Faculty Bibliography

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Homologous recombination (HR) is one of the main pathways for the repair of DNA double strand breaks (DSBs). To investigate whether inherited variants in genes encoding proteins that repair DSBs by HR modulate acute myeloid leukaemia (AML) risk, we have examined the frequency of two variants in the 5' untranslated region (UTR) of RAD51 (RAD51 135 G>C and the RAD51 172 G>T) in a large case-control study of acute myeloid leukaemia (AML). Inheritance of a RAD51 135 C allele was associated with a reduced risk of estimate for AML (odds ratio (OR) 0.56, 95% confidence intervals (CI), 0.38-0.83), while the RAD51 172 T allele was not associated with AML. The RAD51 135 and 172 variants were in strong linkage disequilibrium, with three out of the four possible haplotypes being observed in the population. The protective effect associated with the RAD51 135 C allele was found to be associated with inheritance of the RAD51 135-172 C-G haplotype (cases 3.9% versus controls 6.5%, OR 0.61, 95% CI 0.42-0.90). These data suggest that variants in the RAD51 HR gene may modulate genetic predisposition to AML.

In vivo and in vitro studies suggest human growth hormone (hGH) receptors on bone marrow stem cells may be biologically active and could be exploited to promote haemopoetic recovery after intensive chemotherapy. Patients with haematological malignancies receiving intensive chemotherapy and requiring hospitalization were randomized in a double-blind, placebo-controlled single-centre trial. Patients were randomly assigned to receive either hGH 500 microg/day or placebo, for 6 weeks. There was no significant difference in patient characteristics at baseline between the placebo and treatment arms. Patients treated with hGH showed significantly faster recovery of platelets to 25 x 10(9)/l (median of 16 versus 19 days; P = 0.03) compared to the placebo-controlled arm (hazard ratio 1.47 favouring hGH, 95% confidence interval (CI), 1.03-2.08). Time to relapse did not differ significantly between arms. There was no change in the anthropometric parameters at the start and end of hGH/placebo therapy. The study drug was well tolerated. Treatment with hGH in physiological doses improves platelet recovery, but is not associated with a lower relapse rate or improved anthropometric parameters in patients receiving intensive chemotherapy.

The MRE11-RAD50-NBS1 tri-complex is involved in the cellular response to DNA double strand breaks, detecting DNA damage, activating cell cycle checkpoints and apoptosis. Defects in members of the tri-complex are linked to increased chromosomal instability and in lymphoma predisposition. Using genotyping data from six intronic or gene flanking variants in MRE11, five in NBS1 and six in RAD50 in 461 non-Hodgkin's lymphoma cases and 461 age, sex matched controls, Phase 2.1 was used to impute haplotypes for each of these genes. It was observed that the average variant density (12 kb) was dense enough to capture the majority of genetic variation for each locus examined, encoded by four or five common haplotypes. There were no significant differences in allele or genotype frequency, global haplotype distribution between the cases and control, nor effect for individual haplotypes when analysed by unconditional logistic regression for either RAD50 or NBS1. A protective effect against follicular lymphoma was seen for the MRE11 rs601341 variant, the homozygous T allele being associated with an odds ratio (OR) of 0.50, 95% confidence interval (95% CI) 0.26 - 0.97, while a protective effect was seen for the MRE11 haplotype GCTCA (OR 0.72, 95% CI 0.53 - 0.97) for diffuse large B-cell lymphoma. While reproduction of this data in other datasets is indicated, the results are indicative for a role for MRE11 in non-Hodgkin's lymphoma.

The genetics of multiple myeloma is a vastly studied field in which techniques such as classical cytogenetics, fluorescence in situ hybridization, and comparative genomic hybridization have been used. More recently, single nucleotide polymorphism (SNP)-based mapping arrays have become available that allow the identification of regions of gain or loss as small as 2.5 kb. In addition to the increased resolution of SNP-based arrays, the detection of loss of heterozygosity is also possible. This allows the identification of loss of heterozygosity regions that arise through monosomy and recombination, resulting in uniparental disomy, which cannot be detected by conventional genetic methods. In this review, we discuss the benefits of SNP-based arrays along with some of the drawbacks and how that data can be used in conjunction with expression data to identify genes with altered expression in regions of interest.

Alemtuzumab has been proposed as a therapeutic agent in myeloma. CD52 was detected on plasma cells in 46/106 patients but levels were 30-fold lower than on alemtuzumab-responsive cells (n=138) and 8-fold lower than on alemtuzumab-resistant cells (n=57). The data suggest that myeloma plasma cells are unlikely to be depleted by alemtuzumab in most patients.

A retrospective case-matched study was conducted to compare the oral regimen CTD (cyclophosphamide - thalidomide - dexamethasone) and infusional CVAMP (cyclophosphamide - vincristine - doxorubicin - methylprednisolone) as induction therapy followed by autologous peripheral blood stem-cell transplantation (PBSCT) for newly diagnosed multiple myeloma patients. The response rate after three cycles of treatment was statistically higher with CTD (n = 27) compared to CVAMP (n = 27) (89% vs. 56%, P = 0.016). Toxicity studies showed more neutropenia (grade 3/4) (4% vs. 60%, P = 0.0002) with CVAMP and more thrombotic episodes with CTD (11% vs. 4%). CTD may emerge as the superior induction regimen prior to PBSCT, in terms of high efficacy and better tolerability.