Faculty Bibliography

The advent of molecular biological techniques has led to a radical improvement in the evaluation of haematological disorders and offers exciting future prospects. In particular it has led to the improved recognition of distinct clinicopathological entities defined by a combination of morphological, immunophenotypic and chromosomal features. Antibody-based techniques, namely immunophenotyping and immunocytochemical techniques, are essential to malignant haematological diagnosis. More novel is the diagnostic use of antibodies against novel fusion proteins, for example in AML, M3 and anaplastic lymphoma. PCR-based techniques allow the identification of genetic mutations and translocations including such common translocations as t(14;18), t(9;22), inv 16 and t(8;21). Fish-based techniques are greatly improving the ability to detect genetic abnormalities including those conditions with low cell proliferation and current research-based techniques include the combination of FISH with cell surface markers (FICTION), FIBRE FISH and Comparative Genomic Hybridization. Molecular techniques are essential in monitoring residual disease which is best illustrated in CML. The development of real-time automated PCR offers exciting prospects in this field. The increasing number of tests, the need for an integrated approach to diagnosis and the need for cost effectiveness indicate that such services should be provided by a specialized haematology laboratory.

PURPOSE/OBJECTIVE:Ninety-five percent of children with acute lymphoblastic leukemia (ALL) will achieve a remission, but approximately 25% will relapse. Identifying these patients is difficult, as patients with adverse prognostic features at presentation are rare and the majority are standard risk. Analysis of minimal residual disease (MRD) may be able to determine those at risk of relapse, but the best method by which this can be accomplished has yet to be defined. The object of this study was to determine the predictive value of residual disease detection in a group of standard-risk patients with precursor-B ALL at a fixed point in therapy (week 20) using a simple fluorescent consensus immunoglobulin H (IgH) heavy chain polymerase chain reaction (PCR). PATIENTS AND METHODS/METHODS:Forty-two patients who presented with precursor-B ALL with standard-risk clinical features and treated according to either the Medical Research Council (MRC) UKALL X or XI protocols were assessed using a combination of both fluorescent consensus framework I and framework III Ig heavy-chain PCR. The results of the PCR were analyzed on an ABI 373 gene sequencer with genescan software (Applied Biosystems, Foster City, CA). Clonal rearrangements detected at presentation were looked for at week 20. RESULTS:Of 42 patients, 35 had a clonal population detectable at presentation; of these, seven had more than two clonal rearrangements; this latter group showed a similar disease-free survival (DFS) to the group as a whole. Thirty of 35 patients were analyzed before their second course of intensification therapy at week 20. At this point, nine of 30 had a detectable clonal rearrangement, eight (89%) of whom have since relapsed with a median DFS of 27.5 months. Of the rest of the group (n=21), in whom no clonal rearrangement was detectable, only six (21%) have relapsed. CONCLUSION/CONCLUSIONS:Fluorescent IgH PCR at week 20 provides a sensitive and specific means to predict ultimate relapse (57% and 89%, respectively) and is a simple yet promising technique for the identification of patients at risk of poor outcome.

Autologous transplantation in CML has been a focus of interest over the last few years. Determining the indications, optimal timing and method for this procedure remains controversial. One approach has been the mobilisation of Philadelphia chromosome negative (Ph-) peripheral blood stem cells following high-dose chemotherapy as a method of purging the graft. We have described a mobilisation regimen of 7 days of hydroxyurea followed by G-CSF and have shown it to be substantially less toxic than other methods. We now report further experience with this technique in a total of 18 patients and the outcome of transplantation in seven patients using cells so-derived. Following mobilisation, approximately a third of patients had 100% Ph-collections and half had less than 50% Ph+ collections. All patients were 100% Ph+ prior to mobilisation. Six out of seven transplanted patients showed sustained engraftment and two of these patients became 18 and 34% Ph+ 3 months post-transplant. Five patients remain alive and well 13 to 25 months post-autograft. In conclusion, we have developed a well-tolerated regimen for Ph- PBSC mobilisation and have demonstrated that such cells are capable of sustained engraftment and of producing significant cytogenetic responses.

The diagnosis of haematological malignancies has undergone major conceptual changes in the past decade. Accurate diagnosis now depends on a combination of morphological examination, cell marker studies, and molecular biological techniques. Specialist haematopathology diagnostic services are best provided in an integrated laboratory, able to carry out the full range of techniques and staffed by pathologists and scientists with expertise in the investigation of leukaemia and lymphoma.

Liposomal encapsulation of anthracyclines is a potential method of drug targeting, altering both the antitumour activity and side-effect profile of anthracyclines. Liposomal daunorubicin (daunoxome) shows both altered pharmacokinetics and a potential for reducing dose-limiting cardiotoxicity compared to conventional daunorubicin. Anthracyclines have a common role in the treatment of multiple myeloma, a prevalent and fatal haematological malignancy. Avoiding cumulative anthracycline toxicity in these patients is important. There is also a need for more effective relapse schedules given that many patients have chemosensitive disease at relapse. We have analysed daunoxome in vitro in myeloma cell lines using a thymidine-based cytotoxicity assay and show superior efficacy compared to a pegylated liposomal doxorubicin derivative. Subsequently we have treated seven relapsed myeloma patients with a regime consisting of oral CCNU 25-50 mg/m2 on day 1, 4 days of oral dexamethasone 10 mg/m2 and intravenous daunoxome (liposomal daunorubicin) given for 4 days (total 100 mg/m2). The main toxicity was myelosuppression but non-haematological toxicity was minimal and the regime was well tolerated. Four out of seven of these heavily pretreated patients responded. Together with the in vitro data on its cytotoxicity in myeloma and its favourable pharmacokinetic profile further studies of liposomal daunorubicin in myeloma would be warranted.

The reduced levels of normal immunoglobulin in patients with myeloma may be due to suppression of normal B-cell differentiation. However, reports on the numbers of B cells vary, with some finding decreases consistent with immunoparesis, and others reporting expansions of phenotypically aberrant cells. We have therefore assessed the phenotype and levels of B lymphocytes in patients at presentation (n = 23), in plateau or complete remission (PB n = 42, BM n = 18), and in relapse (PB n = 17, BM n = 14), in comparison to normal individuals (n = 10). Phenotypic analysis was performed using five-parameter flow cytometry, with CD14 used to exclude monocytes where necessary. We found no evidence of a phenotypically distinctive blood or marrow B-cell population in patients with myeloma, nor of an increase in the levels of any B-cell subset. Numbers of blood CD19+ 38+ normal plasma cell precursors were significantly reduced in presentation/relapse patients, but not in patients in plateau/remission. Total CD19+ cells were significantly reduced only in patients with circulating myeloma cells, detected by IgH-PCR. In the marrow, CD19+ B cells expressing CD5, CD10, CD34, CD38, CD45(low) and Syndecan-1 were significantly decreased at presentation/relapse, but not in patients in plateau/remission. The majority of these antigens are expressed by normal B-cell progenitors, indicating that myeloma also affects the early stages of B-cell development. The suppression of progenitor cells was not restricted to B-lymphoid differentiation, as total CD34+ cells were also significantly reduced in the marrow of myeloma patients at presentation. These results indicate that, if neoplastic B cells are present in myeloma, they are low in number and have a phenotype similar to their normal counterparts. Furthermore, there is a reversible suppression of CD19+ B lymphocytes that correlates inversely with disease stage, and specifically affects the early and late stages of normal B-cell differentiation.

Secular trends in the incidence of lymphoproliferative disorders on North and West Yorkshire and Humberside from 1985 to 94 were studied and changes in incidence by tumour subtype were analysed. Population-based data on the incidence of lymphoproliferative disorders were obtained from a specialist registry with a high level of ascertainment. Cases of chronic lymphocytic leukaemia and plasma cell myeloma were excluded and the remaining cases classified as Hodgkin's disease and non-Hodgkin's lymphoma (NHL). NHL were subdivided by site of origin and immunophenotype. Nodal B-cell lymphomas were further classified as diffuse large B-cell lymphoma, follicle centre lymphoma, mantle cell lymphoma and miscellaneous. During the study period there was a significant increase in total lymphoproliferative disorders with an average change of 2.5% per annum equivalent to 0.84/10,0000. Most of this increase was due to an increasing incidence of extranodal B-cell lymphomas and peripheral T-cell lymphomas. A numerically small but significant increase in diffuse large B-cell lymphomas was seen. There was no significant increase in other subtypes. The increased incidence of lymphomas in the area studied is mainly due to changes in two specific subgroups. There are several reasons why changes in extranodal B-cell lymphoma and peripheral T-cell lymphoma may have been particularly affected by changing diagnostic practices. Epidemiological studies of particular subtypes of lymphoproliferative disorder facilitate the identification of environmental factors involved in the pathogenesis of these tumours.