA). MiRNA are small, noncoding molecules which regulate gene expression post-transcriptionally. We performed miRNA expression profiling of a cohort of head and neck tumours with known clinical outcomes. The results showed miR-9 to be significantly downregulated in patients with poor treatment outcome, indicating its role as a potential biomarker in HNSCC. Overexpression of miR-9 in HNSCC cell lines significantly decreased cellular proliferation and inhibited colony formation in soft agar. Conversely, miR-9 knockdown significantly increased both these features. Importantly, endogenous CXCR4 expression levels, a known target of miR-9, inversely correlated with miR-9 expression in a panel of HNSCC cell lines tested. Induced overexpression of CXCR4 in low expressing cells increased proliferation, colony formation and cell cycle progression. Moreover, CXCR4-specific ligand, CXCL12, enhanced cellular proliferation, migration, colony formation and invasion in CXCR4-overexpressing and similarly in miR-9 knockdown cells. CXCR4-specific inhibitor plerixafor abrogated the oncogenic phenotype of CXCR4 overexpression as well as miR-9 knockdown. Our data demonstrate a clear role for miR-9 as a tumour suppressor microRNA in HNSCC, and its role seems to be mediated through CXCR4 suppression. MiR-9 knockdown, similar to CXCR4 overexpression, significantly promoted aggressive HNSCC tumour cell characteristics. Our results suggest CXCR4-specific inhibitor plerixafor as a potential therapeutic agent, and miR-9 as a possible predictive biomarker of treatment response in HNSCC.

miR-181c-BRK1 axis plays a key role in actin cytoskeleton-dependent T cell function.

MicroRNAs are short endogenous noncoding RNAs that play pivotal roles in a diverse range of cellular processes. The miR-181 family is important in T cell development, proliferation, and activation. In this study, we have identified BRK1 as a potential target of miR-181c using a dual selection functional assay and have showed that miR-181c regulates BRK1 by translational inhibition. Given the importance of miR-181 in T cell function and the potential role of BRK1 in the involvement of WAVE2 complex and actin polymerization in T cells, we therefore investigated the influence of miR-181c-BRK1 axis in T cell function. Stimulation of PBMC derived CD3+ T cells resulted in reduced miR-181c expression and up-regulation of BRK1 protein expression, suggesting that miR-181c-BRK1 axis is important in T cell activation. We further showed that overexpression of miR-181c or suppression of BRK1 resulted in inhibition of T cell activation and actin polymerization coupled with defective lamellipodia generation and immunological synapse formation. Additionally, we found that BRK1 silencing led to reduced expressions of other proteins in the WAVE2 complex, suggesting that the impairment of T cell actin dynamics was a result of the instability of the WAVE2 complex following BRK1 depletion. Collectively, we demonstrated that miR-181c reduces BRK1 protein expression level and highlighted the important role of miR-181c-BRK1 axis in T cell activation and actin polymerization-mediated T cell functions.

Retroviral insertional mutagenesis implicates E3 ubiquitin ligase RNF168 in the control of cell proliferation and survival.

The E3 ubiquitin ligase RNF168 is a ring finger protein that has previously been identified to play an important regulatory role in the repair of double-strand DNA breaks.  In the present study, an unbiased forward genetics functional screen in mouse granulocyte/ macrophage progenitor cell line FDCP1 has identified E3 ubiquitin ligase RNF168 as a key regulator of cell survival and proliferation. Our data indicate that RNF168 is an important component of the mechanisms controlling cell fate, not only in human and mouse haematopoietic growth factor-dependent cells, but also in the human breast epithelial cell line MCF-7. These observations therefore suggest that RNF168 provides a connection to key pathways controlling cell fate, potentially through interaction with PML nuclear bodies and/or epigenetic control of gene expression. Our study is the first to demonstrate a critical role for RNF168 in the in the mechanisms regulating cell proliferation and survival, in addition to its well-established role in DNA repair.

RPL27A is a target of miR-595 and may contribute to the myelodysplastic phenotype through ribosomal dysgenesis.

We investigated the functional consequences following deletion of a microRNA (miR) termed miR-595 which resides on chromosome 7q and is localised within one of the commonly deleted regions identified for Myelodysplasia (MDS) with monosomy 7 (-7)/isolated loss of 7q (7q-). We identified several targets for miR-595, including a large ribosomal subunit protein RPL27A. RPL27A downregulation induced p53 activation, apoptosis and inhibited proliferation. Moreover, p53-independent effects were additionally identified secondary to a reduction in the ribosome subunit 60s. We confirmed that RPL27A plays a pivotal role in the maintenance of nucleolar integrity and ribosomal synthesis/maturation. Of note, RPL27A overexpression, despite showing no significant effects on p53 mRNA levels, did in fact enhance cellular proliferation. In normal CD34+ cells, RPL27A knockdown preferentially blocked erythroid proliferation and differentiation. Lastly, we show that miR-595 expression appears significantly downregulated in the majority of primary samples derived from MDS patients with (-7)/(7q-), in association with RPL27A upregulation. This significant downregulation of miR-595 is also apparent when higher risk MDS cases are compared to lower risk cases. The potential clinical importance of these findings requires further validation.

E1A-mediated suppression of EGFR expression and induction of apoptosis in head and neck squamous carcinoma cell lines.

Previous studies have shown early region 1A (E1A) gene to inhibit the proliferation of tumour cells with wild-type, but not mutant, p53. E1A has also been shown to downregulate c-erb-B-2/neu expression, resulting in inhibition of growth in c-erb-B-2/neu overexpressing tumour cells. In this study, we have investigated the effect of E1A expression on four head and neck squamous cell carcinoma (HNSCC) cell lines that do not overexpress c-erb-B-2/neu. Cell cycle and Western blot analysis show E1A-mediated induction of apoptosis in all cell lines examined. This induction of apoptosis was independent of the p53 status as it occurred in the cell lines with wild-type, mutated or deleted p53. However, there was no evidence of E1A-induced apoptosis in a p53(+ve) normal human fibroblast cell line, 1BR3. Analysis of apoptosis in the SCC cell lines demonstrated E1A-mediated downregulation of EGFR, which was overexpressed in each of these cell lines. Overexpression of an exogenously introduced EGFR, under the control of an E1A-insensitive heterologous promoter, blocked E1A induction of apoptosis in these cells. Therefore, E1A-mediated downregulation of EGFR expression appears to be the cause, rather than a consequence of E1A-induced apoptosis in these SCC cell lines. Previous studies have shown downregulation of EGFR expression by PML. Interestingly, E1A expression in the HNSCC cells altered the pattern of PML distribution and induced the level of PML protein, thus suggesting that E1A-mediated downregulation of EGFR may occur via direct or indirect interactions with PML. These findings demonstrate a novel pathway by which E1A can induce apoptosis and identify EGFR as a potential target for the development of therapeutic strategies against epithelial malignancies, the majority of which have abnormal EGFR expression.

TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells.

Apoptin has been described to induce apoptosis in various human cancer cell lines, but not in normal cells, thus making it an interesting candidate for the development of novel therapeutic strategies. Apoptin was generated and cloned into several mammalian expression vectors. Transfection or microinjection of apoptin cDNA resulted in its expression, initially in the cytoplasm with a filamentous pattern. Subsequently, apoptin entered the nucleus and efficiently induced apoptosis in several cancer cell lines. Nuclear localization was shown to be required for induction of apoptosis. Apoptin expression level was found to be an important determinant of the efficiency of induction of apoptosis. Surprisingly, expression of apoptin or GFP-apoptin cDNA induced apoptosis in some normal cells. When fused to the HIV-TAT protein transduction domain and delivered as a protein, TAT-apoptin was transduced efficiently (>90%) into normal and tumour cells. However, TAT-apoptin remained in the cytoplasm and did not kill normal 6689 and 1BR3 fibroblasts. In contrast TAT-apoptin migrated from the cytoplasm to the nucleus of Saos-2 and HSC-3 cancer cells resulting in apoptosis after 24 h. This study shows that apoptin is a powerful apoptosis-inducing protein with a potential for cancer therapy.

CSNK1A1 mutations and isolated del(5q) abnormality in myelodysplastic syndrome: a retrospective mutational analysis.

BACKGROUND: A mechanism for clonal growth advantage in isolated del(5q) disease remains elusive. CSNK1A1 resides on the critically deleted region, and deletion of this gene has been shown in mouse knockout and transplantation studies to produce some characteristics of bone marrow failure, including a proliferative advantage. We aimed to establish the frequency, nature, and clinical association of CSNK1A1 mutations in patients with myelodysplastic syndrome and associated myeloid neoplasms. METHODS: Between June 1, 2004, and May 31, 2014, in King's College (London, UK), we did whole-exome sequencing of five patients with isolated del(5q) followed by targeted screening for CSNK1A1 mutations and 20 myelodysplastic syndrome-associated mutations in 245 additional patients with myeloid neoplasms. All patients met present WHO diagnostic criteria for myelodysplastic syndrome and other related myeloid neoplasms. FINDINGS: 39 (16%) of 250 patients with myeloid neoplasms had isolated del(5q), of whom seven (18%) had CSNK1A1 mutations. All these mutations were missense and presented in a highly conserved region that is implicated in ATP catalysis. Serial sampling and response to lenalidomide treatment showed that CSNK1A1 mutations were highly associated with the del(5q) clone. Only one patient with a CSNK1A1 mutation showed complete cytogenetic response to lenalidomide. Four (57%) of the seven patients carrying a CSNK1A1 mutation showed disease progression coupled with an increase in mutant allele burden (all four were on lenalidomide). We detected coexisting myelodysplastic syndrome-related gene mutations in patients with CSNK1A1 mutations, including TP53. INTERPRETATION: Similar to the effect of TP53 mutations on progression of del(5q) abnormality, mutant CSNK1A1 also gives rise to a poor prognosis in del(5q) abnormality, for which a coupled increase in P53 activation is suggested. CSNK1A1 mutations in del(5q) disease are important in the context of therapeutic manipulation and need incorporation into future prospective studies. FUNDING: Leukaemia and Lymphoma Research.

Crucial roles for protein kinase C isoforms in tumor-specific killing by apoptin.

The chicken anemia virus-derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein-apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34(+) cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cß (PKCß) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCß significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCß, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCß and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma.

Novel TET2 mutations associated with UPD4q24 in myelodysplastic syndrome.

PURPOSE: Cryptic chromosomal aberrations, such as regions of uniparental disomy (UPD), have been shown to harbor homozygous mutations and are a common feature in myelodysplastic syndrome (MDS). We investigated the sequence integrity of 4q24 candidate tumor suppressor gene TET2 in MDS patients with UPD on chromosome 4. PATIENTS AND METHODS: The coding exons of TET2 were analyzed by 454 deep sequencing and Sanger sequencing in nine patients with UPD on 4q. Four patients had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) and UPD4q24, and five patients (refractory anemia with excess blasts-II, n = 1; 5q- syndrome, n = 1; RCMD-RS, n = 1; refractory anemia, n = 1; refractory cytopenia with multilineage dysplasia, n = 1) had no UPD4q24. RESULTS: Mutations on TET2 were identified in all four patients with UPD4q24. These were localized to exons 3, 6, and 9 and resulted in two premature stop codons, one frameshift mutation, and one cysteine to glycine amino acid change. Mutant clone size varied between 30% and 85%. One patient with UPD outside of q24 (UPD4q28.3) displayed additional TET2 mutations, but these were at low clonal levels (13%, 4%, and 4% for a silent mutation, a 180-base pair deletion in exon 3, and a lysine to phenylalanine substitution in exon 11, respectively). The other patients who did not have UPD4q24 did not have verifiable TET2 mutations. CONCLUSION: Our data identify novel TET2 mutations in a dominant clone in patients with UPD4q24. The presence of UPD4q24 and mutations in RCMD-RS patients may suggest specificity to this subtype. Our preliminary results need to be confirmed in a large cohort of all MDS subtypes.

Prevalence and prognostic significance of allelic imbalance by single-nucleotide polymorphism analysis in low-risk myelodysplastic syndromes.

Low-risk myelodysplastic syndrome (MDS) with normal cytogenetics accounts for approximately 50% of MDS patients. There are no pathognomonic markers in these cases and the diagnosis rests on cytomorphologic abnormalities in bone marrow and/or peripheral blood. Affymetrix high-resolution single-nucleotide polymorphism (SNP) genotyping microarrays allow detection of cytogenetically cryptic genomic aberrations. We have studied 119 low-risk MDS patients (refractory anemia [RA] = 22; refractory cytopenia with multilineage dysplasia [RCMD] = 51; refractory anemia with ringed sideroblasts [RARS] = 12; refractory cytopenia with multilineage dysplasia with ringed sideroblasts [RCMD-RS] = 12; 5q- syndrome = 16; refractory anemia with excess blasts [RAEB] = 6) using SNP microarrays to seek chromosomal markers undetected by conventional cytogenetics. Loss of heterozygosity (LOH) detected by 50K arrays was verified using 250K and 500K arrays. We demonstrate the presence of uniparental disomy (UPD) in 46%, deletions in 10%, and amplifications in 8% of cases. Copy number (CN) changes were acquired, whereas UPDs were also detected in constitutional DNA. UPD on 4q was identified in 25% of RARS, 12% of RCMD with normal cytogenetics, 17% of RAEB, and 6% of 5q- syndrome cases. Univariate analysis showed deletions (P = .04) and International Prognostic Scoring System (IPSS; P < .001) scores correlated with overall survival; however, on multivariate analysis only IPSS scores retained prognostic significance (P < .001). We show, for the first time, that SNP microarray analysis in low-risk MDS patients reveals hitherto unrecognized UPD and CN changes that may allow stratification of these patients for early therapeutic interventions.

An immune edited tumour versus a tumour edited immune system: Prospects for immune therapy of acute myeloid leukaemia.

Cell based therapies for acute myeloid leukaemia (AML) have made significant progress in the last decade benefiting the prognosis and survival of patients with this aggressive form of leukaemia. Due to advances in haematopoietic stem cell transplantation (HSCT) and particularly the advent of reduced intensity conditioning (RIC), the scope of transplantation has now extended to those patients previously ineligible due to age and health restrictions and has been associated with a decrease in transplant related mortality. The apparent graft versus leukaemia (GvL) effect observed following HSCT demonstrates the potential of the immune system to target and eradicate AML cells. Building on previously published pre-clinical studies by ourselves and others, we are now initiating a Phase I clinical study in which lentiviral vectors are used to genetically modify AML cells to express B7.1 (CD80) and IL-2. By combining allogeneic HSCT with immunisation, using the autologous AML cells expressing B7.1 and IL-2, we hope to stimulate immune eradication of residual AML cells in poor prognosis patients that have achieved donor chimerism. In this report we describe the background to cell therapy based approaches for AML, and discuss difficulties associated with the deployment of a chronically stimulated, hence exhausted/depleted immune system to eradicate tumour cells that have already escaped immune surveillance.

Prevalence of the activating JAK2 tyrosine kinase mutation V617F in the Budd-Chiari syndrome.

BACKGROUND & AIMS: Budd-Chiari Syndrome (BCS) results from obstruction to hepatic venous outflow, with myeloproliferative disorder (MPD) accounting for up to 40% of cases. A number of BCS cases labelled as "idiopathic" do not fulfill the diagnostic criteria for MPD but have features suggestive of a latent form based on hyperplastic bone marrow and erythroid progenitor cell culture; these cases may subsequently develop overt MPD. A clonal mutation in JAK2 tyrosine kinase (JAK2V617F) occurs in a high proportion of patients with MPD and is of use in the characterization of latent MPD in BCS. METHODS: We performed allele-specific polymerase chain reaction to screen for JAK2V617F in subjects with BCS (n = 41) and polycythemia vera (PV) (n = 20) and in hematologically normal controls (n = 27). RESULTS: AK2V617F was detected in 24 of 41 (58.5%) subjects with BCS, 19 of 20 PV controls, and 0 of 27 hematologically normal controls. Mean hemoglobin concentration and hematocrit were significantly higher in patients with JAK2V617F. Bone marrow was hyperplastic in 16 of 41 subjects (12/16 JAK2V617F positive). Nine of 33 (27.3%) showed endogenous erythroid colony formation (7/9 JAK2V617F positive). Eleven of 41 subjects developed overt MPD (8/11 essential thrombocythemia, 3/11 PV) after the diagnosis of BCS (median, 49 months; range, 8-87 months), and in 90.9% of these JAK2V617F was detected. CONCLUSIONS: JAK2V617F occurs in a high proportion of patients with BCS. Latent MPD was missed in a substantial number of our subjects by using standard techniques. Such cases should be screened for JAK2V617F and carefully observed for the subsequent development of overt MPD.