Lindsey K. Goff

Number of CD4+ Cells and Location of Forkhead Box Protein P3–Positive Cells in Diagnostic Follicular Lymphoma Tissue Microarrays Correlates With Outcome

PURPOSE To examine the immune microenvironment in diagnostic follicular lymphoma (FL) biopsies and evaluate its prognostic significance. PATIENTS AND METHODS Immunohistochemistry was used to study numbers and location of cells staining positive for immune cell markers CD4, CD7, CD8, CD25, CD68, forkhead box protein P3 (FOXP3), T-cell intracellular antigen-1, and Granzyme B in tissue microarrays of paraffin-embedded, diagnostic lymph node biopsies taken from 59 FL patients who lived less than 5 years (short-survival group; n = 34) and more than 15 years (long-survival group; n = 25). RESULTS CD4 and FOXP3 expression were significantly different between the two groups. Samples from the long-survival group were more likely than those from the short-survival group to have CD4+ staining cells and to have FOXP3-positive cells in a perifollicular location. CONCLUSION This study has identified differences in immune cell composition of the diagnostic FL lymph node immune microenvironment and these have the potential for use as prognostic biomarkers in a routine histopathology setting.

Frequency of the Bcl-2/IgH Rearrangement in Normal Individuals: Implications for the Monitoring of Disease in Patients With Follicular Lymphoma

PURPOSE To determine the incidence and frequency of the Bcl-2/IgH rearrangement in the peripheral blood of normal individuals to define the potential complication this may pose for the molecular monitoring of disease in patients with follicular lymphoma (FL). MATERIALS AND METHODS The incidence and frequency of the major breakpoint cluster region rearrangement in DNA extracted from peripheral blood or lymphoblastoid cell lines from 481 normal individuals was determined using a TaqMan real-time polymerase chain reaction assay (PE Applied Biosystems, Foster City, CA). RESULTS Twenty three percent of samples were positive for the Bcl-2/IgH rearrangement, with approximately 3% of these at levels of more than 1 in 10(4) cells. CONCLUSION The presence of circulating Bcl-2/IgH+ cells, other than those derived from the malignant clone, could confound the detection and quantitation of minimal residual disease in patients with FL, particularly at low levels of tumor burden.

Transformation of follicular lymphoma to diffuse large B-cell lymphoma proceeds by distinct oncogenic mechanisms

This study was undertaken to further elucidate the biological mechanisms underlying the frequent event of transformation of follicular lymphoma (FL) to diffuse large B‐cell lymphoma (t‐FL). The gene expression profiles of 20 paired lymph node biopsies, derived from the same patient pre‐ and post‐transformation, were analysed using the Lymphochip cDNA microarray. TP53 mutation analysis was performed and copy number alterations at the c‐REL and CDNK2A examined. Immunohistochemistry was performed on an independent panel of paired transformation paraffin‐embedded samples. Transformed follicular lymphoma was predominantly of the germinal centre B‐like phenotype both at the mRNA and protein level. Despite this homogeneity, transformation proceeded by at least two pathways. One mechanism was characterised by high proliferation, as assessed by the co‐ordinately expressed genes of the proliferation signature. This group was associated with the presence of recurrent oncogenic abnormalities. In the remaining cases, proliferation was not increased and transformation proceeded by alternative routes as yet undetermined. Genes involved in cellular proliferation prevailed amongst those that were significantly increased upon transformation and T cell and follicular dendritic‐associated genes predominated amongst those that decreased. t‐FL is a germinal centre B (GCB)‐like malignancy that evolves by two pathways, one that is similar in proliferation rate to the antecedent FL and the other that has a higher proliferation rate and is characterised by the presence of recognised oncogenic abnormalities.

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.

A limited role for TP53 mutation in the transformation of follicular lymphoma to diffuse large B-cell lymphoma

The role of TP53 mutation in transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (t-FL) was examined in a panel of 91 lymph node biopsies derived from 29 patients pre- and post-transformation. The entire TP53 coding sequence was screened and immunocytochemistry performed to determine expression of p53 and its key regulator MDM2. A total of 10 mutations were detected in eight patients (28%), although none were present at FL diagnosis. Mutations were not detected solely at the time of transformation; in three patients, mutated TP53 arose in at least one antecedent FL sample (6 months, 2.5 years and 4 years prior to transformation). Loss of heterozygosity at the TP53 locus occurred in 2/20 informative patients (only in t-FL samples). p53 staining was positive in 82% (9/11) of available biopsies with a missense mutation, and negative in 71% (45/63) with wtTP53. MDM2 expression was significantly higher in t-FL samples (mean 72% positive; 95% confidence interval (95% CI) 68–76%) than FL (mean 58% positive; 95% CI 54–62%) (P<0.001) but did not correlate with TP53 status. TP53 mutation has only a limited role in the transformation of FL, exerting a heterogeneous influence upon phenotypic change. In contrast, dysregulation of MDM2 is frequent and may provide a more rational therapeutic target.

BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations

The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein overexpression in most follicular lymphomas. However the expression of BCL2 is not always homogeneous and may demonstrate a variable degree of heterogeneity. This study analysed BCL2 protein expression pattern in 33 cases of t(14;18)‐positive follicular lymphomas using antibodies against two different epitopes (i.e. the widely used antibody BCL2/124 and an alternative antibody E17). 16/33 (49%) cases demonstrated strong BCL2 expression. In 10/33 (30%) cases, BCL2 expression was heterogeneous and in some of these, its loss appeared to be correlated with cell proliferation, as indicated by Ki67 expression. Double immunofluorescence labelling confirmed an inverse BCL2/Ki67 relationship, where in 24/28 (86%) cases cellular expression of BCL2 and Ki67 was mutually exclusive. In addition, seven BCL2 ‘pseudo‐negative’ cases were identified in which immunostaining was negative with antibody BCL2/124, but positive with antibody E17. Genomic DNA sequencing of these ‘pseudo‐negative’ cases demonstrated eleven mutations in four cases and nine of these were missense mutations. It can be concluded that in follicular lymphomas, despite carrying the t(14;18) translocations, BCL2 protein expression may be heterogeneous and loss of BCL2 could be related to cell proliferation. Secondly, mutations in translocated BCL2 genes appear to be common and may cause BCL2 pseudo‐negative immunostaining.

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.

Quantitative PCR Analysis for Bcl-2/IgH in a Phase III Study of Yttrium-90 Ibritumomab Tiuxetan As Consolidation of First Remission in Patients With Follicular Lymphoma

PURPOSE The randomized First-Line Indolent Trial (FIT) was conducted in patients with advanced follicular lymphoma (FL), to evaluate the safety and efficacy of yttrium-90 ((90)Y) ibritumomab tiuxetan given as consolidation of complete or partial remission. This study of minimal residual disease was undertaken in parallel, to determine the rate of conversion from bcl-2 polymerase chain reaction (PCR) -detectable to -undetectable status and the corresponding effect on progression-free survival (PFS). PATIENTS AND METHODS Blood samples from 414 patients ((90)Y-ibritumomab, n = 208; control, n = 206) were evaluated using real-time quantitative polymerase chain reaction (RQ-PCR); 186 were found to have the bcl-2 rearrangement and were thus eligible for inclusion in the RQ-PCR analysis. Results Overall, 90% of treated patients converted from bcl-2 PCR-detectable to -undetectable disease status, compared with 36% in the control group. Treatment significantly prolonged median PFS in patients converting to bcl-2 PCR-undetectable status (40.8 v 24.0 months in the control group; P < .01, hazard ratio [HR], 0.399). In patients who had bcl-2 PCR-detectable disease at random assignment, treatment significantly prolonged median PFS (38.4 v 8.2 months in the control group; P < .01, HR, 0.293). CONCLUSION Eradication of PCR-detectable disease occurred more frequently after treatment with (90)Y-ibritumomab tiuxetan and was associated with prolongation of PFS.

Expression profile of wild-type ETV6 in childhood acute leukaemia

Summary. Comparative expression analysis of wild‐typeETV6 in the disease state showed an absence of expression in ETV6–CBFA2 acute lymphoblastic leukaemia (ALL) when compared with non‐ETV6–CBFA2 ALL and acute myeloid leukaemia. Fluorescent in‐situ hybridization and loss of heterozygosity studies showed that 73% of the ETV6–CBFA2 samples had a fully or partially deleted second ETV6 allele, explaining the lack of wild‐type expression in these patients. Although the second ETV6 allele was identified in the remaining patients, no ETV6 expression was detected. These observations support the hypothesis that loss of ETV6 expression is a critical secondary event for leukaemogenesis in ETV6–CBFA2 ALL.

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