Michael Jenner

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.

Development of a quantitative real-time polymerase chain reaction method for monitoring CEBPA mutations in normal karyotype acute myeloid leukaemia

The persistence of minimal residual disease (MRD) posttreatment may provide an early indicator of impending relapse in acute myeloid leukaemia (AML) (Raanani & Ben-Bassat, 2004). The absence of an appropriate molecular signature in 40–60% of AML patients who have normal cytogenetics associated with intermediate risk has limited the analysis in this group. Through advances in mutation analysis a list of recurring gene mutations as potential MRD targets are now being compiled. Many AML cells have more than one recurring mutation, indicating that there is a multistep pathogenesis of disease. Mutation events in AML have been detected by analysis of the blast cells; therefore, chronology of mutational events within the stem cell and/or early progenitor populations has not been established. Many of these mutations result in the insertion or deletion of a small number of additional nucleotides that should allow for the design of a specific primer against the unique sequence generated by the mutation (Pabst et al, 2001). Found in approximately 20% of normal karyotype AML, the presence of CEBPA mutation segregates with favourable risk among this otherwise intermediate risk group. The CEBPA gene encodes the granulocytic differentiation factor CCAAT Enhancer Binding Protein-alpha and plays a crucial role in granulocytic differentiation of haematopoietic stem cells and has antiproliferative activity in various cell types. Mutation in CEBPA was investigated as a potential MRD marker because of its prevalence in normal karyotype AML, the frequency of insertion and deletion mutations enabling a straightforward design of mutation-specific primers and concordance between presentation and relapse (Tiesmeier et al, 2003). Four patients with normal karyotype AML FAB type M1 were selected for study based on the availability of diagnostic (patients 1, 3 and 4) or relapse (patient 2) bone marrow (BM) and on the presence of CEBPA mutations identified during previous screening (Snaddon et al, 2003). Samples were collected and analysed in accordance with the requirements of the local ethics committee. Five real-time quantitative polymerase chain reaction (RQ-PCR) assays were developed that selectively amplified mutated CEBPA DNA from four patients. Figure 1A and B demonstrates the RQ-PCR strategy used in this study. Three patients (1, 2 and 3) had an insertion mutation in the region of the gene that codes for the C-terminal DNA-binding/basic leucine zipper domain of the protein, the fourth contained mutations corresponding to both the Nand C-terminal regions of the C/EBP-a protein (4a and 4b respectively). Mutations in 1, 2, 4a, and 4b were all tandem duplications ranging from 3 to 57 base pairs (bp) in length while patient 3 had a 3-bp insertion. Mutation-specific forward primers were designed by making use of the unique sequences generated by the insertion mutations along with the introduction of single nucleotide changes in the primers to prevent annealing to the wild-type (WT) sequence. Patient-specific primer sequences are given in Fig 1C. Real-time PCR analysis was performed on 25 ll reaction mixture in duplicate using Taqman chemistry on the ABI PRISM 7700 Sequence Detector (Applied Biosystems, Warrington, UK) with default settings. b-2-Microglobulin was used as the endogenous reference gene to assess total cell number per well. The specificity of each RQ-PCR assay was tested by amplifying 750 ng (equivalent to 125 000 cells) of each patient’s diagnostic BM DNA, wt DNA, patient diagnostic BM DNA serially diluted in wt DNA and no template controls, in duplicate. An assay was deemed to be specific if no amplification was observed with wt DNA. To test the sensitivity of each assay a standard curve was generated by five serial dilutions of diagnostic DNA in water (ranging from 125 000 to 4 cells per well). In all amplification plots the same threshold was maintained to allow comparison between experiments. The correlation coefficient of the standard curves was at least 0Æ95, indicating that the assays had a high probability of precise quantification. The mean Ct for DNA equivalent to 125 000 cells was 24Æ6 (range 24–27). The mean slope of the dilution curves was )3Æ2 (range )3Æ5 to )2Æ8), indicating good amplification efficiencies (the perfect theoretical slope is )3Æ3). As shown in Fig 1C, the maximum reproducible sensitivity (defined as the dilution for which duplicates were both positive) for all the patient mutation assays was 10 (8Æ0 · 10; 10 in 125 000) and therefore were of the required order of sensitivity, >10, for an MRD monitoring assay. Insufficient material was available for retrospective MRD monitoring in these patients. Instead we have taken advantage of the assay’s ability to quantify mutated cells for the determination of presence or absence of mutation within the leukaemic stem cell (LSC) population. The LSC generally accounts for approximately 1% of leukaemic cells (Hope et al, 2004). As with the normal haematopoietic stem cell (HSC) compartment, LSCs are not functionally homogeneous and correspondence

Differential and tumor-specific expression of CD160 in B-cell malignancies

CD160 is a human natural killer (NK)-cell-activating receptor that is also expressed on T-cell subsets. In the present study, we examined 811 consecutive cases of B-cell lymphoproliferative disorders (B-LPDs), and demonstrated CD160 expression in 98% (590 of 600) of chronic lymphocytic leukemia (CLL) cases, 100% (32 of 32) of hairy cell leukemia (HCL) cases, 15% (5 of 34) of mantle cell lymphoma (MCL) in the leukemic phase, and 16% (23 of 145) of other B-LPD cases. CD160 transcript and protein were absent in the normal B-cell hierarchy, from stem cells, B-cell precursors, maturing B cells in the germinal center, and circulating B cells, including CD5(+)CD19(+) B1 cells in umbilical cord. CD160 positivity was significantly higher in CLL and HCL in terms of percentage (65.9% and 67.8%, respectively, P < .0001) and median fluorescence intensity (552 and 857, respectively, P < .0001) compared with all other B-LPD cases. Lymph node CLL samples were also CD160(+). Using the disease-specific expression of CD5, CD23, and CD160, a score of 3 characterized CLL (diagnostic odds ratio, 1430); a score of 0 excluded CLL, MCL, and HCL; and the CD23/CD5 ratio differentiated CLL from leukemic CD23(+) MCL. In the B-cell lineage, CD160 is a tumor-specific antigen known to mediate cellular activation signals in CLL, and is a novel target for therapeutic manipulation and monitoring of minimal residual disease.

Reliable detection of clonal IgH/Bcl2 MBR rearrangement in follicular lymphoma: methodology and clinical significance

The prognostic significance of IgH/Bcl2 rearrangement in follicular lymphoma (FL) remains contentious; polymerase chain reaction (PCR) methodology and tissue source variability may account for some inconsistencies. As IgH/Bcl2 major breakpoint region (MBR) sequences may be found in normal blood, an MBR+ result by conventional PCR in blood/bone marrow may not indicate FL. To establish tumour MBR status, 190 lymphoid tissue samples with histologically evident FL (and therefore >1% tumour cells) were examined by real‐time quantifiable PCR; 50% (95/190) had clonal MBR IgH/Bcl2 (MBR was considered clonal when >1%). Overall survival (median = 11·5 years) of MBR+ and MBR− patients was not significantly different.

The relative role of peripheral blood and bone marrow for monitoring molecular evidence of disease in follicular lymphoma by quantitative real‐time polymerase chain reaction

Summary.  Peripheral blood (PB) and bone marrow (BM) are used interchangeably for t(14;18) (IgH/BCL‐2) molecular monitoring in follicular lymphoma (FL) and detection of rearrangement after treatment has been correlated to increased risk of relapse. To determine the relative value of each tissue, MBR t(14;18) was quantified by real‐time polymerase chain reaction in 52 simultaneous paired PB and BM samples from 38 FL patients. In total, 79% of sample pairs taken in remission (n = 19) or when no morphological disease was evident in the BM (n = 29) had t(14;18) copy number within one log difference and the median difference was small. These findings suggest that, in remission, PB may be adequately monitored. In general, however, higher copy number was detected in BM than in the corresponding PB sample.

Successful therapy of transplant-associated veno-occlusive disease with a combination of tissue plasminogen activator and defibrotide

A 36-year-old man underwent matched unrelated donor bone marrow transplantation for chronic myeloid leukaemia. He developed severe hepatic veno-occlusive disease as an early post-transplant complication. Tissue plasminogen activator was initially felt to be contraindicated since the patient had concomitant pericarditis. Defibrotide was therefore commenced as treatment for veno-occlusive disease. The pericarditis improved but the veno-occlusive disease continued to worsen (peak bilirubin 353 μmol/I). Tissue plasminogen activator followed by a heparin infusion was therefore administered. However, he proceeded to develop haemorrhagic cardiac tamponade that required drainage. Thrombolysis was therefore discontinued and treatment with defibrotide resumed after an interval of 48 h. The veno-occlusive disease gradually resolved and defibrotide was discontinued once the bilirubin had plateaued. He was discharged home on day +52 post-transplant.

Patterns of recruitment into acute myeloid leukaemia (AML) 15 and outcome for young patients with AML at a single referral centre.

This study assessed the recruitment to an acute myeloid leukaemia (AML) trial (AML15) in a single centre, evaluated whether outcome was influenced by trial entry and whether the trial population could be considered representative of all AML patients by retrospective comparison of patient characteristics, trial entry and outcome for 81 consecutive patients (<60 years). All patients were considered for trial entry, however the trial was not offered to 12 (15%) patients. These patients had a worse outcome than the 69 (85%) patients that were invited to participate (P = 0·04). Sixteen patients (23%) invited to participate in the trial declined and were treated on equivalent protocols. These patients had a similar outcome to those who accepted entry into the trial (P = 0·2). These results suggested that physicians exert a selection bias when evaluating patients for trial entry. Thus the overall survival estimates generated from large phase III trials may indicate that the outcome for patients with AML is better than the outcome experienced in the ‘real’ world. Furthermore, patients who are considered appropriate for randomization into a trial, but decline entry, experience a similar outcome to those treated on trial when treated in an equivalent manner.

The risk of antimalarials in patients with renal failure

We present here a patient with end stage renal failure who received two weeks antimalarial prophylaxis at full dose leading to life threatening toxicity with severe acute megaloblastic anaemia, symptomatic pancytopenia and exfoliative dermatitis. Prompt recognition and treatment can rapidly reverse these fatal effects but more importantly, education of patients before travel is imperative in preventing such events.