Michael Neat

Mutations of CEBPA in acute myeloid leukemia FAB types M1 and M2

CEBPA encodes the transcription factor C/EBPα and is specifically up‐regulated during granulocytic differentiation. The gene is mutated in approximately 20% of patients with acute myeloid leukemia (AML) FAB type M2 and occurs in the absence of the t(8;21). In much the same way as specific translocations are associated with a particular AML FAB type, the identification of non‐random associations of gene mutation with karyotype or FAB type may be helpful in elucidating the molecular basis of certain forms of leukemia. To confirm these initial findings, 99 patients with AML FAB type M1 or M2 were screened for CEBPA mutations by use of a PCR–single‐strand conformational polymorphism and sequencing approach. Nine CEBPA mutations were identified in eight patients. The mutations were clustered toward the COOH terminal of the protein and occurred exclusively in the intermediate cytogenetic risk group (8/64, 12.5%). Two patients with biallelic mutation, one homozygous for 1137Ins (57 bp) and another with two CEBPA mutations, 1096Ins (27 bp) and 363Ins (GGCC), were observed. There was no evidence for deletion of this region in the other six mutated samples analyzed by fluorescence in situ hybridization with a BAC clone spanning the CEBPA locus. CEBPA mutation status was not demonstrated to be of prognostic importance in this patient group, although this may reflect the selection and size of the AML population studied. In conclusion, mutation of CEBPA is a recurrent finding in AML and appears specific to the intermediate cytogenetic risk group patients.

The use of real-time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma.

Using comparative genomic hybridization (CGH), aberrations in DNA copy number were studied before and after transformation of follicular lymphoma to diffuse large B‐cell lymphoma in six patients (15 lymph node biopsies in total). The most common and also the most discrete and intense amplification occurring in four out of 15 biopsies from three different patients was of 2p13–16. Using real‐time quantitative polymerase chain reaction (RQ‐PCR), REL amplification was found to be implicated at this locus. This technique also identified amplified REL in a further two biopsies, presumably below the detection level of CGH. REL amplification was quantified by comparing it, in most cases, with three endogenous reference genes, albumin, β2‐microglobulin and CD8α, that lie close to REL on 2p. There was no correlation apparent between 2p13–16 amplification or REL amplification and transformation. This study shows the usefulness of coupling CGH, for detecting recurring abnormalities, with the real‐time PCR technique for rapid gene dosage quantification and confirms that the REL gene is a potential candidate in the pathogenesis of a particular subset of follicular lymphomas.

Detection of Chromosome Abnormalities Pre–High-Dose Treatment in Patients Developing Therapy-Related Myelodysplasia and Secondary Acute Myelogenous Leukemia After Treatment for Non-Hodgkin’s Lymphoma

PURPOSE To assess whether pre-high-dose therapy (HDT)-related factors play a critical role in the development of therapy-related myelodysplasia (tMDS) or secondary acute myelogenous leukemia (sAML). PATIENTS AND METHODS Twenty-nine of 230 patients with a primary diagnosis of non-Hodgkins lymphoma (NHL) developed tMDS/sAML after HDT comprising cyclophosphamide and total-body irradiation (TBI) supported by autologous hematopoietic progenitor cells. G-banding and fluorescence in-situ hybridization (FISH) were used to detect clonal cytogenetic abnormalities. RESULTS The majority of patients showed complex karyotypes at diagnosis of tMDS/sAML containing, in particular, complete or partial loss of chromosomes 5 and/or 7. Using single locus-specific FISH probes, significant levels of clonally abnormal cells were found before HDT in 20 of 20 tMDS/sAML patients screened, compared with three of 24 patients screened who currently have not developed tMDS/sAML, at a median follow-up of 5.9 years after HDT. CONCLUSION Prior cytotoxic therapy may play an important etiologic role and may predispose to the development of tMDS/sAML. Using a triple FISH assay designed to detect loss of chromosomal material from 5q31, 7q22, or 13q14, significant levels of abnormal cells can be detected before HDT and may predict which patients are at increased risk of developing secondary disease. Further prospective evaluation of this FISH assay is warranted to determine its predictive power in this setting.

Localisation of a novel region of recurrent amplification in follicular lymphoma to an ∼6.8 Mb region of 13q32‐33

Follicular lymphoma (FL) is characterised by the presence of the t(14;18)(q32;q21) and represents ∼25% of new cases of non‐Hodgkins lymphoma. While the t(14;18) is a well‐documented rearrangement, the role of secondary cytogenetic abnormalities in the development and progression of these tumours remains unclear. Comparative genomic hybridisation was used to characterise changes in DNA copy number in tumour DNA from patients with this malignancy. The mean numbers of deletion and amplification events found in each of the 45 samples studied were 1.8 and 2.3, respectively. Regions of recurrent (>10% tumour samples) gain involved chromosomes 2p13‐16 (16%), 7 (20%), 12 (16%), 13q21‐33 (18%), 18 (27%), and X (36%) and frequent losses localised to 6q (29%) and 17p (20%). Amplification of chromosome 13 represents a novel finding in FL. The minimal amplified region was refined to a 6.8‐Mb interval of 13q32‐33 between the BAC clones 88K16 and 44H20 by fluorescence in situ hybridisation studies using metaphase chromosomes derived from tumour material. There are a number of reports in the literature suggesting that amplification of chromosome 13 also occurs in other human cancers. The location of the putative oncogene on 13q described here in follicular and transformed lymphoma may also be important in the evolution of many other malignancies.

Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multiple time-points reveals high levels of concordance between molecular and immunophenotypic approaches

In this single centre study of childhood acute lymphoblastic leukaemia (ALL) patients treated on the Medical Research Council UKALL 97/99 protocols, it was determined that minimal residual disease (MRD) detected by real time quantitative polymerase chain reaction (RQ‐PCR) and 3‐colour flow cytometry (FC) displayed high levels of qualitative concordance when evaluated at multiple time‐points during treatment (93·38%), and a combined use of both approaches allowed a multi time‐point evaluation of MRD kinetics for 90% (53/59) of the initial cohort. At diagnosis, MRD markers with sensitivity of at least 0·01% were identified by RQ‐PCR detection of fusion gene transcripts, IGH/TRG rearrangements, and FC. Using a combined RQ‐PCR and FC approach, the evaluation of 367 follow‐up BM samples revealed that the detection of MRD >1% at Day 15 (P = 0·04), >0·01% at the end of induction (P = 0·02), >0·01% at the end of consolidation (P = 0·01), >0·01% prior to the first delayed intensification (P = 0·01), and >0·1% prior to the second delayed intensification and continued maintenance (P = 0·001) were all associated with relapse and, based on early time‐points (end of induction and consolidation) a significant log‐rank trend (P = 0·0091) was noted between survival curves for patients stratified into high, intermediate and low‐risk MRD groups.

Development of a human acute myeloid leukaemia screening panel and consequent identification of novel gene mutation in FLT3 and CCND3

A study was undertaken to develop an acute myeloid leukaemia (AML) screening panel to uncover novel recurring gene mutations. Analysis was performed on six genes known to be mutated in AML (RUNX1, FLT3, KIT, CEBPA, PTPN11 and NRAS) and an additional two candidate genes (CCND3 and FES) in a panel of 175 primary human AML samples that included all French–American–British types except M3, and all cytogenetic risk groups. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with two mutations and two patients (1%) with three mutations. Fifty‐five of 88 (63%) patients with normal karyotype AML had at least one mutation. Correlation was observed between KIT mutation and ‘favourable risk’ cytogenetics (P < 0·001), CEBPA mutation and ‘intermediate risk’ cytogenetics (P = 0·045), and PTPN11 mutation and ‘poor risk’ disease (P < 0·001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 were detected (Y589D, D839G, Y842H) that would have been overlooked by conventional gel electrophoresis. A 51‐bp deletion was detected in CCND3 in a patient with normal karyotype AML. This validated panel now provides an important tool to evaluate other candidate genes in the genesis of myeloid malignancy.

Mutation of BRAF is uncommon in AML FAB type M1 and M2.

BRAF (v-raf murine sarcoma viral oncogene homolog B1) is one of three related serine/threonine RAF kinases that form an essential part of the RAS–MAP kinase signalling pathway.1 It has been shown that activating RAS mutations occur in up to 30% of AML cases at presentation2 and that MAP kinase is activated in approximately 50% of AML samples,3 suggesting that this pathway may be important in leukaemogenesis. Recent work by Davies et al has identified BRAF mutations in a variety of cancer cell lines and primary human cancers. These all affect the kinase domain and have been shown to have transforming activity irrespective of RAS mutation status. While the majority of such BRAF mutations was identified in melanoma, colorectal cancer and ovarian tumours, no primary AML samples and only eight AML cell lines were screened. We therefore set out to determine the incidence of BRAF mutations in our AML patient population. DNA was extracted by standard phenol–chloroform method from the peripheral blood or bone marrow of 104 patients with AML-M1 (56) and M2 (48). These patients were referred to St Bartholomew’s Hospital between 1985 and 2001 and were identified from the historical database. Seventy were male and the median age was 53 years (range 18–83). Six cases were of secondary AML. Patients were subdivided according to standard Medical Research Council cytogenetic risk-groups into ‘good’ (20), ‘intermediate’ (67) and ‘poor’ (17). Patients were selected for analysis based on sample availability and assessment of disease infiltration within the sample. Ninety-eight samples were taken at diagnosis and six were taken at relapse. The study was carried out in accordance with the requirements of the local ethical committee. DNA was also extracted from the lung adenocarcinoma cell line NCI-H1395 (ATCC Cat. No. 45508) and the colorectal cancer cell line HT-29 (Central Cell Services Cancer Research UK). These were used as positive controls as they had previously been shown to have BRAF mutations of exon 11 G1403C/G468A (NCIH1395) and exon 15 T1796A/ V599E (HT29), respectively.4 Mutation analysis was restricted to exon 11 and 15 of BRAF as all previously reported mutations cluster to these two exons. Mutation screening was carried out using a PCR-single stranded conformational polymorphism (SSCP) analysis (GenePhor II, Amersham-Pharmacia Biotech, Bucks, UK). Exon 11 (313bp) and exon 15 (224bp) were individually amplified from 50 ng of patient or cell line DNA using an annealing temperature of 52°C. The primers and conditions used have been described previously.4 PCR products were denatured in formamide and electrophoresed at 15°C using the CleanGene system and Disc Buffer Kit pH 8.3. Banding patterns were visualised by silver staining using the Plus One Silver Staining kit (Amersham-Pharmacia Biotech) and a Hoefer automated gel stainer. Aberrant SSCP band shifts were detected for the two positive controls (HT-29 and NCIH1395) and in exon 11 of patient 52. Following removal of nucleotides and primers using Microcon PCR columns (Millipore, Bedford, MA, USA), aberrantly migrating PCR fragments were sequenced directly using an ABI 377 DNA sequencer (Perkin Elmer, Foster City, CA, USA). Sequence analysis for patient 52 identified a novel synonymous G1329A/R443R polymorphism. Representative SSCP results are shown in Figure 1.

Cytogenetic and molecular evidence of marrow involvement in extramedullary acute myeloid leukaemia

A diagnosis of granulocytic sarcoma was made in a 2‐year‐old child based on the detection of myelomonocytic blasts in tissue obtained from a subcutaneous nodule with no evidence of concomitant disease in the bone marrow. The child responded to systemic chemotherapy and is in remission 3 years later. An identical clone with an in frame fusion of the MLL and AF10 genes was identified from both tissue and bone marrow samples. The generation of an in frame MLL–AF10 fusion requires complex intra‐ and interchromosomal exchanges between chromosomes 10 and 11. In this case, an intrachromosomal rearrangement of chromosome 5 was also observed. This case illustrates the presence of systemic disease in extramedullary leukaemia, its response to systemic rather than topical therapy and suggests that the events leading to chromosomal translocations in leukaemia may be part of a generalized intracellular event.

Identification and molecular characterisation of a CALM-AF10 fusion in acute megakaryoblastic leukaemia

The t(10;11)(p13;q14–21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927–2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.

The characterisation of the lymphoma cell line U937, using comparative genomic hybridisation and multi-plex FISH

The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia). Apart from this translocation, very little is known about the other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to characterise fully all of the cytogenetic alterations present in the U937 cell line. M-FISH analysis confirmed the presence of the t(10;11) and an apparently normal copy of both chromosomes 10 and 11. A t(1;5) translocation was observed as well as several unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes could result in fusion gene products involved in carcinogenesis or the positions of putative oncogenes and tumour suppressor genes. A good correlation between conventional G-banding, CGH and M-FISH was observed.